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LA1512

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train_pubmed_ORC_4096_20k

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all subjects , aged 70 years , living in the community of uppsala , sweden , were eligible for the study . the subjects were chosen from the register of community living and were invited in a randomized order from april 2001 to june 2004 . of 2,025 subjects invited , 1,016 subjects were investigated at baseline ( participation rate 50.1% ) . from march 2006 to september 2009 , when the subjects turned 75 years of age , reinvestigation of the cohort was performed with a follow - up rate of 81.4% . among participants at baseline , 992 subjects had valid measurement of pops ; after excluding 3 subjects without information on fasting blood glucose , 989 subjects constituted the study sample for the cross - sectional analyses . the sample size for the prospective analyses was 725 subjects after excluding 112 type 2 diabetes cases at baseline and 152 subjects who were not followed up ( of whom 52 had died ) . the study was approved by the ethics committee of the university of uppsala , and the participants gave written informed consent . the participants were asked about their health behaviors , medical history , and regular medication . all subjects were investigated in the morning after an overnight fast , with no mediation or smoking allowed after midnight . bmi was calculated as weight in kilograms divided by the square of height in meters ( kg / m ) . waist circumference was measured in the supine position midway between the lowest rib and the iliac crest . serum cholesterol and triglyceride concentrations were determined with an enzymatic assay , and fasting blood glucose was analyzed with the hexokinase method using an architect ( abbott laboratories , abbott park , il ) . type 2 diabetes was defined as a fasting blood glucose 6.2 mmol / l or the use of insulin or oral hypoglycemic agents . analyses of pops were performed using a micromass autospec ultima ( waters , mildford , ma ) high - resolution chromatography coupled to a high - resolution mass spectrometry system based on the method by sandau et al . a total of 21 pops were measured : 14 pcb congeners ; 5 organochlorine pesticides ; 1 octachlorodibenzo - p - dioxin ; and 1 brominated diphenyl ether congener ( supplementary table 1 ) . among 21 pops measured , 2 organochlorine pesticides ( trans - chlordane and cis - chlordane ) with a detection rate < 10% were not included in the final analyses . because pops predominantly are carried in the lipid component of the blood , lipid - standardized concentrations , the concentrations of pops divided by total serum lipid content , tended to be reported in epidemiological studies . however , pops can disturb lipid metabolism ( 14,15 ) , and dyslipidemia also is involved in the pathogenesis of type 2 diabetes ( 16 ) . therefore , wet - weight concentrations ( pops concentrations in pg / ml not divided by lipids ) would have greater validity because lipid concentrations may be intermediate in a causal chain linking pops and type 2 diabetes . in addition , one simulation study ( 17 ) reported that wet - weight concentrations adjusting for triglycerides and total cholesterol in a model as covariates induced less bias than lipid - standardized concentrations . therefore , we present wet - weight concentrations ( pg / ml ) , adjusting for triglycerides and total cholesterol by including these two lipid profiles in the models as covariates . in fact , analyses on the basis of lipid - standardized concentrations showed similar results in the current study ( data not shown ) . analyses were done on individual pops and summary pop measures . in the individual analyses , cutoff points of individual pops were provided in the supplementary table 2 . in the summary analyses , the individual ranks were summed ( 1922 ) for each pop , as applied in previous studies ( 5,6 ) , and summed values also were divided into quintiles . odds ratios ( ors ) and 95% cis for the risk of type 2 diabetes were estimated using logistic regression . the covariates were sex , bmi ( continuous ) , cigarette smoking ( current , former , and never ) , alcohol consumption ( grams per week ) , exercise ( no , mild , moderate , and vigorous ) , triglycerides ( continuous ) , and total cholesterol ( continuous ) . the additional value of adding pops to the traditional risk factors was evaluated by c - statistics , and tests of reclassification ( net reclassification index [ nri ] ) and discrimination ( improved discrimination index [ idi ] ) according to the method proposed by pencina et al . ( three risk groups were considered : < 3% ( low risk ) ; 37% ( intermediate risk ) ; and > 7% ( high risk ) risk of diabetes development . the participants were asked about their health behaviors , medical history , and regular medication . all subjects were investigated in the morning after an overnight fast , with no mediation or smoking allowed after midnight . bmi was calculated as weight in kilograms divided by the square of height in meters ( kg / m ) . waist circumference was measured in the supine position midway between the lowest rib and the iliac crest . serum cholesterol and triglyceride concentrations were determined with an enzymatic assay , and fasting blood glucose was analyzed with the hexokinase method using an architect ( abbott laboratories , abbott park , il ) . type 2 diabetes was defined as a fasting blood glucose 6.2 mmol / l or the use of insulin or oral hypoglycemic agents . analyses of pops were performed using a micromass autospec ultima ( waters , mildford , ma ) high - resolution chromatography coupled to a high - resolution mass spectrometry system based on the method by sandau et al . a total of 21 pops were measured : 14 pcb congeners ; 5 organochlorine pesticides ; 1 octachlorodibenzo - p - dioxin ; and 1 brominated diphenyl ether congener ( supplementary table 1 ) . among 21 pops measured , 2 organochlorine pesticides ( trans - chlordane and cis - chlordane ) with a detection rate < 10% were not included in the final analyses . because pops predominantly are carried in the lipid component of the blood , lipid - standardized concentrations , the concentrations of pops divided by total serum lipid content , tended to be reported in epidemiological studies . however , pops can disturb lipid metabolism ( 14,15 ) , and dyslipidemia also is involved in the pathogenesis of type 2 diabetes ( 16 ) . therefore , wet - weight concentrations ( pops concentrations in pg / ml not divided by lipids ) would have greater validity because lipid concentrations may be intermediate in a causal chain linking pops and type 2 diabetes . in addition , one simulation study ( 17 ) reported that wet - weight concentrations adjusting for triglycerides and total cholesterol in a model as covariates induced less bias than lipid - standardized concentrations . therefore , we present wet - weight concentrations ( pg / ml ) , adjusting for triglycerides and total cholesterol by including these two lipid profiles in the models as covariates . in fact , analyses on the basis of lipid - standardized concentrations showed similar results in the current study ( data not shown ) . analyses were done on individual pops and summary pop measures . in the individual analyses , cutoff points of individual pops were provided in the supplementary table 2 . in the summary analyses , the individual ranks were summed ( 1922 ) for each pop , as applied in previous studies ( 5,6 ) , and summed values also were divided into quintiles . odds ratios ( ors ) and 95% cis for the risk of type 2 diabetes were estimated using logistic regression . the covariates were sex , bmi ( continuous ) , cigarette smoking ( current , former , and never ) , alcohol consumption ( grams per week ) , exercise ( no , mild , moderate , and vigorous ) , triglycerides ( continuous ) , and total cholesterol ( continuous ) . the additional value of adding pops to the traditional risk factors was evaluated by c - statistics , and tests of reclassification ( net reclassification index [ nri ] ) and discrimination ( improved discrimination index [ idi ] ) according to the method proposed by pencina et al . ( three risk groups were considered : < 3% ( low risk ) ; 37% ( intermediate risk ) ; and > 7% ( high risk ) risk of diabetes development . among 989 subjects for cross - sectional analyses , approximately one - half of the subjects were women and 22.1% were obese ( bmi 30 kg / m ) ( table 1 ) . among prevalent diabetes cases , subjects eligible for the prospective analyses ( n = 725 ) had baseline characteristics similar to those for the cross - sectional analysis ( table 1 ) . among incident diabetes cases , 11.1 and 30.6% of patients baseline characteristics of study subjects used in cross - sectional and prospective analyses data are means sd or percent . prospective analyses omitted prevalent diabetes at baseline as well as participants who were not followed up . * even though characteristics of all subjects for prospective analyses were baseline characteristics at age 70 years , clinical features of incident diabetes cases were follow - up information at age 75 years . supplementary table 3 shows the cross - sectional associations between pops and prevalent type 2 diabetes ( n = 112 ) . among 14 pcbs , 9 pcbs showed significant ors in the highest quintile , although ptrend was significant only for pcb congeners 105 , 118 , and 153 . the adjusted ors ( 95% cis ) for prevalent type 2 diabetes , according to quintiles of the sum of individual ranks of the 14 pcbs , were 1.0 , 1.6 , 1.3 , and 2.1 ( 1.14.4 ) ( ptrend = 0.20 ) . among organochlorine pesticides , both trans - nonachlor and p , p-2,2-bis(4-chlorophenyl)-1,1-dichloroethene ( dde ) showed statistically significant associations with prevalent type 2 diabetes . the adjusted ors ( 95% cis ) of the summary measure of three organochlorine pesticides were 1.0 , 0.9 , 1.8 , 1.7 , and 2.5 ( 1.25.2 ) ( ptrend < 0.01 ) . neither octachlorodibenzo - p - dioxin nor brominated diphenyl ether 47 was associated with prevalent type 2 diabetes ( supplementary table 4 ) . during the 5-year follow - up , 36 subjects developed diabetes ( cumulative incidence 5.0% ) . plasma concentrations of pops , in particular pcbs , strongly predicted future risk of type 2 diabetes ( table 2 ) . although the adjusted ors from the second to fifth quintiles were substantially different for individual pcbs , five pcbs showed a significant p value for trend ( congeners 74 , 99 , 138 , 194 , and 206 ) . for some pcbs with a nonsignificant ptrend ( for example pcb105 , pcb118 , and pcb156 ) , adjusted ors in the second quintile already were significantly increased but did not further increase in the higher quintiles . when we combined by summing the individual rank of each pcb , adjusted ors ( 95% cis ) were 1.0 , 4.5 , 5.1 , 8.8 ( 1.842.7 ) , and 7.5 ( 1.438.8 ) ( ptrend < 0.01 ) . among organochlorine pesticides , only trans - nonachlor showed a significant ptrend . both p , p-dde and hexachlorobenzene showed nonsignificant positive trends . adjusted ors ( 95% cis ) for the summary measure were 1.0 , 1.1 , 1.6 , 1.5 , and 3.4 ( 1.011.7 ) ( ptrend = 0.03 ) . neither octachlorodibenzo - p - dioxin nor brominated diphenyl ether 47 was associated with incident type 2 diabetes ( supplementary table 5 ) . adjusted * ors ( 95% cis ) of incident diabetes according to quintiles of plasma concentrations of pcbs , organochlorine pesticides , and summary measures of pcbs or organochlorine pesticides * adjusted for sex , bmi , cigarette smoking , exercise , alcohol consumption , triglycerides , and total cholesterol . pcb105 had zero cases in the lowest quintile ; for statistical assessment , we artificially added one case to that quintile so that or estimates were not infinite ; covariates were evaluated at the median values of noncases in the lowest quintile . in a single regression including both the summary measure of pcb and waist circumference , the strength of association of the summary measure of pcbs was as strong as that of waist circumference ( fig . 1 ) ( supplementary table 6 ) . in particular , the adjusted or in the second quintile of summary pcbs was similar to that in the fourth quintile of waist circumference . the summary measure of organochlorine pesticides showed a weaker association with diabetes than waist circumference ( fig . ors per 1-sd increase in summary pcbs and waist circumference were 1.7 ( 95% ci 1.12.5 ) and 2.0 ( 1.42.9 ) , respectively . those figures for summary organochlorine pesticides and waist circumference were 1.5 ( 1.02.2 ) and 1.7 ( 1.22.5 ) . we assessed a possible interaction between pops and adiposity in predicting diabetes ; there was no reportable interaction ( data not shown ) , although power was low for this test given the small number of incident diabetes cases . comparison of adjusted ors and 95% cis between quintiles of the summary measure of pcbs or organochlorine ( oc ) pesticides and quintiles of waist circumference on the risk of future type 2 diabetes . findings in each panel are from a single regression model that includes the sum of either pcbs or organochlorine pesticides and waist circumference , adjusted for sex , cigarette smoking , exercise , alcohol consumption , total cholesterol , and triglycerides . * the addition of the sum of pcbs to a model containing bmi , smoking , serum cholesterol , serum triglycerides , sex , exercise habits , and alcohol intake as risk factors for diabetes development increased the receiver - operating curve area under the curve from 0.68 ( 95% ci 0.580.76 ) to 0.72 ( 0.630.80 ) . adding the sum of organochlorine pesticides to the risk factors increased the receiver - operating curve area under the curve less ( 0.69 [ 0.600.77 ] ) . to evaluate whether the addition of information of pops to risk factors improved the risk classification with regard to diabetes development , idi and nri were used . adding the sum of pcbs to the risk factors described above resulted in significantly improved reclassification with both methods ( estimate 0.013 , se 0.0065 , and p = 0.034 for idi and estimate 0.22 , se 0.11 , and p = 0.045 for nri ) . when we used 7% as the cutoff for high risk and 3% as the cutoff for low risk , nri analysis showed that of 36 cases 10 were reclassified to higher risk versus 5 reclassified to lower risk , by the addition of the sum of pcbs . of 689 noncases , reclassification generally was reversed ; 110 reclassified to higher risk versus 166 reclassified to lower risk ( supplementary table 7 ) . nri was substantially greater within the low - risk and moderate - risk strata ( 0.35 and 0.52 vs. 0.07 in the highest - risk group ) and as an average across risk strata ( 0.32 ) . in a similar analysis , adding the sum of organochlorine pesticides to the traditional risk factors , a similar improvement in reclassification was seen using idi but was less powerful using nri ( estimate 0.0097 , se 0.0045 , and p = 0.034 for idi and estimate 0.16 , se 0.086 , and p = 0.063 for nri ) . supplementary table 3 shows the cross - sectional associations between pops and prevalent type 2 diabetes ( n = 112 ) . among 14 pcbs , 9 pcbs showed significant ors in the highest quintile , although ptrend was significant only for pcb congeners 105 , 118 , and 153 . the adjusted ors ( 95% cis ) for prevalent type 2 diabetes , according to quintiles of the sum of individual ranks of the 14 pcbs , were 1.0 , 1.6 , 1.3 , and 2.1 ( 1.14.4 ) ( ptrend = 0.20 ) . among organochlorine pesticides , both trans - nonachlor and p , p-2,2-bis(4-chlorophenyl)-1,1-dichloroethene ( dde ) showed statistically significant associations with prevalent type 2 diabetes . the adjusted ors ( 95% cis ) of the summary measure of three organochlorine pesticides were 1.0 , 0.9 , 1.8 , 1.7 , and 2.5 ( 1.25.2 ) ( ptrend < 0.01 ) . neither octachlorodibenzo - p - dioxin nor brominated diphenyl ether 47 was associated with prevalent type 2 diabetes ( supplementary table 4 ) . during the 5-year follow - up , 36 subjects developed diabetes ( cumulative incidence 5.0% ) . plasma concentrations of pops , in particular pcbs , strongly predicted future risk of type 2 diabetes ( table 2 ) . although the adjusted ors from the second to fifth quintiles were substantially different for individual pcbs , five pcbs showed a significant p value for trend ( congeners 74 , 99 , 138 , 194 , and 206 ) . for some pcbs with a nonsignificant ptrend ( for example pcb105 , pcb118 , and pcb156 ) , adjusted ors in the second quintile already were significantly increased but did not further increase in the higher quintiles . when we combined by summing the individual rank of each pcb , adjusted ors ( 95% cis ) were 1.0 , 4.5 , 5.1 , 8.8 ( 1.842.7 ) , and 7.5 ( 1.438.8 ) ( ptrend < 0.01 ) . adjusted ors ( 95% cis ) for the summary measure were 1.0 , 1.1 , 1.6 , 1.5 , and 3.4 ( 1.011.7 ) ( ptrend = 0.03 ) . neither octachlorodibenzo - p - dioxin nor brominated diphenyl ether 47 was associated with incident type 2 diabetes ( supplementary table 5 ) . adjusted * ors ( 95% cis ) of incident diabetes according to quintiles of plasma concentrations of pcbs , organochlorine pesticides , and summary measures of pcbs or organochlorine pesticides * adjusted for sex , bmi , cigarette smoking , exercise , alcohol consumption , triglycerides , and total cholesterol . pcb105 had zero cases in the lowest quintile ; for statistical assessment , we artificially added one case to that quintile so that or estimates were not infinite ; covariates were evaluated at the median values of noncases in the lowest quintile . in a single regression including both the summary measure of pcb and waist circumference , the strength of association of the summary measure of pcbs was as strong as that of waist circumference ( fig . the adjusted or in the second quintile of summary pcbs was similar to that in the fourth quintile of waist circumference . the summary measure of organochlorine pesticides showed a weaker association with diabetes than waist circumference ( fig . ors per 1-sd increase in summary pcbs and waist circumference were 1.7 ( 95% ci 1.12.5 ) and 2.0 ( 1.42.9 ) , respectively . those figures for summary organochlorine pesticides and waist circumference were 1.5 ( 1.02.2 ) and 1.7 ( 1.22.5 ) . we assessed a possible interaction between pops and adiposity in predicting diabetes ; there was no reportable interaction ( data not shown ) , although power was low for this test given the small number of incident diabetes cases . comparison of adjusted ors and 95% cis between quintiles of the summary measure of pcbs or organochlorine ( oc ) pesticides and quintiles of waist circumference on the risk of future type 2 diabetes . findings in each panel are from a single regression model that includes the sum of either pcbs or organochlorine pesticides and waist circumference , adjusted for sex , cigarette smoking , exercise , alcohol consumption , total cholesterol , and triglycerides . * the addition of the sum of pcbs to a model containing bmi , smoking , serum cholesterol , serum triglycerides , sex , exercise habits , and alcohol intake as risk factors for diabetes development increased the receiver - operating curve area under the curve from 0.68 ( 95% ci 0.580.76 ) to 0.72 ( 0.630.80 ) . adding the sum of organochlorine pesticides to the risk factors increased the receiver - operating curve area under the curve less ( 0.69 [ 0.600.77 ] ) . to evaluate whether the addition of information of pops to risk factors improved the risk classification with regard to diabetes development , idi and nri were used . adding the sum of pcbs to the risk factors described above resulted in significantly improved reclassification with both methods ( estimate 0.013 , se 0.0065 , and p = 0.034 for idi and when we used 7% as the cutoff for high risk and 3% as the cutoff for low risk , nri analysis showed that of 36 cases 10 were reclassified to higher risk versus 5 reclassified to lower risk , by the addition of the sum of pcbs . of 689 noncases , reclassification generally was reversed ; 110 reclassified to higher risk versus 166 reclassified to lower risk ( supplementary table 7 ) . nri was substantially greater within the low - risk and moderate - risk strata ( 0.35 and 0.52 vs. 0.07 in the highest - risk group ) and as an average across risk strata ( 0.32 ) . in a similar analysis , adding the sum of organochlorine pesticides to the traditional risk factors , a similar improvement in reclassification was seen using idi but was less powerful using nri ( estimate 0.0097 , se 0.0045 , and p = 0.034 for idi and estimate 0.16 , se 0.086 , and p = 0.063 for nri ) . in this study , plasma concentrations of pops , especially pcbs and organochlorine pesticides , strongly predicted incident type 2 diabetes during a 5-year follow - up . although there were some differences in terms of the strengths of the associations , most pops measured in the current study showed positive associations . although pcbs with differing biological characteristics were included in this study , dioxin - like pcbs ( pcb105 , pcb118 , pcb156 , pcb157 , and pcb189 ) and nondioxin - like pcbs showed similar associations . it is important to note that prediction using the summary measure of pcbs improved risk classification compared with traditional risk factors for diabetes . although the cross - sectional associations also showed significant results , the strengths of associations in this study of elderly subjects , in whom selective survivorship could play an important role , were weaker than those of prospective analyses . although obesity is critical in the pathogenesis of type 2 diabetes , one recent prospective study ( 11 ) reported that the association between adiposity and type 2 diabetes was weaker in elderly subjects . when the pathogenesis of type 2 diabetes is classified into two stages , insulin resistance and insulin secretory defects , the latter part seems to be more important among elderly people ( 10 ) . indeed , it is well known that many chemicals , including streptozotocin , alloxan , and vacor , can induce -cell destruction ( 19 ) . although there has been no experimental study on the effects of pops on pancreatic -cells , one experimental study ( 9 ) observed alterations in mitochondrial function and oxidative capacity in the liver of rats exposed to a mixture of pops . similar mitochondrial defects may happen in the pancreatic -cell after long - term exposure to low - dose pops in humans , possibly more severely in elderly people because of the general decrease of antioxidant capacity with aging ( 20 ) . mitochondrial function has been implicated as a key for pancreatic -cells to secrete insulin ( 21 ) . aside from studies performed among people with high exposure to several selected pops in the occupational or accidental setting , there have been three prospective studies that examined associations of type 2 diabetes with organochlorine pesticides and pcbs in general populations ( 68 ) . compared with occupational or accidental exposures , the background exposure in the general population is characterized by low - dose and long - term exposure to a mixture of various pops . consistent with the current findings , all three prospective studies have reported that some organochlorine pesticides and pcbs predicted the future risk of type 2 diabetes , but the details differed among the studies . a prospective study in a cohort of great lakes sport fish consumers in michigan in the u.s . demonstrated a strong association of incident diabetes with p , p-dde but not pcbs ( 7 ) . likewise , in a nested case - control study among a cohort of swedish women , p , p-dde was also a risk factor for developing type 2 diabetes later in life but not pcb153 ( 8) . in these two studies , no other organochlorine pesticides , except p , p-dde , were measured . in a nested case - control study performed in young adults within the coronary artery risk development in young adults ( cardia ) cohort , trans - nonachlor and highly chlorinated pcbs predicted type 2 diabetes , whereas p , p-dde did not ( 6 ) . in the current study , the association between p , p-dde and type 2 diabetes was not as strong as that seen in the michigan study . unlike the cardia study , both moderately and highly chlorinated pcbs predicted future risk of type 2 diabetes . however , trans - nonachlor came out as the most significant among the organochlorine pesticides , similar to the cardia study . the current study and the michigan study showed similar dose - response curves ( 7 ) . the risk of future diabetes was substantially increased with only a slight increase of concentrations of pops . however , after that , only a slight increase in risk was seen when the concentrations of pops became higher . although the cardia study also observed the increased risk at the low dose of some pops , after that the risk even decreased as concentrations of pops increased ( 6 ) . considering all these findings , the bottom line may be that the biological effects of pops already are induced at very low concentrations of certain pops . at present , we do not have any clear explanation for why different pops predicting type 2 diabetes and/or shapes of dose - response relations were different among epidemiological studies . the inconsistency may be related to the idea that pops are involved in the pathogenesis of type 2 diabetes by interfering with endocrine signaling pathways ( 22 ) . first , low - dose effects have been proposed as possible biological responses of endocrine disruptors ( 23 ) . also , endocrine function generally declines with age because hormone receptors become less sensitive and levels of most hormones change with age ( 24 ) . therefore , the different age distribution among the study populations may have led to different results even when comparing similar concentrations of pops . in addition , endocrine - disturbing effects of one specific pop may differ depending on concentrations of other related endocrine disruptors . humans are exposed to a mixture of various pops with unique exposure patterns depending on the study population . for example , concentrations of p , p-dde in the cardia study was higher than in those of the current study and the michigan study . however , concentrations of pcbs with six or seven chlorine atoms were much higher in the current study than in the cardia study , whereas those with four or five chlorine atoms were similar between these two studies . although concentration of any particular pop might be similar between two populations , the strength of association between this pop and diabetes can differ depending on concentrations of other pops . therefore , the cis were wide , and some associations may be chance findings despite strong statistical significance . in addition , although our general hypothesis of associations between pops and incident diabetes were made a priori , we did perform many statistical tests , and caution should be exercised in interpreting the findings . second , some diabetes cases would be misclassified as noncases because we did not perform an oral glucose tolerance test . isolated postprandial hyperglycemia with normal fasting glucose is common among the elderly ( 25 ) . third , because concentrations of pops tend to be highly correlated in the general population , any pop that is strongly correlated with other biologically important pops may come out statistically significant in epidemiological studies . although the measured pops in the current study are a selection of the pops referred to in the stockholm convention and are representative markers for the group of compounds , the real exposure of the subject is , of course , more complex . in conclusion , the current study observed that pcbs and some organochlorine pesticides predicted the future risk of type 2 diabetes . although careful interpretation may be needed because of the small number of incident type 2 diabetes cases , the association between pcbs and incident type 2 diabetes seemed to be at least as strong as the association of adiposity with incident diabetes in this elderly population . the environmental exposure to pcbs and some organochlorine pesticides may play a role in the development of type 2 diabetes .

objectivepersistent organic pollutants ( pops ) , lipophilic chemicals that accumulate mainly in adipose tissue , have recently been linked to type 2 diabetes . however , evidence from prospective studies is sparse . this study was performed to evaluate prospective associations of type 2 diabetes with selected pops among the elderly.research design and methodsnineteen pops ( 14 polychlorinated biphenyl [ pcb ] congeners , 3 organochlorine pesticides , 1 brominated diphenyl ether , and 1 dioxin ) were measured in plasma collected at baseline in 725 participants , aged 70 years , of the prospective investigation of the vasculature in uppsala seniors ( pivus).resultsafter adjusting for known type 2 diabetes risk factors , including obesity , odds ratios ( ors ) ( 95% cis ) for type 2 diabetes at age 75 years ( n = 36 ) according to the quintiles of a summary measure of concentrations of pcbs ( vs. the lowest quintile ) were 4.5 , 5.1 , 8.8 ( 1.842.7 ) , and 7.5 ( 1.438.8 ) ( ptrend < 0.01 ) . among organochlorine pesticides , adjusted ors across concentrations of trans - nonachlor showed that ptrend = 0.03 . adjusted ors ( 95% cis ) across quintiles of the sum of three organochlorine pesticides were 1.1 , 1.6 , 1.5 , and 3.4 ( 1.011.7 ) ( ptrend = 0.03 ) . neither brominated diphenyl ether 47 nor dioxin was significantly associated with incident diabetes . the sum of pcbs improved reclassification significantly when added to traditional risk factors for diabetes.conclusionsdespite the small number of incident cases , this study found that environmental exposure to some pops substantially increased risk of future type 2 diabetes in an elderly population .

RESEARCH DESIGN AND METHODS Measurement POPs analyses Statistical analyses RESULTS Cross-sectional analyses Prospective analyses CONCLUSIONS Supplementary Material

all subjects , aged 70 years , living in the community of uppsala , sweden , were eligible for the study . from march 2006 to september 2009 , when the subjects turned 75 years of age , reinvestigation of the cohort was performed with a follow - up rate of 81.4% . the sample size for the prospective analyses was 725 subjects after excluding 112 type 2 diabetes cases at baseline and 152 subjects who were not followed up ( of whom 52 had died ) . the study was approved by the ethics committee of the university of uppsala , and the participants gave written informed consent . type 2 diabetes was defined as a fasting blood glucose 6.2 mmol / l or the use of insulin or oral hypoglycemic agents . a total of 21 pops were measured : 14 pcb congeners ; 5 organochlorine pesticides ; 1 octachlorodibenzo - p - dioxin ; and 1 brominated diphenyl ether congener ( supplementary table 1 ) . however , pops can disturb lipid metabolism ( 14,15 ) , and dyslipidemia also is involved in the pathogenesis of type 2 diabetes ( 16 ) . therefore , wet - weight concentrations ( pops concentrations in pg / ml not divided by lipids ) would have greater validity because lipid concentrations may be intermediate in a causal chain linking pops and type 2 diabetes . therefore , we present wet - weight concentrations ( pg / ml ) , adjusting for triglycerides and total cholesterol by including these two lipid profiles in the models as covariates . in the summary analyses , the individual ranks were summed ( 1922 ) for each pop , as applied in previous studies ( 5,6 ) , and summed values also were divided into quintiles . odds ratios ( ors ) and 95% cis for the risk of type 2 diabetes were estimated using logistic regression . the covariates were sex , bmi ( continuous ) , cigarette smoking ( current , former , and never ) , alcohol consumption ( grams per week ) , exercise ( no , mild , moderate , and vigorous ) , triglycerides ( continuous ) , and total cholesterol ( continuous ) . the additional value of adding pops to the traditional risk factors was evaluated by c - statistics , and tests of reclassification ( net reclassification index [ nri ] ) and discrimination ( improved discrimination index [ idi ] ) according to the method proposed by pencina et al . type 2 diabetes was defined as a fasting blood glucose 6.2 mmol / l or the use of insulin or oral hypoglycemic agents . a total of 21 pops were measured : 14 pcb congeners ; 5 organochlorine pesticides ; 1 octachlorodibenzo - p - dioxin ; and 1 brominated diphenyl ether congener ( supplementary table 1 ) . among 21 pops measured , 2 organochlorine pesticides ( trans - chlordane and cis - chlordane ) with a detection rate < 10% were not included in the final analyses . however , pops can disturb lipid metabolism ( 14,15 ) , and dyslipidemia also is involved in the pathogenesis of type 2 diabetes ( 16 ) . therefore , wet - weight concentrations ( pops concentrations in pg / ml not divided by lipids ) would have greater validity because lipid concentrations may be intermediate in a causal chain linking pops and type 2 diabetes . therefore , we present wet - weight concentrations ( pg / ml ) , adjusting for triglycerides and total cholesterol by including these two lipid profiles in the models as covariates . in the summary analyses , the individual ranks were summed ( 1922 ) for each pop , as applied in previous studies ( 5,6 ) , and summed values also were divided into quintiles . odds ratios ( ors ) and 95% cis for the risk of type 2 diabetes were estimated using logistic regression . the additional value of adding pops to the traditional risk factors was evaluated by c - statistics , and tests of reclassification ( net reclassification index [ nri ] ) and discrimination ( improved discrimination index [ idi ] ) according to the method proposed by pencina et al . among prevalent diabetes cases , subjects eligible for the prospective analyses ( n = 725 ) had baseline characteristics similar to those for the cross - sectional analysis ( table 1 ) . among incident diabetes cases , 11.1 and 30.6% of patients baseline characteristics of study subjects used in cross - sectional and prospective analyses data are means sd or percent . prospective analyses omitted prevalent diabetes at baseline as well as participants who were not followed up . * even though characteristics of all subjects for prospective analyses were baseline characteristics at age 70 years , clinical features of incident diabetes cases were follow - up information at age 75 years . supplementary table 3 shows the cross - sectional associations between pops and prevalent type 2 diabetes ( n = 112 ) . the adjusted ors ( 95% cis ) for prevalent type 2 diabetes , according to quintiles of the sum of individual ranks of the 14 pcbs , were 1.0 , 1.6 , 1.3 , and 2.1 ( 1.14.4 ) ( ptrend = 0.20 ) . among organochlorine pesticides , both trans - nonachlor and p , p-2,2-bis(4-chlorophenyl)-1,1-dichloroethene ( dde ) showed statistically significant associations with prevalent type 2 diabetes . the adjusted ors ( 95% cis ) of the summary measure of three organochlorine pesticides were 1.0 , 0.9 , 1.8 , 1.7 , and 2.5 ( 1.25.2 ) ( ptrend < 0.01 ) . neither octachlorodibenzo - p - dioxin nor brominated diphenyl ether 47 was associated with prevalent type 2 diabetes ( supplementary table 4 ) . plasma concentrations of pops , in particular pcbs , strongly predicted future risk of type 2 diabetes ( table 2 ) . although the adjusted ors from the second to fifth quintiles were substantially different for individual pcbs , five pcbs showed a significant p value for trend ( congeners 74 , 99 , 138 , 194 , and 206 ) . for some pcbs with a nonsignificant ptrend ( for example pcb105 , pcb118 , and pcb156 ) , adjusted ors in the second quintile already were significantly increased but did not further increase in the higher quintiles . when we combined by summing the individual rank of each pcb , adjusted ors ( 95% cis ) were 1.0 , 4.5 , 5.1 , 8.8 ( 1.842.7 ) , and 7.5 ( 1.438.8 ) ( ptrend < 0.01 ) . among organochlorine pesticides , only trans - nonachlor showed a significant ptrend . adjusted ors ( 95% cis ) for the summary measure were 1.0 , 1.1 , 1.6 , 1.5 , and 3.4 ( 1.011.7 ) ( ptrend = 0.03 ) . neither octachlorodibenzo - p - dioxin nor brominated diphenyl ether 47 was associated with incident type 2 diabetes ( supplementary table 5 ) . adjusted * ors ( 95% cis ) of incident diabetes according to quintiles of plasma concentrations of pcbs , organochlorine pesticides , and summary measures of pcbs or organochlorine pesticides * adjusted for sex , bmi , cigarette smoking , exercise , alcohol consumption , triglycerides , and total cholesterol . pcb105 had zero cases in the lowest quintile ; for statistical assessment , we artificially added one case to that quintile so that or estimates were not infinite ; covariates were evaluated at the median values of noncases in the lowest quintile . in a single regression including both the summary measure of pcb and waist circumference , the strength of association of the summary measure of pcbs was as strong as that of waist circumference ( fig . the summary measure of organochlorine pesticides showed a weaker association with diabetes than waist circumference ( fig . ors per 1-sd increase in summary pcbs and waist circumference were 1.7 ( 95% ci 1.12.5 ) and 2.0 ( 1.42.9 ) , respectively . those figures for summary organochlorine pesticides and waist circumference were 1.5 ( 1.02.2 ) and 1.7 ( 1.22.5 ) . we assessed a possible interaction between pops and adiposity in predicting diabetes ; there was no reportable interaction ( data not shown ) , although power was low for this test given the small number of incident diabetes cases . comparison of adjusted ors and 95% cis between quintiles of the summary measure of pcbs or organochlorine ( oc ) pesticides and quintiles of waist circumference on the risk of future type 2 diabetes . findings in each panel are from a single regression model that includes the sum of either pcbs or organochlorine pesticides and waist circumference , adjusted for sex , cigarette smoking , exercise , alcohol consumption , total cholesterol , and triglycerides . * the addition of the sum of pcbs to a model containing bmi , smoking , serum cholesterol , serum triglycerides , sex , exercise habits , and alcohol intake as risk factors for diabetes development increased the receiver - operating curve area under the curve from 0.68 ( 95% ci 0.580.76 ) to 0.72 ( 0.630.80 ) . adding the sum of organochlorine pesticides to the risk factors increased the receiver - operating curve area under the curve less ( 0.69 [ 0.600.77 ] ) . to evaluate whether the addition of information of pops to risk factors improved the risk classification with regard to diabetes development , idi and nri were used . adding the sum of pcbs to the risk factors described above resulted in significantly improved reclassification with both methods ( estimate 0.013 , se 0.0065 , and p = 0.034 for idi and estimate 0.22 , se 0.11 , and p = 0.045 for nri ) . when we used 7% as the cutoff for high risk and 3% as the cutoff for low risk , nri analysis showed that of 36 cases 10 were reclassified to higher risk versus 5 reclassified to lower risk , by the addition of the sum of pcbs . in a similar analysis , adding the sum of organochlorine pesticides to the traditional risk factors , a similar improvement in reclassification was seen using idi but was less powerful using nri ( estimate 0.0097 , se 0.0045 , and p = 0.034 for idi and estimate 0.16 , se 0.086 , and p = 0.063 for nri ) . supplementary table 3 shows the cross - sectional associations between pops and prevalent type 2 diabetes ( n = 112 ) . the adjusted ors ( 95% cis ) for prevalent type 2 diabetes , according to quintiles of the sum of individual ranks of the 14 pcbs , were 1.0 , 1.6 , 1.3 , and 2.1 ( 1.14.4 ) ( ptrend = 0.20 ) . among organochlorine pesticides , both trans - nonachlor and p , p-2,2-bis(4-chlorophenyl)-1,1-dichloroethene ( dde ) showed statistically significant associations with prevalent type 2 diabetes . the adjusted ors ( 95% cis ) of the summary measure of three organochlorine pesticides were 1.0 , 0.9 , 1.8 , 1.7 , and 2.5 ( 1.25.2 ) ( ptrend < 0.01 ) . neither octachlorodibenzo - p - dioxin nor brominated diphenyl ether 47 was associated with prevalent type 2 diabetes ( supplementary table 4 ) . plasma concentrations of pops , in particular pcbs , strongly predicted future risk of type 2 diabetes ( table 2 ) . for some pcbs with a nonsignificant ptrend ( for example pcb105 , pcb118 , and pcb156 ) , adjusted ors in the second quintile already were significantly increased but did not further increase in the higher quintiles . when we combined by summing the individual rank of each pcb , adjusted ors ( 95% cis ) were 1.0 , 4.5 , 5.1 , 8.8 ( 1.842.7 ) , and 7.5 ( 1.438.8 ) ( ptrend < 0.01 ) . adjusted ors ( 95% cis ) for the summary measure were 1.0 , 1.1 , 1.6 , 1.5 , and 3.4 ( 1.011.7 ) ( ptrend = 0.03 ) . neither octachlorodibenzo - p - dioxin nor brominated diphenyl ether 47 was associated with incident type 2 diabetes ( supplementary table 5 ) . adjusted * ors ( 95% cis ) of incident diabetes according to quintiles of plasma concentrations of pcbs , organochlorine pesticides , and summary measures of pcbs or organochlorine pesticides * adjusted for sex , bmi , cigarette smoking , exercise , alcohol consumption , triglycerides , and total cholesterol . in a single regression including both the summary measure of pcb and waist circumference , the strength of association of the summary measure of pcbs was as strong as that of waist circumference ( fig . the summary measure of organochlorine pesticides showed a weaker association with diabetes than waist circumference ( fig . ors per 1-sd increase in summary pcbs and waist circumference were 1.7 ( 95% ci 1.12.5 ) and 2.0 ( 1.42.9 ) , respectively . those figures for summary organochlorine pesticides and waist circumference were 1.5 ( 1.02.2 ) and 1.7 ( 1.22.5 ) . we assessed a possible interaction between pops and adiposity in predicting diabetes ; there was no reportable interaction ( data not shown ) , although power was low for this test given the small number of incident diabetes cases . comparison of adjusted ors and 95% cis between quintiles of the summary measure of pcbs or organochlorine ( oc ) pesticides and quintiles of waist circumference on the risk of future type 2 diabetes . findings in each panel are from a single regression model that includes the sum of either pcbs or organochlorine pesticides and waist circumference , adjusted for sex , cigarette smoking , exercise , alcohol consumption , total cholesterol , and triglycerides . * the addition of the sum of pcbs to a model containing bmi , smoking , serum cholesterol , serum triglycerides , sex , exercise habits , and alcohol intake as risk factors for diabetes development increased the receiver - operating curve area under the curve from 0.68 ( 95% ci 0.580.76 ) to 0.72 ( 0.630.80 ) . adding the sum of organochlorine pesticides to the risk factors increased the receiver - operating curve area under the curve less ( 0.69 [ 0.600.77 ] ) . adding the sum of pcbs to the risk factors described above resulted in significantly improved reclassification with both methods ( estimate 0.013 , se 0.0065 , and p = 0.034 for idi and when we used 7% as the cutoff for high risk and 3% as the cutoff for low risk , nri analysis showed that of 36 cases 10 were reclassified to higher risk versus 5 reclassified to lower risk , by the addition of the sum of pcbs . in a similar analysis , adding the sum of organochlorine pesticides to the traditional risk factors , a similar improvement in reclassification was seen using idi but was less powerful using nri ( estimate 0.0097 , se 0.0045 , and p = 0.034 for idi and estimate 0.16 , se 0.086 , and p = 0.063 for nri ) . in this study , plasma concentrations of pops , especially pcbs and organochlorine pesticides , strongly predicted incident type 2 diabetes during a 5-year follow - up . although pcbs with differing biological characteristics were included in this study , dioxin - like pcbs ( pcb105 , pcb118 , pcb156 , pcb157 , and pcb189 ) and nondioxin - like pcbs showed similar associations . it is important to note that prediction using the summary measure of pcbs improved risk classification compared with traditional risk factors for diabetes . although obesity is critical in the pathogenesis of type 2 diabetes , one recent prospective study ( 11 ) reported that the association between adiposity and type 2 diabetes was weaker in elderly subjects . when the pathogenesis of type 2 diabetes is classified into two stages , insulin resistance and insulin secretory defects , the latter part seems to be more important among elderly people ( 10 ) . aside from studies performed among people with high exposure to several selected pops in the occupational or accidental setting , there have been three prospective studies that examined associations of type 2 diabetes with organochlorine pesticides and pcbs in general populations ( 68 ) . consistent with the current findings , all three prospective studies have reported that some organochlorine pesticides and pcbs predicted the future risk of type 2 diabetes , but the details differed among the studies . demonstrated a strong association of incident diabetes with p , p-dde but not pcbs ( 7 ) . in these two studies , no other organochlorine pesticides , except p , p-dde , were measured . in a nested case - control study performed in young adults within the coronary artery risk development in young adults ( cardia ) cohort , trans - nonachlor and highly chlorinated pcbs predicted type 2 diabetes , whereas p , p-dde did not ( 6 ) . in the current study , the association between p , p-dde and type 2 diabetes was not as strong as that seen in the michigan study . unlike the cardia study , both moderately and highly chlorinated pcbs predicted future risk of type 2 diabetes . however , trans - nonachlor came out as the most significant among the organochlorine pesticides , similar to the cardia study . the risk of future diabetes was substantially increased with only a slight increase of concentrations of pops . although the cardia study also observed the increased risk at the low dose of some pops , after that the risk even decreased as concentrations of pops increased ( 6 ) . the inconsistency may be related to the idea that pops are involved in the pathogenesis of type 2 diabetes by interfering with endocrine signaling pathways ( 22 ) . for example , concentrations of p , p-dde in the cardia study was higher than in those of the current study and the michigan study . however , concentrations of pcbs with six or seven chlorine atoms were much higher in the current study than in the cardia study , whereas those with four or five chlorine atoms were similar between these two studies . in addition , although our general hypothesis of associations between pops and incident diabetes were made a priori , we did perform many statistical tests , and caution should be exercised in interpreting the findings . isolated postprandial hyperglycemia with normal fasting glucose is common among the elderly ( 25 ) . in conclusion , the current study observed that pcbs and some organochlorine pesticides predicted the future risk of type 2 diabetes . although careful interpretation may be needed because of the small number of incident type 2 diabetes cases , the association between pcbs and incident type 2 diabetes seemed to be at least as strong as the association of adiposity with incident diabetes in this elderly population . the environmental exposure to pcbs and some organochlorine pesticides may play a role in the development of type 2 diabetes .

this study focused on the motivational process proposed by the jd - r model , whereby individual job resources exert a motivational potential and lead to high work engagement . job resources refer to those physical , psychological , social , or organizational aspects of the job that may either ( 1 ) be functional in achieving work goals ; ( 2 ) reduce job demands and associated physiological and psychological costs ; or ( 3 ) stimulate personal growth , learning , and development . work engagement is defined as a positive , fulfilling , work - related state of mind that is characterized by vigor ( i.e. , high levels of energy and mental resilience while working ) , dedication ( i.e. , sense of significance , enthusiasm , inspiration , and challenge ) , and absorption ( i.e. , being fully involved and happily engrossed in one 's work ) . the positive effect of job resources on work engagement has been supported in cross - sectional as well as longitudinal studies . the relationships between job resources and work engagement , however , can not be generalized to apply to cross - level relationships to answer the present research question because these studies were conducted at an individual level of analysis . it can be assumed that job resources are shared at the level of the immediate work group to a certain extent . accordingly , one can expect some minimal agreement on the perceptions of job resources within groups because group members are exposed to shared psychosocial context factors or group characteristics , such as similar work tasks , common supervisors and colleagues , and a group climate . likewise , it can be assumed that work groups significantly differ with respect to their mean levels of job resources . in conceptualizing the construct of group - level job resources , the present study applied an additive composition model , also referred to as a summary index model , and aggregated individual - level job resources to the work group level . in doing so , it was assumed that the aggregated group - level job resource construct represents a proxy for a resource - rich psychosocial work group environment . the following hypotheses were formulated : hypothesis 1a : group - level job resources emerge as a contextual resources construct from aggregated individual perceptions of job resources . hypothesis 1b : group - level job resources subfactors , i.e. , group - level manager behavior , group - level peer behavior , group - level task - related resources , emerge as contextual resource constructs from aggregated individual perceptions of job resource subfactors . as research has shown , the social context influences individual group members . previous research on the relationships between psychosocial work characteristics at group level and individual outcomes such as well - being , however , is scarce . gavin and hofmann aggregated individual perceptions of task significance , a core job resource , to the group level and found evidence for an additional contextual influence of group - level task significance on individual - level hostility after controlling for individual - level task significance . with regard to the positive side of work characteristics and employee health and well - being , referred to as the motivational process in the jd - r model , there is one study that examined the clustering of group - level job resources and their relationships with individual well - being in terms of work engagement . that study showed that team - level support from co - workers and supervisors was positively related to individual work engagement . however , this effect was not controlled for the individual - level support from co - workers and supervisors . thus , that study does not indicate whether team - level support is helpful for work engagement over and above individual support . what we know so far is that perceiving high individual job resources is beneficial for feeling engaged in one 's job . however , what we do not know is whether it is additionally beneficial for employees ' work engagement if they work in a resource - rich psychosocial work group environment , i.e. , where their co - workers experience high job resources on average . on the basis of the limited existing evidence summarized above , we expected that a resource - rich work group environment would have an additionally motivation - enhancing effect on one 's work engagement and thus formulated the following hypotheses : hypothesis 2a : group - level job resources have an additive positive effect on individual work engagement over and above that of individual - level job resources . hypothesis 2b : group - level job resource subfactors , i.e. , group - level manager behavior , group - level peer behavior , group - level task - related resources , have an additive positive effect on individual work engagement over and above that of individual - level job resource subfactors . when analyzing the effects of group - level job resources , it is important to control for the effects of variability of job resources within groups , which has been shown in a study on leadership climate . variability within groups can suppress irrelevant variance in group - level job resources and thus ensures an unbiased estimation of the effect of group - level job resources on individual engagement . not only a direct effect of variability within groups on engagement but also an interaction effect of variability and group - level job resources is possible and should therefore be controlled for in the analyses to facilitate an unbiased estimation of the cross - level effect . these methodological considerations led to the formulation of the following hypothesis : hypothesis 2c : the cross - level relationships assumed in hypotheses 2a and 2b remain significant even when a possible competing effect of and interaction with the variability of job resources within groups is controlled for . this study employed data collected in the baseline employee survey of a large stress - management intervention project ( see acknowledgments ) . the study sample consisted of 1,219 employees without supervisory function from six medium- and large - sized swiss organizations in diverse sectors ( three industrial production companies , one food processing company , one public administration service , and one hospital ) . the sample consisted of slightly more male than female employees ( females : 47.7% ) , the mean age of our respondents was 38 years ( sd=11 ) , the mean organizational tenure was 7.7 years ( sd=8.7 ) , and the mean job tenure was 4.8 years ( sd=6.2 ) . this study employed data collected in the baseline employee survey of a large stress - management intervention project ( see acknowledgments ) . the study sample consisted of 1,219 employees without supervisory function from six medium- and large - sized swiss organizations in diverse sectors ( three industrial production companies , one food processing company , one public administration service , and one hospital ) . the sample consisted of slightly more male than female employees ( females : 47.7% ) , the mean age of our respondents was 38 years ( sd=11 ) , the mean organizational tenure was 7.7 years ( sd=8.7 ) , and the mean job tenure was 4.8 years ( sd=6.2 ) . the independent variable at individual level , i.e. , individual job resources , was assessed using eight scales , which were clustered into three subfactors of job resources : manager behavior , peer behavior , and task - related resources . these subfactors and also the total individual job resources factor were previously used in two other studies . the independent variable at work group level , i.e. , group - level job resources , was assessed by calculating the means of individual job resources of all employees who participated from each work group . this was accordingly performed for each subfactor and the overall job resources factor , resulting in group - level manager behavior , group - level peer behavior , group - level task - related resources , and the overall group - level job resources construct . in addition to the variable of interest , i.e. , group - level job resources , we controlled for their variability within groups to ensure that the effect of group - level job resources on engagement is not biased by variability within groups . applying a dispersion composition model , group - level job resources variability was assessed by calculating the standard deviation of individual job resources scores of all employees who participated from each work group . again , this was performed for each job resources subfactor and the overall job resources factor . the dependent variable of this study , i.e. , work engagement , was assessed at an individual level ( see table 1 ) . in the analyses , we controlled for a number of covariates both at individual and group levels ( see table 1 ) . in addition to sociodemographic variables , we controlled for job demands , thus following a plea for a better understanding of the motivational process of the jd - r framework . to reduce the data set to a smaller subset of variables , we calculated factor scores ( regression method ) for the three job resources subfactors of manager behavior , peer behavior , and task - related resources in the first step and for the overall job resources factor in the second step . the same factors were used in two other studies and were supported using exploratory and confirmatory factor analyses ( results can be obtained from the corresponding author ) . to test the hypotheses , we employed multilevel random coefficient modeling using the nlme package in r. model fits were estimated by the restricted maximum likelihood ( reml ) method . we assumed minimal within - group agreement of individual ratings and significant differences across work groups in the mean level of job resources reflecting the shared psychosocial work group environment ( hypotheses 1a and 1b ) . we assessed the intra - class correlation coefficient icc(1 ) to identify the proportion of the variance explained by the grouping structure of the data . an icc(1 ) value of 1% , 10% , or 25% indicates a small , medium , or large effect of the group context , respectively . further , we calculated icc(2 ) , which indicates the reliability of the group mean and the james , demaree and wolf mean rwg ( j ) agreement index that indicates within - group agreement in the corresponding measures . for the multilevel analyses , all variables with no meaningful zero point were centered according to the recommendations of enders and tofighi . to test for the presumed cross - level main effect of group - level job resources ( hypotheses 2a and 2b ) , we estimated , first , a model with no explanatory variables ( intercept - only model ) , which served as a benchmark value of deviance for comparison with competing models ( step 1 ) . second , we added all group mean - centered individual - level predictors and covariates fixed ( step 2 ) . this means that relationships between individual - level predictors and work engagement were not allowed to vary between groups . covariates with no explanatory value were then omitted from the model before the next step . in the third step , the group level covariate and a group - level job resources factor were included ( step 3 ) . in this step , individual and group - level job resources were grand mean centered to detect an additional explanatory value of group - level job resources on individual engagement . again , covariates with no explanatory value were omitted from the model before the next step . following the recommended procedure of cole et al . to ensure unbiased estimates of relationships with group - level constructs , two more steps in model building were performed . this is particularly indicated in cases where icc(2 ) and rwg ( j ) estimates indicate substantive variability within groups . thus , fourth , we included the corresponding group - level job resources variability variable to control for the varying dispersion of job resources at group level ( step 4 ) . in a final step , we examined a possible interaction effect of group - level job resources and their variability within groups to examine if the hypothesized cross - level effect of group - level job resources is independent of the level of variability of job resources within groups ( step 5 ) . to reduce the data set to a smaller subset of variables , we calculated factor scores ( regression method ) for the three job resources subfactors of manager behavior , peer behavior , and task - related resources in the first step and for the overall job resources factor in the second step . the same factors were used in two other studies and were supported using exploratory and confirmatory factor analyses ( results can be obtained from the corresponding author ) . to test the hypotheses , we employed multilevel random coefficient modeling using the nlme package in r. model fits were estimated by the restricted maximum likelihood ( reml ) method . we assumed minimal within - group agreement of individual ratings and significant differences across work groups in the mean level of job resources reflecting the shared psychosocial work group environment ( hypotheses 1a and 1b ) . we assessed the intra - class correlation coefficient icc(1 ) to identify the proportion of the variance explained by the grouping structure of the data . an icc(1 ) value of 1% , 10% , or 25% indicates a small , medium , or large effect of the group context , respectively . further , we calculated icc(2 ) , which indicates the reliability of the group mean and the james , demaree and wolf mean rwg ( j ) agreement index that indicates within - group agreement in the corresponding measures . for the multilevel analyses , all variables with no meaningful zero point were centered according to the recommendations of enders and tofighi . to test for the presumed cross - level main effect of group - level job resources ( hypotheses 2a and 2b ) , we estimated , first , a model with no explanatory variables ( intercept - only model ) , which served as a benchmark value of deviance for comparison with competing models ( step 1 ) . second , we added all group mean - centered individual - level predictors and covariates fixed ( step 2 ) . this means that relationships between individual - level predictors and work engagement were not allowed to vary between groups . covariates with no explanatory value were then omitted from the model before the next step . in the third step , the group level covariate and a group - level job resources factor were included ( step 3 ) . in this step , individual and group - level job resources were grand mean centered to detect an additional explanatory value of group - level job resources on individual engagement . again , covariates with no explanatory value were omitted from the model before the next step . following the recommended procedure of cole et al . to ensure unbiased estimates of relationships with group - level constructs , two more steps in model building were performed . this is particularly indicated in cases where icc(2 ) and rwg ( j ) estimates indicate substantive variability within groups . thus , fourth , we included the corresponding group - level job resources variability variable to control for the varying dispersion of job resources at group level ( step 4 ) . in a final step , we examined a possible interaction effect of group - level job resources and their variability within groups to examine if the hypothesized cross - level effect of group - level job resources is independent of the level of variability of job resources within groups ( step 5 ) . table 2 shows the means , standard deviations , and bivariate correlations of the study variables at individual level , and table 3 shows those of the study variables at group level . results of an analysis of variance of work engagement with work groups as the grouping variable indicated significant between - group differences , f ( 102 , 1079 ) = 1.45 , p < .01 . icc(1 ) shows that 4% of the variance in individual - level work engagement depended on group membership , representing a small effect of the grouping structure . the data thus indicates that a multilevel structure is confirmed , and multilevel analyses are indicated . means , standard deviations , and bivariate correlations of study variables at individual level ( n=1,219 ) means , standard deviations , and bivariate correlations of study variables at group level ( n=103 ) the first hypothesis stated that group - level job resources emerge as a contextual resources construct from aggregated individual perceptions of job resources . results of an analysis of variance of job resources with work groups as the grouping variable indicated significant between - group differences [ f ( 102 , 990 ) = 1.78 , p < .001 ] . the calculated icc(1 ) shows that 7% of the variance in individual - level job resources depended on group membership , representing a small to medium effect of the grouping structure . finally , the mean within - group agreement coefficient rwg ( j ) was.97 ( range=.91-.99 ) , indicating very strong agreement within groups . group differences were significant for all subfactors with icc(1 ) values indicating that 5% , 8% , and 19% of the variance in manager behavior , peer behavior , and task - related resources , respectively , depended on group membership . this represents a small to medium effect of the grouping structure in manager and peer behavior and a medium to large effect in task - related resources . the corresponding icc(2 ) values were.38,.51 , and.73 for manager behavior , peer behavior , and task - related resources , respectively , indicating a low reliability of the group mean for manager behavior , a moderate reliability for peer behavior , and a good reliability of the group mean for task - related resources . finally , the mean within - group agreement coefficients rwg ( j ) for manager behavior , peer behavior , and task - related resources were.93 ( range=.81-.98),.82 ( range=.00-.98 ) , and.90 ( range=.65-.98 ) , respectively . in summary , these results seem to support the aggregation of individual - level job resources to the work group level , forming a contextual group - level job resource construct . therefore , concerning the supposed influence of the work group context on individual job resources subfactors , hypothesis 1b was supported . hypothesis 2a stated that group - level job resources have an additionally positive effect on individual work engagement over and above individual - level job resources . to test for this presumed cross - level main effect of group - level job resources all control variables that did not contribute to the prediction of work engagement were omitted from the models in step 2 for the individual - level covariates education ( unstandardized parameter estimate =0.02 , p=.67 ) and job tenure ( =0.00 , p=.96 ) and in step 3 for the group - level covariate group size ( =0.00 , p=.71 ) . however , as table 4 indicates , there was a significant relationship between sex , age , and work engagement . women ( =0.17 , p < .05 ) and older employees ( =0.01 , p < .001 ) indicated higher levels of work engagement . furthermore , individual job demands negatively relate with work engagement ( =0.12 , p < .01 ) and individual job resources positively relate ( =0.44 , p < .001 ) ( see step 2 in table 4 ) . with regard to the cross - level main effect predicted in hypothesis 2a , the results indicate an unexpectedly negative effect of group - level job resources on individual work engagement ( =0.30 , p < .05 ) over and above individual job resources ( see step 3 in table 4 ) . hypothesis 2c stated that the cross - level relationship should sustain even when possible competing effects of and interaction with the variability of job resources within groups are added to the model . the results show that the negative relationship remains marginally significant when group - level variability of job resources is controlled for ( see step 4 in table 4 ) . therefore , group - level job resources had an opposite relationship to work engagement compared with individual - level job resources . step 5 in table 4 indicates that group - level job resources interact with their variability within groups ( =1.69 , p < .05 ) , indicating that the cross - level relationship of group - level job resources and work engagement is dependent on the variability within groups . in case of such a significant interaction effect , cole et al . recommend to test for curvilinear effects of group - level job resources and their variability . results however indicate no curvilinear relationships with work engagement , whereupon a significant interaction effect can be interpreted . it seems as if the combination of high group - level job resources with low variability is associated with low work engagement . we further tested for random slopes in the individual - level relationships specified in the model described above . the results indicate that the random - slopes model does not suit the data better than the random intercept - fixed slope models ( ( 1)=3.15 , p=.21 ) . therefore , the relationship between individual job resources and work engagement did not significantly vary between groups . additionally , we calculated the likelihood ratio - based pseudo r with nagelkerke adjustments to obtain an estimate for variance explanation in work engagement . the final and best - fitting model consequently explains 39% of the variance in work engagement ( see step 5 in table 4 ) . to summarize , with regard to the analyses conducted with the group - level job resources total factor , hypotheses 2a and therefore 2c were not supported . multilevel analysis examining overall group - level job resources , group - level job resources variability , and their interaction ( dependent variable : individual work engagement ) work engagement as a function of group - level job resources and their variability within groups . the analyses for the job resources subfactors were conducted according to the same procedure described above . again , the control variables that did not contribute to the prediction of work engagement were omitted from the models : education , = 0.02 , p=.64 and job tenure , = 0.00 , p=.80 both in step 2 and group size , =0.00 , p=.70 in step 3 . all three individual job resources subfactors were significantly positively associated with individual work engagement ( see step 2 in table 5 ) . with regard to the group - level job resources subfactors , the picture looks slightly different than in the analyses with the total job resources factor . as step 3 in table 5 indicates , only the two subfactors , peer behavior ( =0.24 , p < .05 ) and task - related job resources ( =0.38 , p < .001 ) , yield negative relationships with work engagement , whereas manager behavior is not associated with engagement ( =0.10 , p=.41 ) . we further tested for random slopes in the individual - level relationships of the three job resources subfactors and work engagement . the results again indicate that the random - slope models do not suit the data better than the random intercept - fixed slope models ( manager behavior : ( 2)=1.03 , p=.60 ; peer behavior : ( 2)=3.79 , p=.15 ; task - related resources : ( 2)=2.92 , p=.23 ) . therefore , the relationship between individual job resources subfactors and work engagement did not significantly vary between the groups . according to hypothesis 2c , we further controlled for the corresponding variability in job resources subfactors within groups , as recommended by cole et al .. only task - related resources remain a significant negative predictor of work engagement . moreover , variability of task - related resources itself positively predicts work engagement ( =0.67 , p < .01 ) ( see step 4 in table 5 ) . thus , the lower the group - level task - related resources and the higher their variance within groups , the higher employees ' work engagement is . according to hypothesis 2c we further tested for possible interactions of group means and variability within groups and found no effects ( see step 5 in table 5 ) . compared with the model including only individual - level variables ( step 2 ) , the final model explains 1% more variance in individual work engagement . to summarize , the results of the analyses conducted with the job resources subfactors confirm the negative relationship of group - level job resources but are limited to the subfactor of task - related resources . furthermore , the interaction effect yielded in the analyses with the total job resources factor did not appear when using the subfactors . instead , results indicate that , above all , group - level task - related resources and their variability explain variance in individual work engagement over and above individual job resources . therefore , on the basis of the results of the analyses conducted with the subfactors of group - level job resources , hypothesis 2b and therefore 2c were not supported . multilevel analysis examining group - level job resources subfactors , group - level job resources subfactors variability , and their interactions ( dependent variable : individual work engagement ) because of the unexpected negative relationships found , we conducted three kinds of supplementary analyses to ensure that collinearity did not influence the results and inferences drawn by the multilevel analyses . a detailed report of the post - hoc analyses is omitted with regard to space restrictions and can be obtained from the corresponding author . overall , post - hoc analyses indicate that multicollinearity does not pose a problem in the analyses . the first hypothesis stated that group - level job resources emerge as a contextual resources construct from aggregated individual perceptions of job resources . results of an analysis of variance of job resources with work groups as the grouping variable indicated significant between - group differences [ f ( 102 , 990 ) = 1.78 , p < .001 ] . the calculated icc(1 ) shows that 7% of the variance in individual - level job resources depended on group membership , representing a small to medium effect of the grouping structure . finally , the mean within - group agreement coefficient rwg ( j ) was.97 ( range=.91-.99 ) , indicating very strong agreement within groups . group differences were significant for all subfactors with icc(1 ) values indicating that 5% , 8% , and 19% of the variance in manager behavior , peer behavior , and task - related resources , respectively , depended on group membership . this represents a small to medium effect of the grouping structure in manager and peer behavior and a medium to large effect in task - related resources . the corresponding icc(2 ) values were.38,.51 , and.73 for manager behavior , peer behavior , and task - related resources , respectively , indicating a low reliability of the group mean for manager behavior , a moderate reliability for peer behavior , and a good reliability of the group mean for task - related resources . finally , the mean within - group agreement coefficients rwg ( j ) for manager behavior , peer behavior , and task - related resources were.93 ( range=.81-.98),.82 ( range=.00-.98 ) , and.90 ( range=.65-.98 ) , respectively . in summary , these results seem to support the aggregation of individual - level job resources to the work group level , forming a contextual group - level job resource construct . therefore , concerning the supposed influence of the work group context on individual job resources subfactors , hypothesis 1b was supported . hypothesis 2a stated that group - level job resources have an additionally positive effect on individual work engagement over and above individual - level job resources . to test for this presumed cross - level main effect of group - level job resources , multilevel analyses were conducted following the procedure explained in the method section . all control variables that did not contribute to the prediction of work engagement were omitted from the models in step 2 for the individual - level covariates education ( unstandardized parameter estimate =0.02 , p=.67 ) and job tenure ( =0.00 , p=.96 ) and in step 3 for the group - level covariate group size ( =0.00 , p=.71 ) . however , as table 4 indicates , there was a significant relationship between sex , age , and work engagement . women ( =0.17 , p < .05 ) and older employees ( =0.01 , p < .001 ) indicated higher levels of work engagement . furthermore , individual job demands negatively relate with work engagement ( =0.12 , p < .01 ) and individual job resources positively relate ( =0.44 , p < .001 ) ( see step 2 in table 4 ) . with regard to the cross - level main effect predicted in hypothesis 2a , the results indicate an unexpectedly negative effect of group - level job resources on individual work engagement ( =0.30 , p < .05 ) over and above individual job resources ( see step 3 in table 4 ) . hypothesis 2c stated that the cross - level relationship should sustain even when possible competing effects of and interaction with the variability of job resources within groups are added to the model . the results show that the negative relationship remains marginally significant when group - level variability of job resources is controlled for ( see step 4 in table 4 ) . therefore , group - level job resources had an opposite relationship to work engagement compared with individual - level job resources . this result was further validated . step 5 in table 4 indicates that group - level job resources interact with their variability within groups ( =1.69 , p < .05 ) , indicating that the cross - level relationship of group - level job resources and work engagement is dependent on the variability within groups . in case of such a significant interaction effect , cole et al . recommend to test for curvilinear effects of group - level job resources and their variability . results however indicate no curvilinear relationships with work engagement , whereupon a significant interaction effect can be interpreted . it seems as if the combination of high group - level job resources with low variability is associated with low work engagement . we further tested for random slopes in the individual - level relationships specified in the model described above . the results indicate that the random - slopes model does not suit the data better than the random intercept - fixed slope models ( ( 1)=3.15 , p=.21 ) . therefore , the relationship between individual job resources and work engagement did not significantly vary between groups . additionally , we calculated the likelihood ratio - based pseudo r with nagelkerke adjustments to obtain an estimate for variance explanation in work engagement . the final and best - fitting model consequently explains 39% of the variance in work engagement ( see step 5 in table 4 ) . to summarize , with regard to the analyses conducted with the group - level job resources total factor , hypotheses 2a and therefore 2c were not supported . multilevel analysis examining overall group - level job resources , group - level job resources variability , and their interaction ( dependent variable : individual work engagement ) work engagement as a function of group - level job resources and their variability within groups . the analyses for the job resources subfactors were conducted according to the same procedure described above . again , the control variables that did not contribute to the prediction of work engagement were omitted from the models : education , = 0.02 , p=.64 and job tenure , = 0.00 , p=.80 both in step 2 and group size , =0.00 , p=.70 in step 3 . all three individual job resources subfactors were significantly positively associated with individual work engagement ( see step 2 in table 5 ) . with regard to the group - level job resources subfactors , the picture looks slightly different than in the analyses with the total job resources factor . as step 3 in table 5 indicates , only the two subfactors , peer behavior ( =0.24 , p < .05 ) and task - related job resources ( =0.38 , p < .001 ) , yield negative relationships with work engagement , whereas manager behavior is not associated with engagement ( =0.10 , p=.41 ) . we further tested for random slopes in the individual - level relationships of the three job resources subfactors and work engagement . the results again indicate that the random - slope models do not suit the data better than the random intercept - fixed slope models ( manager behavior : ( 2)=1.03 , p=.60 ; peer behavior : ( 2)=3.79 , p=.15 ; task - related resources : ( 2)=2.92 , p=.23 ) . therefore , the relationship between individual job resources subfactors and work engagement did not significantly vary between the groups . according to hypothesis 2c , we further controlled for the corresponding variability in job resources subfactors within groups , as recommended by cole et al .. only task - related resources remain a significant negative predictor of work engagement . moreover , the lower the group - level task - related resources and the higher their variance within groups , the higher employees ' work engagement is . according to hypothesis 2c we further tested for possible interactions of group means and variability within groups and found no effects ( see step 5 in table 5 ) . the final model therefore explains 40% of the variance in work engagement . compared with the model including only individual - level variables ( step 2 ) , the final model explains 1% more variance in individual work engagement . to summarize , the results of the analyses conducted with the job resources subfactors confirm the negative relationship of group - level job resources but are limited to the subfactor of task - related resources . furthermore , the interaction effect yielded in the analyses with the total job resources factor did not appear when using the subfactors . instead , results indicate that , above all , group - level task - related resources and their variability explain variance in individual work engagement over and above individual job resources . therefore , on the basis of the results of the analyses conducted with the subfactors of group - level job resources , hypothesis 2b and therefore 2c were not supported . multilevel analysis examining group - level job resources subfactors , group - level job resources subfactors variability , and their interactions ( dependent variable : individual work engagement ) because of the unexpected negative relationships found , we conducted three kinds of supplementary analyses to ensure that collinearity did not influence the results and inferences drawn by the multilevel analyses . a detailed report of the post - hoc analyses is omitted with regard to space restrictions and can be obtained from the corresponding author . overall , post - hoc analyses indicate that multicollinearity does not pose a problem in the analyses . a recent overview of the state of the art of the jd - r model encouraged the integration of multilevel thinking into it . this study followed the call by investigating the following : first , whether and to what extent job resources cluster within work groups and second , whether it is additionally beneficial for employee engagement if they work in a resource - rich psychosocial work group environment , i.e. , where co - workers experience on average high job resources . the results supported the first hypothesis , as group membership indeed accounted for 7% of the variance in individual job resources , which represents a small to medium effect . as stated in the introduction , this is reasonable because employees in a group share variance in individual job resources because of their group membership and therefore shared psychosocial context factors or group characteristics such as similar work tasks , common supervisors and colleagues , and a group climate . with regard to the three job resources subfactors , group membership yielded a small to medium effect on manager and peer behavior ( 5% and 8% , respectively ) and a medium to large effect ( 19% ) on task - related resources . these values are comparable to those found in the literature on work characteristics and well - being . studies reporting higher icc(1 ) values used a different approach in operationalizing group - level constructs . for example , constructs have a different meaning if they directly refer to the overall level of support within the team , i.e. , team support , which has been performed in a study by vera et al . , or whether individually perceived support is aggregated to a mean level of support in teams , which is the case in this study . an explanation for this pattern could be found in the nature of medium- and large - sized organizations that participated in this study . medium- and large - sized organizations are more likely to have work groups with similar job tasks ( in structural terms ) clustered in these . moreover , the more personal job resources , such as manager and peer behavior , are more likely to individually vary as relationships are more affected by individual characteristics than more structural aspects of the work characteristics , such as job control and task identity . the results of the study did not support the second hypothesis : although group - level job resources had a significant cross - level relationship with work engagement over and above individual - level job resources , the relationship was in the opposite direction than assumed . in addition , the amount of variance explained in work engagement at group level was very small , particularly when compared with the variance explained by individual job resources . moreover , the interaction with job resources variability at group level indicates that the combination of overall high group - level job resources and a low dispersion within the group is not favorable for employee engagement either . the results for the three subfactors of group - level job resources yield a more detailed picture . when controlled for the variability at group level , only task - related resources ( comprised of job control and task identity ) are significantly negatively related to work engagement . furthermore , the analyses indicate that the dispersion of task - related resources within work groups plays a role because a positive cross - level main effect was found on work engagement . to conclude , it seems not only not additionally favorable but even detrimental for employees ' work engagement , first , if their work group colleagues on average perceive high job control and task identity and second , when there is a small dispersion in these perceptions , i.e. , work group members perceive their task - related resources very similarly . this negative relationship is unexpected and contrary to the positive cross - level relationship reported in one study where team coworker and manager support were positively related to individual work engagement . that study , however , was based on a more narrow study population of nursing teams in one hospital . nursing teams are supposedly more cohesive and interdependent than the more diverse work groups from different sectors in the present study . moreover , the nursing team study used a different operationalization of team job resources , referring to all team members in general and not to the individuals . furthermore , the study of vera et al . only focused on social team resources , which in our case with the subfactors peer and manager behavior did not yield a significant ( negative ) relationship with work engagement . to summarize , the nursing teams study did not examine the same research question as this study ; vera et al . were not interested in mean levels of job resources and their effect on work engagement over and above individual job resources because they did not include the individual support variables into their analyses as well . in summary , because the results of the present study do not support the initial hypotheses and because there are not sufficient studies on the topic to provide a clear picture , future research is required to further explore the unexpected relationships found in this study . if this result pattern is replicated in future studies , alternative explanations should be investigated . we suggest some ideas and directions about possible alternative explanatory approaches , which could be explored in future studies to shed light on these somewhat counterintuitive results . considering the observed change in the direction of the relationships between engagement and individual- and group - level job resources , our assumption - of whether one can consider group - level job resources as a proxy for a resource - rich work group environment - bliese offers an alternative approach in describing the fuzzy composition model and associated emergent processes and effects at group level , implying that the aggregate variable at group level and the lower - level variable have a ( slightly ) different meaning . as a result , the aggregate - level variables often tap more or rather other constructs than the individual - level variables so that the aggregated variable contains a higher level of contextual influences not captured by the individual - level construct . consequently , bliese states that by applying fuzzy composition processes , " analyses involving higher - level constructs are likely to reveal relationships that differ from those at lower levels " ( p. 371 ) . in line with this reasoning , it has , for example , been suggested " that a supportive team atmosphere is a clear resource at the individual level but at the team level it can represent a factor that restricts individual freedom . in this way , the same construct could have different functions at different levels of analysis " ( p. 5 ) . the finding of a negative cross - level relationship of group - level job resources and engagement thus supports the notion of change in the meaning of constructs across levels . following this line of thought , we can speculate about what factors may manifest in the group - level job resources construct , particularly in the task - related resources subfactor , which may explain the unexpected negative relationship with work engagement . work groups with high levels of task - related resources are characterized by employees having high job control and task identity , which means they can perform their tasks in an independent and autonomous way . when we disregard the group context , this situation enhances the engagement of the individual . however , taking into account the work group context and its influence , this situation leads to a picture of a work group of lone fighters , where people do not need to coordinate and interact a lot to fulfill their tasks . from an employee 's perspective , working in a group of lone fighters with low task interdependence and no common goals is rather demotivating and engagement derogating . therefore , in comparing themselves with their co - workers in their immediate work context , employees may well consider their prospects of gaining resources . consequently , we assume that employees working in groups with high group - level job resources and low dispersion of job resources could perceive that there is not that much room for improvement left . in contrast , employees working in groups with lower group - level job resources on average combined with high dispersion could perceive a potential for improvement that is worth aspiring for . social comparisons , particularly their results , influence many outcomes such as one 's self - concept , aspiration level , and subjective well - being . some limitations of this study should be acknowledged . first of all , we can only speculate about the nature of group characteristics , underlying group processes , or context factors that could manifest in the construct of group - level job resources . according to bliese and colleagues , shared group characteristics , such as cohesion , and/or clustering of individual attributes by work groups , such as intelligence , could influence individual reports of engagement and consequently their group averages . thus , future research should include specific group constructs , such as the need for cooperation and communication , and group cohesion or collective control to gain further insight into the emergent meaning of job resources at group level . moreover , it would be interesting to examine whether the relative position of an individual 's job resources within a group has an effect on his / her work engagement in the sense of a singled out or frog pond hypothesis . another limitation relates to the weakness of single item measures , which we used to assess appreciation from colleagues and supervisors , social support from the supervisor , and task identity . however , as we have a theoretically grounded selection of measures and structure because we subsequently built factor scores of job resources , which was supported by exploratory and confirmatory factor analyses , we partly counter the potential drawback of single - item measures . a third limitation concerns the cross - sectional design of the current study , which does not allow us to draw causal relationships between the study variables . thus , longitudinal research and cross - lagged designs would be useful to examine causal relationships between group - level job resources and work engagement . a final limitation of the present study relates to the restriction to two - level models . although our data had more than two hierarchical levels , the limitations of the study sample of six organizations precluded the integration of a third level of the organization itself . the data employed by this study was collected in the context of the swing project financed by health promotion switzerland and the swiss assurance association . the first author was supported by the swiss national science foundation ( snsf ) . first of all , we can only speculate about the nature of group characteristics , underlying group processes , or context factors that could manifest in the construct of group - level job resources . according to bliese and colleagues , shared group characteristics , such as cohesion , and/or clustering of individual attributes by work groups , such as intelligence , could influence individual reports of engagement and consequently their group averages . thus , future research should include specific group constructs , such as the need for cooperation and communication , and group cohesion or collective control to gain further insight into the emergent meaning of job resources at group level . moreover , it would be interesting to examine whether the relative position of an individual 's job resources within a group has an effect on his / her work engagement in the sense of a singled out or frog pond hypothesis . another limitation relates to the weakness of single item measures , which we used to assess appreciation from colleagues and supervisors , social support from the supervisor , and task identity . however , as we have a theoretically grounded selection of measures and structure because we subsequently built factor scores of job resources , which was supported by exploratory and confirmatory factor analyses , we partly counter the potential drawback of single - item measures . a third limitation concerns the cross - sectional design of the current study , which does not allow us to draw causal relationships between the study variables . thus , longitudinal research and cross - lagged designs would be useful to examine causal relationships between group - level job resources and work engagement . a final limitation of the present study relates to the restriction to two - level models . although our data had more than two hierarchical levels , the limitations of the study sample of six organizations precluded the integration of a third level of the organization itself . the data employed by this study was collected in the context of the swing project financed by health promotion switzerland and the swiss assurance association . the first author was supported by the swiss national science foundation ( snsf ) .

objectives : this study adds a multilevel perspective to the well - researched individual - level relationship between job resources and work engagement . in addition , we explored whether individual job resources cluster within work groups because of a shared psychosocial environment and investigated whether a resource - rich psychosocial work group environment is beneficial for employee engagement over and above the beneficial effect of individual job resources and independent of their variability within groups . methods : data of 1,219 employees nested in 103 work groups were obtained from a baseline employee survey of a large stress management intervention project implemented in six medium and large - sized organizations in diverse sectors . a variety of important job resources were assessed and grouped to an overall job resource factor with three subfactors ( manager behavior , peer behavior , and task - related resources ) . data were analyzed using multilevel random coefficient modeling . results : the results indicated that job resources cluster within work groups and can be aggregated to a group - level job resources construct . however , a resource - rich environment , indicated by high group - level job resources , did not additionally benefit employee work engagement but on the contrary , was negatively related to it . conclusions : on the basis of this unexpected result , replication studies are encouraged and suggestions for future studies on possible underlying within - group processes are discussed . the study supports the presumed value of integrating work group as a relevant psychosocial environment into the motivational process and indicates a need to further investigate emergent processes involved in aggregation procedures across levels .

Job resources and their relationship with work engagement Aggregated group-level job resources Cross-level relationship between group-level job resources and individual work engagement Method Participants Measures Data analyses Results Total job resources factor (H1a) Three job resources subfactors (H1b) Total job resources factor (H2a+c) Three job resources subfactors (H2b+c) Post-hoc analyses Discussion Study limitations and directions for future research

this study focused on the motivational process proposed by the jd - r model , whereby individual job resources exert a motivational potential and lead to high work engagement . the relationships between job resources and work engagement , however , can not be generalized to apply to cross - level relationships to answer the present research question because these studies were conducted at an individual level of analysis . accordingly , one can expect some minimal agreement on the perceptions of job resources within groups because group members are exposed to shared psychosocial context factors or group characteristics , such as similar work tasks , common supervisors and colleagues , and a group climate . in conceptualizing the construct of group - level job resources , the present study applied an additive composition model , also referred to as a summary index model , and aggregated individual - level job resources to the work group level . in doing so , it was assumed that the aggregated group - level job resource construct represents a proxy for a resource - rich psychosocial work group environment . , group - level manager behavior , group - level peer behavior , group - level task - related resources , emerge as contextual resource constructs from aggregated individual perceptions of job resource subfactors . gavin and hofmann aggregated individual perceptions of task significance , a core job resource , to the group level and found evidence for an additional contextual influence of group - level task significance on individual - level hostility after controlling for individual - level task significance . with regard to the positive side of work characteristics and employee health and well - being , referred to as the motivational process in the jd - r model , there is one study that examined the clustering of group - level job resources and their relationships with individual well - being in terms of work engagement . however , what we do not know is whether it is additionally beneficial for employees ' work engagement if they work in a resource - rich psychosocial work group environment , i.e. on the basis of the limited existing evidence summarized above , we expected that a resource - rich work group environment would have an additionally motivation - enhancing effect on one 's work engagement and thus formulated the following hypotheses : hypothesis 2a : group - level job resources have an additive positive effect on individual work engagement over and above that of individual - level job resources . , group - level manager behavior , group - level peer behavior , group - level task - related resources , have an additive positive effect on individual work engagement over and above that of individual - level job resource subfactors . when analyzing the effects of group - level job resources , it is important to control for the effects of variability of job resources within groups , which has been shown in a study on leadership climate . variability within groups can suppress irrelevant variance in group - level job resources and thus ensures an unbiased estimation of the effect of group - level job resources on individual engagement . not only a direct effect of variability within groups on engagement but also an interaction effect of variability and group - level job resources is possible and should therefore be controlled for in the analyses to facilitate an unbiased estimation of the cross - level effect . this study employed data collected in the baseline employee survey of a large stress - management intervention project ( see acknowledgments ) . the study sample consisted of 1,219 employees without supervisory function from six medium- and large - sized swiss organizations in diverse sectors ( three industrial production companies , one food processing company , one public administration service , and one hospital ) . this study employed data collected in the baseline employee survey of a large stress - management intervention project ( see acknowledgments ) . the study sample consisted of 1,219 employees without supervisory function from six medium- and large - sized swiss organizations in diverse sectors ( three industrial production companies , one food processing company , one public administration service , and one hospital ) . , individual job resources , was assessed using eight scales , which were clustered into three subfactors of job resources : manager behavior , peer behavior , and task - related resources . , group - level job resources , was assessed by calculating the means of individual job resources of all employees who participated from each work group . this was accordingly performed for each subfactor and the overall job resources factor , resulting in group - level manager behavior , group - level peer behavior , group - level task - related resources , and the overall group - level job resources construct . , group - level job resources , we controlled for their variability within groups to ensure that the effect of group - level job resources on engagement is not biased by variability within groups . applying a dispersion composition model , group - level job resources variability was assessed by calculating the standard deviation of individual job resources scores of all employees who participated from each work group . to reduce the data set to a smaller subset of variables , we calculated factor scores ( regression method ) for the three job resources subfactors of manager behavior , peer behavior , and task - related resources in the first step and for the overall job resources factor in the second step . we assumed minimal within - group agreement of individual ratings and significant differences across work groups in the mean level of job resources reflecting the shared psychosocial work group environment ( hypotheses 1a and 1b ) . to test for the presumed cross - level main effect of group - level job resources ( hypotheses 2a and 2b ) , we estimated , first , a model with no explanatory variables ( intercept - only model ) , which served as a benchmark value of deviance for comparison with competing models ( step 1 ) . in this step , individual and group - level job resources were grand mean centered to detect an additional explanatory value of group - level job resources on individual engagement . in a final step , we examined a possible interaction effect of group - level job resources and their variability within groups to examine if the hypothesized cross - level effect of group - level job resources is independent of the level of variability of job resources within groups ( step 5 ) . to reduce the data set to a smaller subset of variables , we calculated factor scores ( regression method ) for the three job resources subfactors of manager behavior , peer behavior , and task - related resources in the first step and for the overall job resources factor in the second step . we assumed minimal within - group agreement of individual ratings and significant differences across work groups in the mean level of job resources reflecting the shared psychosocial work group environment ( hypotheses 1a and 1b ) . to test for the presumed cross - level main effect of group - level job resources ( hypotheses 2a and 2b ) , we estimated , first , a model with no explanatory variables ( intercept - only model ) , which served as a benchmark value of deviance for comparison with competing models ( step 1 ) . in this step , individual and group - level job resources were grand mean centered to detect an additional explanatory value of group - level job resources on individual engagement . in a final step , we examined a possible interaction effect of group - level job resources and their variability within groups to examine if the hypothesized cross - level effect of group - level job resources is independent of the level of variability of job resources within groups ( step 5 ) . means , standard deviations , and bivariate correlations of study variables at individual level ( n=1,219 ) means , standard deviations , and bivariate correlations of study variables at group level ( n=103 ) the first hypothesis stated that group - level job resources emerge as a contextual resources construct from aggregated individual perceptions of job resources . group differences were significant for all subfactors with icc(1 ) values indicating that 5% , 8% , and 19% of the variance in manager behavior , peer behavior , and task - related resources , respectively , depended on group membership . the corresponding icc(2 ) values were.38,.51 , and.73 for manager behavior , peer behavior , and task - related resources , respectively , indicating a low reliability of the group mean for manager behavior , a moderate reliability for peer behavior , and a good reliability of the group mean for task - related resources . finally , the mean within - group agreement coefficients rwg ( j ) for manager behavior , peer behavior , and task - related resources were.93 ( range=.81-.98),.82 ( range=.00-.98 ) , and.90 ( range=.65-.98 ) , respectively . in summary , these results seem to support the aggregation of individual - level job resources to the work group level , forming a contextual group - level job resource construct . hypothesis 2a stated that group - level job resources have an additionally positive effect on individual work engagement over and above individual - level job resources . to test for this presumed cross - level main effect of group - level job resources all control variables that did not contribute to the prediction of work engagement were omitted from the models in step 2 for the individual - level covariates education ( unstandardized parameter estimate =0.02 , p=.67 ) and job tenure ( =0.00 , p=.96 ) and in step 3 for the group - level covariate group size ( =0.00 , p=.71 ) . with regard to the cross - level main effect predicted in hypothesis 2a , the results indicate an unexpectedly negative effect of group - level job resources on individual work engagement ( =0.30 , p < .05 ) over and above individual job resources ( see step 3 in table 4 ) . therefore , group - level job resources had an opposite relationship to work engagement compared with individual - level job resources . step 5 in table 4 indicates that group - level job resources interact with their variability within groups ( =1.69 , p < .05 ) , indicating that the cross - level relationship of group - level job resources and work engagement is dependent on the variability within groups . recommend to test for curvilinear effects of group - level job resources and their variability . therefore , the relationship between individual job resources and work engagement did not significantly vary between groups . multilevel analysis examining overall group - level job resources , group - level job resources variability , and their interaction ( dependent variable : individual work engagement ) work engagement as a function of group - level job resources and their variability within groups . as step 3 in table 5 indicates , only the two subfactors , peer behavior ( =0.24 , p < .05 ) and task - related job resources ( =0.38 , p < .001 ) , yield negative relationships with work engagement , whereas manager behavior is not associated with engagement ( =0.10 , p=.41 ) . the results again indicate that the random - slope models do not suit the data better than the random intercept - fixed slope models ( manager behavior : ( 2)=1.03 , p=.60 ; peer behavior : ( 2)=3.79 , p=.15 ; task - related resources : ( 2)=2.92 , p=.23 ) . according to hypothesis 2c , we further controlled for the corresponding variability in job resources subfactors within groups , as recommended by cole et al .. only task - related resources remain a significant negative predictor of work engagement . thus , the lower the group - level task - related resources and the higher their variance within groups , the higher employees ' work engagement is . to summarize , the results of the analyses conducted with the job resources subfactors confirm the negative relationship of group - level job resources but are limited to the subfactor of task - related resources . instead , results indicate that , above all , group - level task - related resources and their variability explain variance in individual work engagement over and above individual job resources . therefore , on the basis of the results of the analyses conducted with the subfactors of group - level job resources , hypothesis 2b and therefore 2c were not supported . multilevel analysis examining group - level job resources subfactors , group - level job resources subfactors variability , and their interactions ( dependent variable : individual work engagement ) because of the unexpected negative relationships found , we conducted three kinds of supplementary analyses to ensure that collinearity did not influence the results and inferences drawn by the multilevel analyses . the first hypothesis stated that group - level job resources emerge as a contextual resources construct from aggregated individual perceptions of job resources . group differences were significant for all subfactors with icc(1 ) values indicating that 5% , 8% , and 19% of the variance in manager behavior , peer behavior , and task - related resources , respectively , depended on group membership . the corresponding icc(2 ) values were.38,.51 , and.73 for manager behavior , peer behavior , and task - related resources , respectively , indicating a low reliability of the group mean for manager behavior , a moderate reliability for peer behavior , and a good reliability of the group mean for task - related resources . finally , the mean within - group agreement coefficients rwg ( j ) for manager behavior , peer behavior , and task - related resources were.93 ( range=.81-.98),.82 ( range=.00-.98 ) , and.90 ( range=.65-.98 ) , respectively . in summary , these results seem to support the aggregation of individual - level job resources to the work group level , forming a contextual group - level job resource construct . hypothesis 2a stated that group - level job resources have an additionally positive effect on individual work engagement over and above individual - level job resources . to test for this presumed cross - level main effect of group - level job resources , multilevel analyses were conducted following the procedure explained in the method section . all control variables that did not contribute to the prediction of work engagement were omitted from the models in step 2 for the individual - level covariates education ( unstandardized parameter estimate =0.02 , p=.67 ) and job tenure ( =0.00 , p=.96 ) and in step 3 for the group - level covariate group size ( =0.00 , p=.71 ) . with regard to the cross - level main effect predicted in hypothesis 2a , the results indicate an unexpectedly negative effect of group - level job resources on individual work engagement ( =0.30 , p < .05 ) over and above individual job resources ( see step 3 in table 4 ) . step 5 in table 4 indicates that group - level job resources interact with their variability within groups ( =1.69 , p < .05 ) , indicating that the cross - level relationship of group - level job resources and work engagement is dependent on the variability within groups . it seems as if the combination of high group - level job resources with low variability is associated with low work engagement . therefore , the relationship between individual job resources and work engagement did not significantly vary between groups . multilevel analysis examining overall group - level job resources , group - level job resources variability , and their interaction ( dependent variable : individual work engagement ) work engagement as a function of group - level job resources and their variability within groups . as step 3 in table 5 indicates , only the two subfactors , peer behavior ( =0.24 , p < .05 ) and task - related job resources ( =0.38 , p < .001 ) , yield negative relationships with work engagement , whereas manager behavior is not associated with engagement ( =0.10 , p=.41 ) . the results again indicate that the random - slope models do not suit the data better than the random intercept - fixed slope models ( manager behavior : ( 2)=1.03 , p=.60 ; peer behavior : ( 2)=3.79 , p=.15 ; task - related resources : ( 2)=2.92 , p=.23 ) . therefore , the relationship between individual job resources subfactors and work engagement did not significantly vary between the groups . according to hypothesis 2c , we further controlled for the corresponding variability in job resources subfactors within groups , as recommended by cole et al .. only task - related resources remain a significant negative predictor of work engagement . moreover , the lower the group - level task - related resources and the higher their variance within groups , the higher employees ' work engagement is . to summarize , the results of the analyses conducted with the job resources subfactors confirm the negative relationship of group - level job resources but are limited to the subfactor of task - related resources . instead , results indicate that , above all , group - level task - related resources and their variability explain variance in individual work engagement over and above individual job resources . therefore , on the basis of the results of the analyses conducted with the subfactors of group - level job resources , hypothesis 2b and therefore 2c were not supported . multilevel analysis examining group - level job resources subfactors , group - level job resources subfactors variability , and their interactions ( dependent variable : individual work engagement ) because of the unexpected negative relationships found , we conducted three kinds of supplementary analyses to ensure that collinearity did not influence the results and inferences drawn by the multilevel analyses . this study followed the call by investigating the following : first , whether and to what extent job resources cluster within work groups and second , whether it is additionally beneficial for employee engagement if they work in a resource - rich psychosocial work group environment , i.e. as stated in the introduction , this is reasonable because employees in a group share variance in individual job resources because of their group membership and therefore shared psychosocial context factors or group characteristics such as similar work tasks , common supervisors and colleagues , and a group climate . with regard to the three job resources subfactors , group membership yielded a small to medium effect on manager and peer behavior ( 5% and 8% , respectively ) and a medium to large effect ( 19% ) on task - related resources . the results of the study did not support the second hypothesis : although group - level job resources had a significant cross - level relationship with work engagement over and above individual - level job resources , the relationship was in the opposite direction than assumed . moreover , the interaction with job resources variability at group level indicates that the combination of overall high group - level job resources and a low dispersion within the group is not favorable for employee engagement either . the results for the three subfactors of group - level job resources yield a more detailed picture . when controlled for the variability at group level , only task - related resources ( comprised of job control and task identity ) are significantly negatively related to work engagement . furthermore , the analyses indicate that the dispersion of task - related resources within work groups plays a role because a positive cross - level main effect was found on work engagement . were not interested in mean levels of job resources and their effect on work engagement over and above individual job resources because they did not include the individual support variables into their analyses as well . considering the observed change in the direction of the relationships between engagement and individual- and group - level job resources , our assumption - of whether one can consider group - level job resources as a proxy for a resource - rich work group environment - bliese offers an alternative approach in describing the fuzzy composition model and associated emergent processes and effects at group level , implying that the aggregate variable at group level and the lower - level variable have a ( slightly ) different meaning . the finding of a negative cross - level relationship of group - level job resources and engagement thus supports the notion of change in the meaning of constructs across levels . following this line of thought , we can speculate about what factors may manifest in the group - level job resources construct , particularly in the task - related resources subfactor , which may explain the unexpected negative relationship with work engagement . consequently , we assume that employees working in groups with high group - level job resources and low dispersion of job resources could perceive that there is not that much room for improvement left . thus , longitudinal research and cross - lagged designs would be useful to examine causal relationships between group - level job resources and work engagement . thus , longitudinal research and cross - lagged designs would be useful to examine causal relationships between group - level job resources and work engagement .

microbiological strategies for the immobilization of dissolved uranium in aquifers focus on the addition of exogenous electron donors to stimulate growth of the indigenous microbial community . u(iv ) products observed in laboratory and field u(vi ) reduction experiments include biogenic uraninite [ uo2+x(s ) ] nanoparticles and a variety of noncrystalline u(iv ) products , including u(iv ) coordination polymers associated with biomass and u(iv ) monomers associated with fe(ii)-bearing minerals and titanium dioxide . the discovery of non - uraninite forms of u(iv ) in bioreduced field sediments is a key development in the field of uranium bioremediation because these species are less stable than uraninite in the presence of dissolved carbonate species and oxygen . although the products of uranium reduction are increasingly well documented in the laboratory , their dependence upon specific types of geochemical conditions and microbial metabolisms prevalent in the field has received comparatively less attention . bargar et al . examined the products of u(vi ) reduction in whole sediments bioreduced in the aquifer at the u.s . department of energy field research site at rifle , colorado , after 90 days of biostimulation , at which point deep sulfate - reducing conditions had been achieved ( i.e. , conditions characterized by reduction of ca . acetate stimulation of the rifle aquifer leads to the onset of predominantly metal - reducing conditions , followed by development of sulfate - reducing conditions after a lag time of ca . 1040 days , with shorter periods observed if the well gallery had previously been biostimulated . microbial community structure and groundwater / sediment geochemistry under these conditions contrast strongly . for example , in metal - reducing conditions , aqueous fe(ii ) species accumulate to concentrations exceeding 100 m , and planktonic metal - reducing bacteria are abundant , whereas in sulfate - reducing conditions fe(ii ) is sequestered in solid iron sulfide precipitates while sulfate - reducing bacteria and aqueous sulfide species begin to dominate . in particular , abiotic reduction often produces uraninite , whereas biological u(vi ) reduction in axenic culture often produces a mixture of noncrystalline u(iv ) and uraninite . these differences led us to hypothesize that u(iv ) products may vary in structure and stability in response to microbial community structure and redox chemistry . ultimately the reactivity of u(iv ) in the field is controlled by its speciation . thus , characterizing u(iv ) products formed as aquifer biogeochemical conditions progress is critical to designing effective field remediation schemes and provides insights to understand naturally occurring u(vi ) bioreduction . the objective of this study was to determine the effect of the predominant biogeochemical redox regime on the products of u(vi ) bioreduction under field - relevant conditions . to do so , we deployed reactors filled with fresh sediments from rifle , co , into groundwater wells at the field research site during the 2010 super 8 acetate biostimulation field experiment . the reactors were continuously eluted with the surrounding groundwater prior to and throughout the field experiment to biogeochemically couple them to the surrounding aquifer . reactors were sacrificed during late iron reduction and early and mid sulfate reduction , allowing us to compare the u(iv ) products and their reactivity under contrasting biogeochemical conditions . extended x - ray absorption fine structure ( exafs ) , chemical extraction , and electron microscopy were used to characterize the uranium products formed . sediments were collected at the integrated field research challenge ( ifrc , rifle , colorado , usa ) field site from below the water table with a backhoe on july 28 , 2010 , sieved in the field to < 2 mm , and homogenized in the process . sediments were stored moist in nitrogen - purged aluminized mylar bags at 4 c before being packed into borosilicate glass chromatography columns ( kontes ) measuring 2.6 cm in diameter and 15 cm in length ( approximately 80 cm of sediments ) . acid digestion of the super 8 composite ( vide infra ) showed that it contains only trace concentrations of u ( ( 8.0 2.4 ) 10 mol u g sediment ; see figure 3 ) . the in - well column field experiments were conducted using a design similar to those reported by moon et al . and bargar et al . specifically , reactors were deployed into groundwater well cd-04 in the experimental plot c well gallery on august 4 , 2010 ( supporting information , figure si-1 ) , located approximately 100 m from the super 8 composite collection site . the water table depth at the rifle site varies between approximately 9 and 12 ft below ground surface ( bgs ) , and the columns were placed at 1417 ft bgs to guarantee they remained submerged throughout the experiment . the rifle aquifer has low but measurable amounts of dissolved oxygen ( do ) , typically < 0.2 ppm . once deployed below the water table and prior to acetate amendment , cd-04 groundwater from a depth of approximately 17 ft bgs was pumped through the reactors at a nominal rate of 16.8 ml h , representing a darcy velocity of 9.5 10 m s , for 19 d to recondition the sediments to the aquifer chemistry and microbiology . subsequently , on august 23 , 2010 , amendment of the entire experimental plot c well gallery with 3 mm acetate commenced ( table 1 ; supporting information , figure si-2 ) . added acetate and uranium ( < 1 m ) present within the aquifer reached the columns via groundwater eluted through the columns . to ensure that sediment uranium concentrations would be adequate for spectroscopic and microscopic analysis , the reactors received supplemental acetate ( 3 mm ) and uranyl ( 20 m ) amendments beginning on august 24 , 2010 . this uranium concentration falls within the range of historical groundwater u(vi ) values at contaminated sites in the colorado river basin ( < ca . amendments were made by mixing acetate and a 200 m stock solution of uranyl acetate in groundwater at a 1:10 dilution to the influent groundwater . columns received up to 3 mm acetate from groundwater during the field acetate amendment . the columns were harvested from the well at three times , designated as t1t3 ( table 1 ) , corresponding to increasing time in the well , increasing acetate amendment duration , and therefore more reducing conditions . prior acetate biostimulation experiments at the rifle ifrc site provided valuable information about when redox transitions would occur in the aquifer ( e.g. , refs ( 7 ) , ( 17 ) , ( 23 ) , ( 24 ) ) , which was used to pre - plan harvest dates . t1 reactors received acetate and uranium amendments for 24 d , after which uranyl additions were halted , and they were eluted with groundwater for 7 d at 60 ml h to flush out unreduced u(vi ) species . all t1 reactors were harvested at the peak of fe(ii ) concentrations ( i.e. , under late iron - reducing conditions ) ( 8 mg l , figure si-2 ) and shipped to stanford synchrotron radiation lightsource ( ssrl ) under anoxic conditions for further analysis . reactors t2 and t3 , targeted for sulfate - reducing conditions , were initially amended with the field experiment acetate plume to advance them through the iron - reducing phase and into early sulfate reduction . subsequently , they were amended with 13 mm acetate and 20 m uranyl acetate , starting on october 9 , 2010 , 47 d after acetate amendment began in the experimental plot c gallery and when hs was present in well cd-04 . by waiting to amend with uranyl until sulfate - reducing conditions had already begun , we were able to avoid significant contamination of the column sediments by u(iv ) produced under preceding iron - reducing conditions . we are thus able to make a head - to - head comparison of iron - reduced and sulfate - reduced , we were able to ensure that sulfate - reducing bacteria ( srb ) did not consume all of the added acetate , and therefore geobacter could be available to reduce u(vi ) throughout . this design was chosen as a foundation for a future experiment ( not yet performed ) in which lower acetate conditions will be completely consumed by srb . the reactor t2 amendment lasted 15 d , after which uranyl amendments were halted , and t2 was eluted with groundwater as described above and recovered . the reactor t3 amendment extended for a total of 56 d , and subsequently t3 was flushed with groundwater for 6 d prior to recovery . during recovery from the aquifer , pre - installed valves located directly above and below the columns were closed to isolate the colums from inlet / outlet lines for removal . within 510 min , the entire intact columns were placed in hermetically sealed stainless steel shipping containers , which were purged with n2 gas . the glass column walls , end - caps , and rapid handling procedures prevented oxygen intrusion . column sediments were divided into top ( effluent ) and bottom ( influent ) sections , transferred into stoppered serum bottles under 5% h2/95% n2 atmosphere at ssrl , and frozen at 40 c to halt any further biogeochemical reactions . groundwater samples were collected from well cd-04 and other wells in the experimental plot c gallery several times per week during the experimental period ( august to november , 2010 ) and analyzed to determine the concentrations of a broad suite of anions and cations , dissolved acetate ( figure si-2 ) , electrical conductivity , do , and ph . details of these analytical methods can be found in the work of williams et al . total uranium in the sediments was determined by digestion using aqua regia , comprised of a 1:3 mixture of 15 m hno3 and 12 m hcl . prior to digestion , sediments were dried in a n2(g ) environment for 24 h at ambient temperatures to normalize water content . approximately 1 g of dried sediment ( weighed precisely in each case ) was placed into 5 ml of oxic aqua regia and allowed to react for 48 h. duplicate digestions were performed for each condition . aliquots of the digest were filtered through 0.22 m membranes and diluted as needed in 0.1 n hno3 , and total u was measured using inductively coupled plasma mass spectrometry ( icp - ms ; perkin - elmer elan drc ii ) . the noncrystalline u(iv ) fraction of the total reduced u pool in the sediments was determined by extraction with 1 m sodium bicarbonate solutions . in an anaerobic chamber containing 97% n2/3% h2 , a precisely weighed sample of each sediment ( approximately 1 g in all cases ) was reacted with 10 ml of anoxic 1 m bicarbonate for 24 h. bicarbonate extractions were performed in triplicate . an aliquot of each supernatant was filtered through a 0.22 m membrane , and 0.5 ml of supernatant was dissolved into 4.5 ml of oxic 1 m hno3 to rapidly oxidize noncrystalline u(iv ) extracted into the bicarbonate solution . the resulting solution was further diluted into 0.1 m hno3 as necessary for icp - ms measurements of total u. the noncrystalline u(iv ) fraction of the total u pool was taken to be the u measured in the bicarbonate extractions . remaining u , calculated by subtracting the noncrystalline u(iv ) fraction from the total u determined by aqua regia digestion , was interpreted as crystalline u(iv ) or uo2(s ) . an aqua regia digest of the initial column sediment , prior to acetate and uranyl amendment , showed that it contains a negligible amount of uranium ( see figure 3 ) . . showed that in some cases u(iv ) species extracted from sediments using a 1 m anoxic bicarbonate solution may be rapidly oxidized , even in the absence of oxygen . to test for the possibility of u(iv ) oxidation following bicarbonate extraction , the potential presence of adsorbed u(vi ) on the sediments , or a solid u(vi ) phase associated with reactor sediments , a separate 20 l aliquot of the filtered anoxic supernatants ( see above ) was mixed with 1.98 ml of anoxic 1 m hno3 and immediately analyzed using a kinetic phosphoresence analyzer . in these control experiments , no uranyl was detected as extracted from the sediments . this result correlates well with x - ray absorption near - edge structure ( xanes ) measurements of the same sediments ( supporting information , figure si-4 ) , which are commonly accepted to have an error of approximately 10% . genomic dna was extracted from 5 g of sediment per sample and amplified using universal bacterial 16s rrna gene primers 27f and 1492r , following methods described by handley et al . after amplification , leptospirillum ferrodiazotrophum(28 ) 16s rrna gene amplicons were spiked into samples at 0.5% final concentration to serve as a control . amplicons were fragmented to an average size of 300 bp , and illumina libraries were prepared as described by handley et al . barcoded libraries were pooled and sequenced on one - sixth of an illumina hiseq2000 flow cell ( illumina , inc . , san diego , ca , usa ) , and 100 bp long paired - end reads were collected . reads were trimmed to remove low - quality bases and assembled into ( nearly ) full - length 16s rrna gene sequences using the emirge ( expectation maximization iterative reconstruction of genes from the environment ) method for 16s rrna gene amplicons . briefly , 1 million randomly sampled reads per sample were reconstructed into full - length 16s rrna genes over 100 emirge iterations , and after initial read mapping to a de - replicated version of the silva 108 16s rrna database ( http://www.arb-silva.de/ ) . during reconstruction , sequences were clustered into operational taxonomic units ( otus ) based on similarities of 97% . otu abundances , calculated on the basis of the number of mapped reads , were normalized to account for varying sequence lengths . to exclude unreliable rare sequences , only otus with raw relative abundances 0.01% were used in analyses . representative otu sequences were analyzed using the ribosomal database project ( rdp , release 10 ) nave baysian classifier , in order to assign taxonomy . for comparison , otus were blasted against the silva 16s rrna database in order to determine sequence identities . sequences from all samples were aligned together using ssu - align 0.1 ( http://selab.janelia.org/software/ssu-align ) and masked to remove inserted columns ( which do not contain aligned nucleotides ) as well as regions of low alignment confidence , which were determined by the calculation of posterior probabilities based on alignment to a covariance model . an approximately maximum - likelihood phylogenetic tree was generated using fasttree 2.0.1 . sample communities were compared using phylogenetic ( tree - based ) and otu abundance information with weighted fast unifrac . data were analyzed with and without normalization , which accounts for potential differences in otu branch length owing to different rates of organism evolution . unreacted super 8 composite sediment and sediment samples from reactors t1t3 were fixed in epoxy ( epotek 301 ) in an anaerobic chamber , from which 30 m thick petrographic thin sections mounted on glass were made ( spectrum petrographics , vancouver , wa ) . scanning electron microscopy ( sem ) and x - ray energy - dispersive spectrometry ( eds ) were performed on a carl zeiss merlin ( oberkochen , germany ) microscope at 310 kv accelerating voltages in secondary electron imaging mode for sediment grains imaging and their chemical analysis . quantification of elements was done using an inca x - ray eds system ( oxford ) . sem images were collected from more than 80 individual grains , and from those , eds measurements of elemental composition were made on more than 350 individual points . for column t3 , which entered into sulfate - reducing conditions , 137 measurements were made at grain boundaries , and fe and s were co - present at 107 , or 77.4% of these points . u l - edge exafs spectra were measured at beamline 11 - 2 of the ssrl at the stanford linear accelerator laboratory ( slac ) to characterize the average molecular structure around uranium in bulk sediments . sediments were mounted into aluminum holders covered with kapton windows in an anaerobic chamber and mounted in a cryostat cooled with liquid nitrogen to 77 k to improve data quality . a double - crystal si ( 220 ) monochromator was used for energy selection , detuned 1530% to reject higher harmonic intensities . beamline energy resolution was controlled at much less than the u liii- or lii - edge line width ( 8.67 ev ) using vertical slits . exafs oscillations were subtracted by fitting a smoothly varying function ( spline ) to remove contributions below 1.4 , which may result in non - physical pair correlations , using the sixpack and horae analysis packages . feff8.4 was used to calculate backscattering phase and amplitude functions used to fit the spectra . changes in the fe and s mineralogy and the microbial communities in reactors t1t3 demonstrate the shift to more reducing conditions . sem images of silicate grains accompanied by eds data ( figure 1 ) show that unstimulated super 8 composite sediment has low concentrations of s ( figure 1a , b ) around the grain surfaces , and no correlation is observed between these elements . in contrast , grains from reactor t3 exhibit well - developed sulfidic coatings in which fe : s are well correlated at a ratio just below unity ( figure 1c , d ) . the ratio of fe : s in grain coatings in t3 less than 1:1 may be explained by the accumulation of elemental sulfur resulting from the oxidation of sulfide by u(vi ) or the reaction between aqueous hydrogen sulfide species and iron oxides . additionally , a significant change in sulfide concentrations in the effluent of reactor t3 , increasing from 0.014 mg l , 30 days following field acetate amendment , to 0.733 mg l after 70 days of field acetate amendment , was observed ( supporting information , table si-2 ) . williams et al . reported that elemental sulfur may form in rifle sediments via abiotic oxidation of aqueous sulfide coupled to the reduction of goethite or other fe(iii)-bearing refractory minerals in the sediments . furthermore , the development of similar grain coatings was observed in biostimulated sediments at the rifle site by bargar et al . sem images with selected area energy - dispersive x - ray spectroscopy ( eds ) data , evidencing the development of fes coatings on silica mineral grains in biostimulated sediments : ( a , b ) preamendment or super 8 composite sediment and ( c , d ) sulfate - reducing conditions in column t3 sediments ( influent end of column ) . a well - developed fe : s ratio is observed in sulfate - reducing conditions ( inset plots of eds data ) . microbial community analyses ( figure 2 ) record the transition in redox conditions observed in sem imaging of sediment grains . clear differences among communities correlate with the duration of acetate amendment and/or whether collected from the top ( effluent ) or bottom ( influent ) of columns ( figure 2 ) . communities collected from t1 reactors grouped together and separated from communities in reactors t2 and t3 ( figure 2 ; supporting information , figure si-3a , b ) . in contrast , communities collected at the infl and effl ends of reactor t2 were distinct from one another . the t2 infl community was evidently more similar to the later t3 communities , in particular the t3 effl community . the rifle ifrc site is far from thermodynamic equilibrium ; thus , nonequilibrium , kinetics - based approaches must be invoked to explain u transport at the site . the column reactor sediments are also far from equilibrium conditions , which allows for redox heterogeneity along the length of the column ( 15 cm ) and at the micrometer pore scale during acetate amendment . microbial community analyses showing community composition of the most abundant taxa ( > 1% relative abundance ) at the genus ( or nearest taxonomic ) level . effl and infl denote the top ( effluent ) and bottom ( influent ) ends the columns , respectively black boxes demark potential fe(iii)- and sulfate - reducing taxa ( i.e. , geobacter and other desulfuromonadales ; rhodoferax ; peptococcaceae such as desulfosporosinus and desulfotomaculum ; and desulfocapsa and desulfobulbaceae ) . abbreviations : cfb , cytophaga flavobacterium bacteroidetes ; betaprot , betaproteobacteria ; deltaprot , deltaproteobacteria . in terms of composition , there was an overall decrease in proteobacteria and increase in firmicutes with time ( figure si-3c ) . specifically , bacteria most closely related to geobacter , other desulfuromonadales , and rhodoferax ( betaproteobacteria ) were abundant at t1 . at later time points these bacteria decreased in relative abundance , and firmicutes most closely related to desulfotomaculum and other peptococcaceae increased ( figure 2 ; figure si-3c ) . the un - amended sediment community differs substantially from the acetate - amended communities and does not contain abundant ( > 1% ) desulfuromonadales or peptococcaceae . the results demonstrate the presence of abundant bacteria that are phylogenetically similar to known fe(iii ) reducers ( geobacter and other desulfuromonadales bacteria , rhodoferax , desulfosporosinus ) in the t1 reactors . in reactors t2 and t3 , the proportion of bacteria closely related to known sulfate - reducers increased substantially . potential srb include the deltaproteobacteria desulfocapsa and desulfobulbaceae and the firmicutes within the peptococcaceae , desulfosporosinus and desulfotomaculum . the progression from fe(iii)- to sulfate - reducing community occurred earlier in the bottoms ( infl ) section of columns than in the tops ( effl ) , as acetate - amended groundwater was pumped up through the columns from bottom to top . consequently , the community at the base of each column had access to higher concentrations of acetate throughout the experiment . while there is no unique gene indicative of uranium reduction , acetate - enriched geobacter species , which persisted throughout the amendment period , represent strong candidates for enzymatic fe(iii ) and uranium reduction within the aquifer ( e.g. , ref ( 45 ) ) . this is particularly likely during early amendment , when they were relatively more abundant . aqua regia sediment digestions revealed large differences in total uranium accumulated in sediments as a function of both reactor ( t1t3 ) and position within an individual reactor ( top or bottom ) . total uranium concentrations in the sediments vary from near zero to approximately 1 mol of u per gram of sediment . generally , more uranium was accumulated in the bottom sections of the reactors , where the influent groundwater containing acetate and uranium entered ( figure 3 ) , and where conditions were more reducing than in the effluent sections of the same columns . there was also more uranium as columns became more reduced , i.e. , t3 > t2 > t1 . this phenomenon is likely due to a combination of longer reaction times and faster u accumulation rates under sulfate reduction possibly involving biogenic iron sulfide minerals such as mackinawite . uranium quantification in sediments , including bicarbonate - extracted noncrystalline u(iv ) species ( red bars ) and total u ( blue bars ) from aqua regia digests , reported in mol of u per g of sediment . the uranium quantification for reactor t1 represents an average over the entire column , whereas t2 and t3 were divided into influent and effluent sediment u extractions . the fraction of total u present as noncrystalline u(iv ) species ( black circles ) is nearly constant for all sediments . uranium xas data were collected for the influent and effluent sections of reactors t1 and t3 and the influent section of t2 . xanes data indicate that between 75 and 100% of the total uranium pool is present as u(iv ) species ( figure si-4 ) . despite large differences in the total sediment uranium concentration ( figure 3 ) , time of reaction , and redox conditions ( figures 1 and 2 ) among the reacted sediments , the u exafs spectra are similar to one another ( figure 4 ; supporting information , table si-1 , figures si-5 and si-6 ) . in particular , all spectra contain a clear and reasonably strong signature of p ( or c ) shells at ca . 3.1 and 3.7 that is consistent with noncrystalline u(iv ) associated with biomass . u peak at 3.83.9 implies that a small fraction of u(iv ) in the samples is present as uraninite . uraninite is produced following the reaction of u(vi ) with a variety of sulfate - reducing , metal - reducing , and other bacteria and with abiotic reductants including mackinawite and other ferrous sulfides and oxides . hs(aq ) is expected to be insignificant as a reductant for u(vi ) in rifle groundwater because ( bi)carbonate concentrations in the millimolar range , such as are observed in rifle groundwater , inhibit the reduction of aqueous u(vi ) . uranium lii - edge exafs and fourier transforms for ( a ) column t1 effl , ( b ) column t1 infl , ( c ) column t2 infl , ( d ) column t3 effl , and ( e ) column t3 infl . the noncrystalline u(iv ) fraction of the total u(iv ) pool as estimated by bicarbonate extraction is constant within analytical and replicate error for the top and bottom sediment sections of all three reactors and averages approximately 65% . this result is also consistent with other studies of bioreduced u(iv ) species formed in laboratory and field experiments that use sediments from the rifle field site . sharp et al . conducted laboratory column experiments with rifle ifrc sediments and amended a solution containing 15 mm acetate , 55 m uranyl acetate , and 30 mm bicarbonate until iron - reducing conditions were achieved . sulfate reduction was not reached because sulfate was not added to the influent solution . they observed an immobilized u pool in the sediments dominated by noncrystalline u(iv ) , suggesting that laboratory column studies may be reasonable proxies for field studies such as ours . the extraction and exafs results suggest that the u(iv ) species remain dominated by noncrystalline u(iv ) through metal - reducing and deep into sulfate - reducing conditions . the apparent constancy in u(iv ) speciation implies a constancy in the mechanism(s ) of reduction . since geobacter and potentially other u(vi)-reducing species are active across widely ranging redox conditions , the u(vi ) removal mechanism(s ) they employ may be operable and produce similar u(iv ) products throughout . however , the presence of geobacter can not explain the strongly increased uranium accumulation under sulfate - reducing conditions . while we can not deduce a specific reaction mechanism or set of mechanisms to reconcile these results , we note that the presence of bacterial biomass is a constant throughout the experimental conditions . bargar et al . proposed a generalized reaction pathway in which the presence of bacterial biomass played a key role as a strong u(vi ) binding substrate . according to this model , u(vi ) bound to phosphoryl sites or phosphate groups in biomass could be reduced to u(iv ) by electrons conferred via shuttles ( soluble inorganic or organic shuttles or conductive pilli , or combinations thereof ) . u(iv ) reduced in this fashion would remain complexed to biomass functional groups as noncrystalline u(iv ) . , using single species cultures , further demonstrated that solution geochemistry controlled the viability of shewanella cells and their ability to produce exopolymers during u(vi ) reduction in rifle composition groundwater . exopolymer production was directly related to a larger fraction of non - crystalline u(iv ) in the overall reduced uranium pool . this model could help to explain the relative abundance of noncrystalline u(iv ) observed in the present study across strongly contrasting biogeochemical regimes . in any case , so long as reducing agents , electron shuttles , and microbial exopolymers are present that can bind and reduce u(vi ) , then noncrystalline u(iv ) is likely to be produced . although groundwater geochemistry has been shown to influence the speciation of u(iv ) formed in sediments ( e.g. , refs ( 6 ) , ( 13 ) ) , this study indicates that factors including the sub - surface redox conditions , dominant microbial community and physiology , grain - coating mineralogy , inorganic reducing agents , and total u accumulated in the sediments do not dictate the appearance of noncrystalline u(iv ) within the range of geochemical and microbial conditions tested here . driving an aquifer into sulfate - reducing conditions during bioremediation may be a sound strategy to increase the stability of u(vi ) reduction to u(iv ) species , i.e. , via association with fes , particularly as this does not appear to impact the speciation of the resulting u(iv ) products . as long as reducing conditions are maintained and in the absence of elevated aqueous carbonate concentrations , u(iv ) should remain immobile , even if present as noncrystalline u(iv ) species . however , if the groundwater chemistry changes , the high fraction of noncrystalline u(iv ) species , shown to be more easily mobilized and oxidized than uraninite u(iv ) , could be of concern . future studies should examine the impact of time and of different aquifer geochemistry and mineralogy on u(iv ) speciation , to further constrain field parameters that impact u(iv ) speciation after aquifer biostimulation .

in this study , we report the results of in situ u(vi ) bioreduction experiments at the integrated field research challenge site in rifle , colorado , usa . columns filled with sediments were deployed into a groundwater well at the site and , after a period of conditioning with groundwater , were amended with a mixture of groundwater , soluble u(vi ) , and acetate to stimulate the growth of indigenous microorganisms . individual reactors were collected as various redox regimes in the column sediments were achieved : ( i ) during iron reduction , ( ii ) just after the onset of sulfate reduction , and ( iii ) later into sulfate reduction . the speciation of u retained in the sediments was studied using x - ray absorption spectroscopy , electron microscopy , and chemical extractions . circa 90% of the total uranium was reduced to u(iv ) in each reactor . noncrystalline u(iv ) comprised about two - thirds of the u(iv ) pool , across large changes in microbial community structure , redox regime , total uranium accumulation , and reaction time . a significant body of recent research has demonstrated that noncrystalline u(iv ) species are more suceptible to remobilization and reoxidation than crystalline u(iv ) phases such as uraninite . our results highlight the importance of considering noncrystalline u(iv ) formation across a wide range of aquifer parameters when designing in situ remediation plans .

Introduction Methods Results and Discussion

microbiological strategies for the immobilization of dissolved uranium in aquifers focus on the addition of exogenous electron donors to stimulate growth of the indigenous microbial community . u(iv ) products observed in laboratory and field u(vi ) reduction experiments include biogenic uraninite [ uo2+x(s ) ] nanoparticles and a variety of noncrystalline u(iv ) products , including u(iv ) coordination polymers associated with biomass and u(iv ) monomers associated with fe(ii)-bearing minerals and titanium dioxide . the discovery of non - uraninite forms of u(iv ) in bioreduced field sediments is a key development in the field of uranium bioremediation because these species are less stable than uraninite in the presence of dissolved carbonate species and oxygen . examined the products of u(vi ) reduction in whole sediments bioreduced in the aquifer at the u.s . department of energy field research site at rifle , colorado , after 90 days of biostimulation , at which point deep sulfate - reducing conditions had been achieved ( i.e. acetate stimulation of the rifle aquifer leads to the onset of predominantly metal - reducing conditions , followed by development of sulfate - reducing conditions after a lag time of ca . microbial community structure and groundwater / sediment geochemistry under these conditions contrast strongly . in particular , abiotic reduction often produces uraninite , whereas biological u(vi ) reduction in axenic culture often produces a mixture of noncrystalline u(iv ) and uraninite . these differences led us to hypothesize that u(iv ) products may vary in structure and stability in response to microbial community structure and redox chemistry . ultimately the reactivity of u(iv ) in the field is controlled by its speciation . thus , characterizing u(iv ) products formed as aquifer biogeochemical conditions progress is critical to designing effective field remediation schemes and provides insights to understand naturally occurring u(vi ) bioreduction . the objective of this study was to determine the effect of the predominant biogeochemical redox regime on the products of u(vi ) bioreduction under field - relevant conditions . to do so , we deployed reactors filled with fresh sediments from rifle , co , into groundwater wells at the field research site during the 2010 super 8 acetate biostimulation field experiment . reactors were sacrificed during late iron reduction and early and mid sulfate reduction , allowing us to compare the u(iv ) products and their reactivity under contrasting biogeochemical conditions . extended x - ray absorption fine structure ( exafs ) , chemical extraction , and electron microscopy were used to characterize the uranium products formed . sediments were collected at the integrated field research challenge ( ifrc , rifle , colorado , usa ) field site from below the water table with a backhoe on july 28 , 2010 , sieved in the field to < 2 mm , and homogenized in the process . acid digestion of the super 8 composite ( vide infra ) showed that it contains only trace concentrations of u ( ( 8.0 2.4 ) 10 mol u g sediment ; see figure 3 ) . specifically , reactors were deployed into groundwater well cd-04 in the experimental plot c well gallery on august 4 , 2010 ( supporting information , figure si-1 ) , located approximately 100 m from the super 8 composite collection site . the water table depth at the rifle site varies between approximately 9 and 12 ft below ground surface ( bgs ) , and the columns were placed at 1417 ft bgs to guarantee they remained submerged throughout the experiment . this uranium concentration falls within the range of historical groundwater u(vi ) values at contaminated sites in the colorado river basin ( < ca . the columns were harvested from the well at three times , designated as t1t3 ( table 1 ) , corresponding to increasing time in the well , increasing acetate amendment duration , and therefore more reducing conditions . prior acetate biostimulation experiments at the rifle ifrc site provided valuable information about when redox transitions would occur in the aquifer ( e.g. , refs ( 7 ) , ( 17 ) , ( 23 ) , ( 24 ) ) , which was used to pre - plan harvest dates . t1 reactors received acetate and uranium amendments for 24 d , after which uranyl additions were halted , and they were eluted with groundwater for 7 d at 60 ml h to flush out unreduced u(vi ) species . all t1 reactors were harvested at the peak of fe(ii ) concentrations ( i.e. reactors t2 and t3 , targeted for sulfate - reducing conditions , were initially amended with the field experiment acetate plume to advance them through the iron - reducing phase and into early sulfate reduction . subsequently , they were amended with 13 mm acetate and 20 m uranyl acetate , starting on october 9 , 2010 , 47 d after acetate amendment began in the experimental plot c gallery and when hs was present in well cd-04 . by waiting to amend with uranyl until sulfate - reducing conditions had already begun , we were able to avoid significant contamination of the column sediments by u(iv ) produced under preceding iron - reducing conditions . we are thus able to make a head - to - head comparison of iron - reduced and sulfate - reduced , we were able to ensure that sulfate - reducing bacteria ( srb ) did not consume all of the added acetate , and therefore geobacter could be available to reduce u(vi ) throughout . the reactor t2 amendment lasted 15 d , after which uranyl amendments were halted , and t2 was eluted with groundwater as described above and recovered . the reactor t3 amendment extended for a total of 56 d , and subsequently t3 was flushed with groundwater for 6 d prior to recovery . column sediments were divided into top ( effluent ) and bottom ( influent ) sections , transferred into stoppered serum bottles under 5% h2/95% n2 atmosphere at ssrl , and frozen at 40 c to halt any further biogeochemical reactions . groundwater samples were collected from well cd-04 and other wells in the experimental plot c gallery several times per week during the experimental period ( august to november , 2010 ) and analyzed to determine the concentrations of a broad suite of anions and cations , dissolved acetate ( figure si-2 ) , electrical conductivity , do , and ph . total uranium in the sediments was determined by digestion using aqua regia , comprised of a 1:3 mixture of 15 m hno3 and 12 m hcl . aliquots of the digest were filtered through 0.22 m membranes and diluted as needed in 0.1 n hno3 , and total u was measured using inductively coupled plasma mass spectrometry ( icp - ms ; perkin - elmer elan drc ii ) . the noncrystalline u(iv ) fraction of the total reduced u pool in the sediments was determined by extraction with 1 m sodium bicarbonate solutions . an aliquot of each supernatant was filtered through a 0.22 m membrane , and 0.5 ml of supernatant was dissolved into 4.5 ml of oxic 1 m hno3 to rapidly oxidize noncrystalline u(iv ) extracted into the bicarbonate solution . the resulting solution was further diluted into 0.1 m hno3 as necessary for icp - ms measurements of total u. the noncrystalline u(iv ) fraction of the total u pool was taken to be the u measured in the bicarbonate extractions . remaining u , calculated by subtracting the noncrystalline u(iv ) fraction from the total u determined by aqua regia digestion , was interpreted as crystalline u(iv ) or uo2(s ) . showed that in some cases u(iv ) species extracted from sediments using a 1 m anoxic bicarbonate solution may be rapidly oxidized , even in the absence of oxygen . to test for the possibility of u(iv ) oxidation following bicarbonate extraction , the potential presence of adsorbed u(vi ) on the sediments , or a solid u(vi ) phase associated with reactor sediments , a separate 20 l aliquot of the filtered anoxic supernatants ( see above ) was mixed with 1.98 ml of anoxic 1 m hno3 and immediately analyzed using a kinetic phosphoresence analyzer . this result correlates well with x - ray absorption near - edge structure ( xanes ) measurements of the same sediments ( supporting information , figure si-4 ) , which are commonly accepted to have an error of approximately 10% . , san diego , ca , usa ) , and 100 bp long paired - end reads were collected . otu abundances , calculated on the basis of the number of mapped reads , were normalized to account for varying sequence lengths . scanning electron microscopy ( sem ) and x - ray energy - dispersive spectrometry ( eds ) were performed on a carl zeiss merlin ( oberkochen , germany ) microscope at 310 kv accelerating voltages in secondary electron imaging mode for sediment grains imaging and their chemical analysis . quantification of elements was done using an inca x - ray eds system ( oxford ) . sem images were collected from more than 80 individual grains , and from those , eds measurements of elemental composition were made on more than 350 individual points . u l - edge exafs spectra were measured at beamline 11 - 2 of the ssrl at the stanford linear accelerator laboratory ( slac ) to characterize the average molecular structure around uranium in bulk sediments . changes in the fe and s mineralogy and the microbial communities in reactors t1t3 demonstrate the shift to more reducing conditions . additionally , a significant change in sulfide concentrations in the effluent of reactor t3 , increasing from 0.014 mg l , 30 days following field acetate amendment , to 0.733 mg l after 70 days of field acetate amendment , was observed ( supporting information , table si-2 ) . reported that elemental sulfur may form in rifle sediments via abiotic oxidation of aqueous sulfide coupled to the reduction of goethite or other fe(iii)-bearing refractory minerals in the sediments . sem images with selected area energy - dispersive x - ray spectroscopy ( eds ) data , evidencing the development of fes coatings on silica mineral grains in biostimulated sediments : ( a , b ) preamendment or super 8 composite sediment and ( c , d ) sulfate - reducing conditions in column t3 sediments ( influent end of column ) . the rifle ifrc site is far from thermodynamic equilibrium ; thus , nonequilibrium , kinetics - based approaches must be invoked to explain u transport at the site . the column reactor sediments are also far from equilibrium conditions , which allows for redox heterogeneity along the length of the column ( 15 cm ) and at the micrometer pore scale during acetate amendment . microbial community analyses showing community composition of the most abundant taxa ( > 1% relative abundance ) at the genus ( or nearest taxonomic ) level . the results demonstrate the presence of abundant bacteria that are phylogenetically similar to known fe(iii ) reducers ( geobacter and other desulfuromonadales bacteria , rhodoferax , desulfosporosinus ) in the t1 reactors . the progression from fe(iii)- to sulfate - reducing community occurred earlier in the bottoms ( infl ) section of columns than in the tops ( effl ) , as acetate - amended groundwater was pumped up through the columns from bottom to top . total uranium concentrations in the sediments vary from near zero to approximately 1 mol of u per gram of sediment . generally , more uranium was accumulated in the bottom sections of the reactors , where the influent groundwater containing acetate and uranium entered ( figure 3 ) , and where conditions were more reducing than in the effluent sections of the same columns . this phenomenon is likely due to a combination of longer reaction times and faster u accumulation rates under sulfate reduction possibly involving biogenic iron sulfide minerals such as mackinawite . uranium quantification in sediments , including bicarbonate - extracted noncrystalline u(iv ) species ( red bars ) and total u ( blue bars ) from aqua regia digests , reported in mol of u per g of sediment . the fraction of total u present as noncrystalline u(iv ) species ( black circles ) is nearly constant for all sediments . xanes data indicate that between 75 and 100% of the total uranium pool is present as u(iv ) species ( figure si-4 ) . despite large differences in the total sediment uranium concentration ( figure 3 ) , time of reaction , and redox conditions ( figures 1 and 2 ) among the reacted sediments , the u exafs spectra are similar to one another ( figure 4 ; supporting information , table si-1 , figures si-5 and si-6 ) . u peak at 3.83.9 implies that a small fraction of u(iv ) in the samples is present as uraninite . uraninite is produced following the reaction of u(vi ) with a variety of sulfate - reducing , metal - reducing , and other bacteria and with abiotic reductants including mackinawite and other ferrous sulfides and oxides . hs(aq ) is expected to be insignificant as a reductant for u(vi ) in rifle groundwater because ( bi)carbonate concentrations in the millimolar range , such as are observed in rifle groundwater , inhibit the reduction of aqueous u(vi ) . uranium lii - edge exafs and fourier transforms for ( a ) column t1 effl , ( b ) column t1 infl , ( c ) column t2 infl , ( d ) column t3 effl , and ( e ) column t3 infl . the noncrystalline u(iv ) fraction of the total u(iv ) pool as estimated by bicarbonate extraction is constant within analytical and replicate error for the top and bottom sediment sections of all three reactors and averages approximately 65% . conducted laboratory column experiments with rifle ifrc sediments and amended a solution containing 15 mm acetate , 55 m uranyl acetate , and 30 mm bicarbonate until iron - reducing conditions were achieved . they observed an immobilized u pool in the sediments dominated by noncrystalline u(iv ) , suggesting that laboratory column studies may be reasonable proxies for field studies such as ours . the extraction and exafs results suggest that the u(iv ) species remain dominated by noncrystalline u(iv ) through metal - reducing and deep into sulfate - reducing conditions . the apparent constancy in u(iv ) speciation implies a constancy in the mechanism(s ) of reduction . since geobacter and potentially other u(vi)-reducing species are active across widely ranging redox conditions , the u(vi ) removal mechanism(s ) they employ may be operable and produce similar u(iv ) products throughout . according to this model , u(vi ) bound to phosphoryl sites or phosphate groups in biomass could be reduced to u(iv ) by electrons conferred via shuttles ( soluble inorganic or organic shuttles or conductive pilli , or combinations thereof ) . u(iv ) reduced in this fashion would remain complexed to biomass functional groups as noncrystalline u(iv ) . , using single species cultures , further demonstrated that solution geochemistry controlled the viability of shewanella cells and their ability to produce exopolymers during u(vi ) reduction in rifle composition groundwater . exopolymer production was directly related to a larger fraction of non - crystalline u(iv ) in the overall reduced uranium pool . this model could help to explain the relative abundance of noncrystalline u(iv ) observed in the present study across strongly contrasting biogeochemical regimes . in any case , so long as reducing agents , electron shuttles , and microbial exopolymers are present that can bind and reduce u(vi ) , then noncrystalline u(iv ) is likely to be produced . although groundwater geochemistry has been shown to influence the speciation of u(iv ) formed in sediments ( e.g. , refs ( 6 ) , ( 13 ) ) , this study indicates that factors including the sub - surface redox conditions , dominant microbial community and physiology , grain - coating mineralogy , inorganic reducing agents , and total u accumulated in the sediments do not dictate the appearance of noncrystalline u(iv ) within the range of geochemical and microbial conditions tested here . driving an aquifer into sulfate - reducing conditions during bioremediation may be a sound strategy to increase the stability of u(vi ) reduction to u(iv ) species , i.e. , via association with fes , particularly as this does not appear to impact the speciation of the resulting u(iv ) products . as long as reducing conditions are maintained and in the absence of elevated aqueous carbonate concentrations , u(iv ) should remain immobile , even if present as noncrystalline u(iv ) species . however , if the groundwater chemistry changes , the high fraction of noncrystalline u(iv ) species , shown to be more easily mobilized and oxidized than uraninite u(iv ) , could be of concern .

there is an increasing recognition of comorbidity between intellectual disability ( i d ) and mental health problems , emotional disorders , and deficits in socialization skills.13 literature reveals that the aging process commences younger in people with i d at approximately 4050 years of age.4 as the previously mentioned problems may further lead to long - term health conditions and mortality,5 effective psychosocial interventions need to be developed for older adults with i d in addition to the pharmacological treatment . life story work ( lsw ) is : the construction , or reconstruction of an individual s life - story and involves the integration of the individual s internal process , as well as the relationships and values with the family , community , and culture in which the individual has developed.6 it creates an opportunity for the person to tell others about their past experiences and then use this life story to benefit them in their present situation.7,8 a variety of forms ( book , digital video disc , or collection of personal items ) can be used , and the content may contain photographs , written biographies , drawings , art pieces , and other aids for understanding a person s own memorable experiences . the lsw program ( p ) in particular , life story book was originally applied to children who were under adoption and foster care services.9 it helped children to develop a sense of identity and continuity in the new setting . lsw was then modified and introduced to a variety of different settings , including children with prior exposure to trauma , people with long - term illnesses,10 older people with or without dementia,11 and recently people with intellectual disabilities.12,13 our past experience and relationships shape our identity and make us who are today.14 this also applies to people with i d and lswp as a way of keeping their past history alive . there is evidence that lswp may encourage people with i d to present and express themselves13 and , hence , improve communication and relationships.15,16 therefore , lswp is like a bridge to create connections among clients , family caregivers , and support workers.17,18 research also indicates that lswp has a positive impact on participants mood . this is especially important for older adults with i d , who are at an increased risk of having mood disorders , particularly depression.19 similar to other aging people , older adults with i d carry with them many unpleasant past life events and face a great deal of unexpected changes , which they may not be able to cope with.20,21 they also have more difficulties in developing and maintaining a stable social support network,22 which is an essential element in fighting mood problems.23 older adults with i d are predisposed to have reduced subjective well - being , given their physical disabilities and related psychological stress.24 however , with a strengthened sense of identity , improved social interaction , and increased pleasure and enjoyment about life , it is reasonable to expect that lswp may enhance the participants quality of life ( qol ) by engaging them in an appropriate level of activities and contacts.2527 hence , it is hypothesized that lswp , when applied to older adults with i d , will have a positive impact on their mood , socialization , and ultimately their qol . thus , the evidence on the use of lswp is immature , and there is a great need for quantitative research evidence to substantiate the potential value of lsw and other cognitive behavioral treatments for patients with id,6,8,28 especially for people with id.16 in addition , to our knowledge , there are very few lswp studies on older adults with i d that have a well - established protocol tailor - made to them , despite their marked disparity from the norm . to address these issues , the present study aimed to develop a training protocol of lswp especially designed for older adults with mild - to - moderate levels of i d and to evaluate the effectiveness of lsw in this group of older adults on enhancing mood , socialization , and qol from a quantitative perspective participants were recruited from nine hostels , day service centers , and sheltered workshops of the hong chi association . the inclusion criteria were : 1 ) > 50 years of age ; 2 ) mild - to - moderate grade i d , according to the service admission record in their personal case files ; and 3 ) without severe psychiatric disorder or behavioral problem . it is suggested that gaining person - centered consent from such participants.29 this was achieved in this study via an ongoing evaluation of both the verbal and nonverbal cues of the participant during the research process , alongside written consent obtained from the guardian of the participant . only participants with both their guardians written informed consent and those who agreed to participate in the program joined the study . this study complied with the ethical standard stipulated by the survey and behavioural research ethics committee of the chinese university of hong kong and ethical approval was granted prior to the data collection . participants were assigned to either the intervention group ( n=32 ) or the control group ( n=28 ) . the intervention group received the lswp intervention led by trained lswp instructors , in addition to their usual daily activities for approximately 6 months . the control group received their usual activities ( ie , training on self - care and daily living skills , basic work skill training , activities that developed their hobbies and interests , physical exercises to maintain or strengthen their physical fitness , vocational training in a sheltered environment ) during this period . assessments on the participants mood , socialization , and qol were conducted at baseline and immediately after the intervention . the protocol of lswp for older people without i d could not fit the special needs of population with i d . therefore , a group of clinical psychologists developed a life story book protocol which was designed specifically for this population . in developing this protocol , the following recommendations were taken into consideration : 1 ) the effective features of a life story intervention design for nursing home residents ; and 2 ) suggestions on the best format for compiling life history resources for older people living in institutional settings.16,30 this life story book protocol consisted of 16 structured one - to - one or group sessions , each lasting for 1.52 hours . the entire program spanned a period of approximately 6 months . it comprised a series of activities of various psychosocial elements , such as field visits , outings , production of life story book with photos , presentation , group sharing , and collecting feedback from the caregiver . lswp instructors work with participants and their family members to collect information and photos that tell the participants life stories . lswp instructors also help participants to express their feelings on their life stories in a caring and accepting atmosphere . participants are encouraged to use the life story books to share their life stories and achievements with other people . participants were guided by trained lswp instructors , who were experienced tutors for people with i d and received training and regular supervision from the clinical psychologists . the protocol provided step - by - step guidelines for the instructors to produce an individualized life story book for each client . the life story book enabled clients to gather current information about themselves ( the present ) and their history ( the past ) . moreover , it was a useful tool that could assist the clients to express themselves and tell their own story to people around them . , each participant will have a personalized life story book that includes the recording of significant people , places , and events for themselves ( table 1 ) . participants were recruited from nine hostels , day service centers , and sheltered workshops of the hong chi association . the inclusion criteria were : 1 ) > 50 years of age ; 2 ) mild - to - moderate grade i d , according to the service admission record in their personal case files ; and 3 ) without severe psychiatric disorder or behavioral problem . it is suggested that gaining person - centered consent from such participants.29 this was achieved in this study via an ongoing evaluation of both the verbal and nonverbal cues of the participant during the research process , alongside written consent obtained from the guardian of the participant . only participants with both their guardians written informed consent and those who agreed to participate in the program joined the study . this study complied with the ethical standard stipulated by the survey and behavioural research ethics committee of the chinese university of hong kong and ethical approval was granted prior to the data collection . participants were assigned to either the intervention group ( n=32 ) or the control group ( n=28 ) . the intervention group received the lswp intervention led by trained lswp instructors , in addition to their usual daily activities for approximately 6 months . the control group received their usual activities ( ie , training on self - care and daily living skills , basic work skill training , activities that developed their hobbies and interests , physical exercises to maintain or strengthen their physical fitness , vocational training in a sheltered environment ) during this period . assessments on the participants mood , socialization , and qol were conducted at baseline and immediately after the intervention . the protocol of lswp for older people without i d could not fit the special needs of population with i d . therefore , a group of clinical psychologists developed a life story book protocol which was designed specifically for this population . in developing this protocol , the following recommendations were taken into consideration : 1 ) the effective features of a life story intervention design for nursing home residents ; and 2 ) suggestions on the best format for compiling life history resources for older people living in institutional settings.16,30 this life story book protocol consisted of 16 structured one - to - one or group sessions , each lasting for 1.52 hours . the entire program spanned a period of approximately 6 months . it comprised a series of activities of various psychosocial elements , such as field visits , outings , production of life story book with photos , presentation , group sharing , and collecting feedback from the caregiver . lswp instructors work with participants and their family members to collect information and photos that tell the participants life stories . lswp instructors also help participants to express their feelings on their life stories in a caring and accepting atmosphere . participants are encouraged to use the life story books to share their life stories and achievements with other people . participants were guided by trained lswp instructors , who were experienced tutors for people with i d and received training and regular supervision from the clinical psychologists . the protocol provided step - by - step guidelines for the instructors to produce an individualized life story book for each client . the life story book enabled clients to gather current information about themselves ( the present ) and their history ( the past ) . moreover , it was a useful tool that could assist the clients to express themselves and tell their own story to people around them . , each participant will have a personalized life story book that includes the recording of significant people , places , and events for themselves ( table 1 ) . validated assessment tools were used to collect data for both intervention and control groups at both pretest and posttest periods . as mood , socialization , and qol were the outcomes of interest , corresponding assessment tools of mood interest and pleasure questionnaire ( mipq),31 vineland adaptive behavior scales , second edition ( vineland - ii),32 and personal well - being index intellectual disability ( cantonese ) , third edition ( pwi - id),33 were used respectively . these standardized measurements were chosen as the most appropriate to detect any change in the outcomes of interest for the subjects over the study period . the selected assessment tools demonstrate good reliability and validity . for the purpose of this study , the analysis of vineland - ii mainly focused on the socialization domain , while the communication abilities were perceived as a background and independent variable . data were analyzed using the statistical package for social sciences version 15 ( spss inc . , chicago , il , usa ) . independent samples t - tests ( and chi - square when appropriate ) were used to compare demographic characteristics between the control and intervention groups at baseline . two - way repeated measures of analysis of variance was further used to examine the effectiveness of the lswp . the difference in each function or well - being was calculated by subtracting participants score measured at baseline from their score measured 2 weeks after the intervention . independent samples t - tests were further conducted to compare the direction and magnitude of change between the control and intervention groups . table 2 summarizes the baseline demographics of the participants . a total of 60 participants with i d were recruited into the study and were assigned to either the lswp intervention group ( n=32 ) or control group ( n=28 ) . results showed that there were no significant differences between the control and intervention groups in terms of demographic factors , including age , sex , education level , communication abilities , marital status , and types of services received . similarly , no significant differences were detected between the two groups on baseline measurements of personal well - being , mood , interest , and pleasure or adaptive behavior on socialization , as measured by the pwi , mipq , and vineland - ii scales , respectively . the lswp had the potential to prevent the deterioration of mood ( as measured by mipq ) of older adults with i d ( table 2 ) . results of repeated measures analysis of variance showed a marginally significant main effect of time ( p=0.07 ) , but the main effect of intervention / control group on the mipq score was insignificant ( p=0.85 ) . the overall interaction effect of time and intervention was also found to be marginally significant ( p=0.09 ) . further analysis showed that the mean mipq score declined from 67.3662.27 in the control group ; whereas , the mean mipq score for the intervention group remained almost the same from 65.6265.35 , indicating the effectiveness of lswp in preventing the negative change in mood , interest , and pleasure of the participants . independent samples t - tests were further conducted on the mean difference in the subdomains of mipq between the control and the intervention groups ( table 3 ) . interest and pleasure ( p=0.04 ) , while no differences were evident in other domains . the lswp showed promise in improving the socialization skills ( as measured by vineland - ii ) of older adults with i d ( table 3 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.54 ) or intervention / control group ( p=0.58 ) on the socialization skills . similarly , the overall interaction effect of time and intervention was not significant ( p=0.56 ) . independent samples t - tests were also conducted on the mean difference in adaptive behaviors and other subscales of the vineland - ii scale between the control and the intervention groups , but there were no significant differences . as stated in table 4 , concerning their socialization skills , the intervention group improved a little ( m = 3.19 ; sd = 20.66 ) while their counterparts in the control group remained the same ( m = 0.07 ; sd = 19.32 ) . the lswp enhanced qol ( as measured by the pwi - id ) of older adults with i d ( table 4 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.77 ) or intervention / control group ( p=0.96 ) on the pwi score . but the overall interaction effect of time and intervention was marginally significant ( p=0.09 ) . the mean pwi score declined from 82.04 to 76.24 in the control group while that for the intervention group increased from 75.24 to 81.31 , indicating the lswp tended to enhance the qol among older adults with i d . the mean scores of both the intervention and control groups were higher than that of the norm in hong kong which is 63.99.33 independent samples t - tests were further conducted on the change of mean ( m ) in different pwi life domains between the control and the intervention groups ( table 5 ) . particularly , the personal health status of participants in the intervention group improved after receiving the lsw treatment ( m = 7.60 ) , but it deteriorated in the control group ( m = -8.57 ) . there was marked improvement concerning community - connectedness among the intervention group ( m = 6.20 ; standard deviation [ sd ] = 29.91 ) ; whereas , a decrease in community - connectedness was detected in the control group ( m = -21.43 ; sd = 34.97 ) . the difference between the two groups was statistically significant ( p=0.01 ; table 5 ) . although there were no significant differences in the other five life domains between the control and the intervention group , the outcome of the intervention group seemed to be better than that of the control group in all of these domains , as demonstrated by the mean differences ( table 5 ) . the effectiveness of lsw tended to vary , depending on the participants communication abilities . to examine whether the effectiveness of lswp was dependent on the participants communication abilities , we divided the participants who received the intervention into two groups based on their communication abilities . a mean cut - off score of 27.7 ( according to the vineland - ii manual on adults with moderate - grade mental retardation ) was used to categorize participants into a low communication group ( mean score , 27.7 ) , and a high communication group ( mean score , > 27.7 ) . as a result , 18 participants were assigned to the low communication group , and 14 participants were assigned to the high communication group . independent samples t - tests were conducted on the change of mean ( m ) in different outcome variables between the low and high communication groups . compared to the low communication score group ( m = -0.95 ; sd = 29.06 ) , the lswp seemed to be more effective in improving the pwi of the group with the higher communication score ( m = 10.60 ; sd = 15.89 ) and its subdomains except for personal safety although this did not reach statistical significance . a similar pattern was observed for the socialization skills ( low communication score group , m = 0.44 , sd = 6.30 ; high communication score group , m = 6.71 , sd = 30.69 ) . however , all of these observed differences did not reach statistical significance , partly due to a relatively small sample in our study . table 2 summarizes the baseline demographics of the participants . a total of 60 participants with i d were recruited into the study and were assigned to either the lswp intervention group ( n=32 ) or control group ( n=28 ) . results showed that there were no significant differences between the control and intervention groups in terms of demographic factors , including age , sex , education level , communication abilities , marital status , and types of services received . similarly , no significant differences were detected between the two groups on baseline measurements of personal well - being , mood , interest , and pleasure or adaptive behavior on socialization , as measured by the pwi , mipq , and vineland - ii scales , respectively . the lswp had the potential to prevent the deterioration of mood ( as measured by mipq ) of older adults with i d ( table 2 ) . results of repeated measures analysis of variance showed a marginally significant main effect of time ( p=0.07 ) , but the main effect of intervention / control group on the mipq score was insignificant ( p=0.85 ) . the overall interaction effect of time and intervention was also found to be marginally significant ( p=0.09 ) . further analysis showed that the mean mipq score declined from 67.3662.27 in the control group ; whereas , the mean mipq score for the intervention group remained almost the same from 65.6265.35 , indicating the effectiveness of lswp in preventing the negative change in mood , interest , and pleasure of the participants . independent samples t - tests were further conducted on the mean difference in the subdomains of mipq between the control and the intervention groups ( table 3 ) . interest and pleasure ( p=0.04 ) , while no differences were evident in other domains . the lswp showed promise in improving the socialization skills ( as measured by vineland - ii ) of older adults with i d ( table 3 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.54 ) or intervention / control group ( p=0.58 ) on the socialization skills . similarly , the overall interaction effect of time and intervention was not significant ( p=0.56 ) . independent samples t - tests were also conducted on the mean difference in adaptive behaviors and other subscales of the vineland - ii scale between the control and the intervention groups , but there were no significant differences . as stated in table 4 , concerning their socialization skills , the intervention group improved a little ( m = 3.19 ; sd = 20.66 ) while their counterparts in the control group remained the same ( m = 0.07 ; sd = 19.32 ) . the lswp enhanced qol ( as measured by the pwi - id ) of older adults with i d ( table 4 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.77 ) or intervention / control group ( p=0.96 ) on the pwi score . but the overall interaction effect of time and intervention was marginally significant ( p=0.09 ) . the mean pwi score declined from 82.04 to 76.24 in the control group while that for the intervention group increased from 75.24 to 81.31 , indicating the lswp tended to enhance the qol among older adults with i d . the mean scores of both the intervention and control groups were higher than that of the norm in hong kong which is 63.99.33 independent samples t - tests were further conducted on the change of mean ( m ) in different pwi life domains between the control and the intervention groups ( table 5 ) . particularly , the personal health status of participants in the intervention group improved after receiving the lsw treatment ( m = 7.60 ) , but it deteriorated in the control group ( m = -8.57 ) . there was marked improvement concerning community - connectedness among the intervention group ( m = 6.20 ; standard deviation [ sd ] = 29.91 ) ; whereas , a decrease in community - connectedness was detected in the control group ( m = -21.43 ; sd = 34.97 ) . the difference between the two groups was statistically significant ( p=0.01 ; table 5 ) . although there were no significant differences in the other five life domains between the control and the intervention group , the outcome of the intervention group seemed to be better than that of the control group in all of these domains , as demonstrated by the mean differences ( table 5 ) . the effectiveness of lsw tended to vary , depending on the participants communication abilities . to examine whether the effectiveness of lswp was dependent on the participants communication abilities , we divided the participants who received the intervention into two groups based on their communication abilities . a mean cut - off score of 27.7 ( according to the vineland - ii manual on adults with moderate - grade mental retardation ) was used to categorize participants into a low communication group ( mean score , 27.7 ) , and a high communication group ( mean score , > 27.7 ) . as a result , 18 participants were assigned to the low communication group , and 14 participants were assigned to the high communication group . independent samples t - tests were conducted on the change of mean ( m ) in different outcome variables between the low and high communication groups . compared to the low communication score group ( m = -0.95 ; sd = 29.06 ) , the lswp seemed to be more effective in improving the pwi of the group with the higher communication score ( m = 10.60 ; sd = 15.89 ) and its subdomains except for personal safety although this did not reach statistical significance . a similar pattern was observed for the socialization skills ( low communication score group , m = 0.44 , sd = 6.30 ; high communication score group , m = 6.71 , sd = 30.69 ) . however , all of these observed differences did not reach statistical significance , partly due to a relatively small sample in our study . the present study represents one of the first attempts , using quantitative assessments , to evaluate the effectiveness of lswp in enhancing mood , socialization , and qol in older adults with i d . the results of the study showed that our lswp was generally effective in improving qol . it had the potential to prevent the deterioration of mood and showed promise in improving the socialization skills in older adults with i d . in addition , we also found that the effectiveness of lsw tended to vary , depending on the participants communication abilities . first , consistent with the results of the previous studies , our lswp improved the personal well - being of the participants , and it especially enhanced participants perceived personal health and sense of community - connectedness.8,34 since there is evidence that happiness and life satisfaction prospectively predict self - rated health,35 and people s self - rated health status is significantly and positively correlated with their actual physical and mental health , which may further contribute to better qol , the improvement of the participants perceived personal health was quite a desirable outcome of lswp.36 to some extent , it indicated participants ameliorated health status and qol after receiving lswp . second , although a significant improvement in mood was not detected in the participants of lswp , the results were encouraging in that , compared to the control group who experienced a drop in the mipq score , those who participated in the lswp had a relatively stable mood reflected by the mipq score . the lswp participants were more emotionally stable , showed interest , and actively participated in the lswp . a previous study has shown that emotional competence is positively and significantly associated with happiness and life satisfaction.37 as a matter of fact , it has been shown that people with i d are more likely to suffer from emotional disorders,1,19 and the severity of emotional problems including depressed mood and the loss of interest was positively correlated with age.5,38 these findings partly explain why a natural decline in the mipq score was detected in the control group in the present study . since previous studies have also found that emotional disorders may further affect long - term health conditions and mortality , effective interventions are needed.5 this study discovered the potential of our lswp to act as a protective factor in preventing the deterioration of mood in people with i d . third , results showed a positive trend of improvement among subjects in the intervention group in terms of their socialization skills , although this was not statistically significant . it could be that participants need some time to practice their improved social skills and , therefore , the improvement may not be immediately observable . in the program , it was believed that the tailor - made life story book with photos from lsw could assist the recall of past memories of the participants , help them to express themselves , and promote sharing with others . this aspect of change could be regarded as the improvement in socialization skills and community - connectedness . to some extent , the stronger sense of community - connectedness , the increased interest and pleasure , and the improved socialization skills can be perceived as enhanced participation , which has been found to be a valuable experience for people with different types of disabilities.39 last but not least participants with better communication skills seemed to benefit more from the program . compared to the group with a lower communication score , the lswp seemed to be more effective for the high communication group in improving their socialization skills and qol although statistical significance was not reached . a possible explanation was that those who had better expressive and comprehension skills were more actively involved in the lswp as compared to those with relatively limited verbal abilities . although the current study represents the first attempt to comprehensively and quantitatively evaluate the effectiveness of lswp on older adults with i d , the results might be affected by the relatively small sample size . in addition , as all of the participants were recruited from the same rehabilitation organization and the intervention and control group were not equally distributed in their living conditions for the ease of administration in the delivery of lswp , this might weaken the rigor and generalizability of the findings . therefore , further studies with larger sample sizes are warranted to provide more convincing evidence . . it would be better if regular booster trainings could be provided after the completion of the lsw , so that participants could update their lsw and refresh their memory and learning . the long - term effect of lsw could be further examined if booster trainings were incorporated into the program .

objectivethis study examines the effectiveness of a life story work program ( lswp ) in older adults with mild - to - moderate levels of intellectual disability ( id).methodsusing a quasiexperimental design , this study assigned 60 older adults who were between 5090 years old with mild - to - moderate levels of i d to receive either the lswp ( intervention group , n=32 ) or usual activities ( control group , n=28 ) during a period of 6 months . evaluation was made based on the outcomes assessed by the mood interest and pleasure questionnaire , the vineland adaptive behavior scales , and the personal wellbeing index id.results and conclusionlswp shows potential for improving the quality of life and preventing the loss of interest and pleasure in older adults with i d . it also shows promise in enhancing their socialization skills . patients with better communication abilities seemed to benefit more from the lswp .

Introduction Methods Participants Study design Content of the LSWp Measurement Data analysis Results Demographics and baseline measures Discussion and conclusion Limitations

there is an increasing recognition of comorbidity between intellectual disability ( i d ) and mental health problems , emotional disorders , and deficits in socialization skills.13 literature reveals that the aging process commences younger in people with i d at approximately 4050 years of age.4 as the previously mentioned problems may further lead to long - term health conditions and mortality,5 effective psychosocial interventions need to be developed for older adults with i d in addition to the pharmacological treatment . life story work ( lsw ) is : the construction , or reconstruction of an individual s life - story and involves the integration of the individual s internal process , as well as the relationships and values with the family , community , and culture in which the individual has developed.6 it creates an opportunity for the person to tell others about their past experiences and then use this life story to benefit them in their present situation.7,8 a variety of forms ( book , digital video disc , or collection of personal items ) can be used , and the content may contain photographs , written biographies , drawings , art pieces , and other aids for understanding a person s own memorable experiences . the lsw program ( p ) in particular , life story book was originally applied to children who were under adoption and foster care services.9 it helped children to develop a sense of identity and continuity in the new setting . there is evidence that lswp may encourage people with i d to present and express themselves13 and , hence , improve communication and relationships.15,16 therefore , lswp is like a bridge to create connections among clients , family caregivers , and support workers.17,18 research also indicates that lswp has a positive impact on participants mood . this is especially important for older adults with i d , who are at an increased risk of having mood disorders , particularly depression.19 similar to other aging people , older adults with i d carry with them many unpleasant past life events and face a great deal of unexpected changes , which they may not be able to cope with.20,21 they also have more difficulties in developing and maintaining a stable social support network,22 which is an essential element in fighting mood problems.23 older adults with i d are predisposed to have reduced subjective well - being , given their physical disabilities and related psychological stress.24 however , with a strengthened sense of identity , improved social interaction , and increased pleasure and enjoyment about life , it is reasonable to expect that lswp may enhance the participants quality of life ( qol ) by engaging them in an appropriate level of activities and contacts.2527 hence , it is hypothesized that lswp , when applied to older adults with i d , will have a positive impact on their mood , socialization , and ultimately their qol . thus , the evidence on the use of lswp is immature , and there is a great need for quantitative research evidence to substantiate the potential value of lsw and other cognitive behavioral treatments for patients with id,6,8,28 especially for people with id.16 in addition , to our knowledge , there are very few lswp studies on older adults with i d that have a well - established protocol tailor - made to them , despite their marked disparity from the norm . to address these issues , the present study aimed to develop a training protocol of lswp especially designed for older adults with mild - to - moderate levels of i d and to evaluate the effectiveness of lsw in this group of older adults on enhancing mood , socialization , and qol from a quantitative perspective participants were recruited from nine hostels , day service centers , and sheltered workshops of the hong chi association . the inclusion criteria were : 1 ) > 50 years of age ; 2 ) mild - to - moderate grade i d , according to the service admission record in their personal case files ; and 3 ) without severe psychiatric disorder or behavioral problem . participants were assigned to either the intervention group ( n=32 ) or the control group ( n=28 ) . the intervention group received the lswp intervention led by trained lswp instructors , in addition to their usual daily activities for approximately 6 months . the control group received their usual activities ( ie , training on self - care and daily living skills , basic work skill training , activities that developed their hobbies and interests , physical exercises to maintain or strengthen their physical fitness , vocational training in a sheltered environment ) during this period . therefore , a group of clinical psychologists developed a life story book protocol which was designed specifically for this population . in developing this protocol , the following recommendations were taken into consideration : 1 ) the effective features of a life story intervention design for nursing home residents ; and 2 ) suggestions on the best format for compiling life history resources for older people living in institutional settings.16,30 this life story book protocol consisted of 16 structured one - to - one or group sessions , each lasting for 1.52 hours . the entire program spanned a period of approximately 6 months . it comprised a series of activities of various psychosocial elements , such as field visits , outings , production of life story book with photos , presentation , group sharing , and collecting feedback from the caregiver . participants were guided by trained lswp instructors , who were experienced tutors for people with i d and received training and regular supervision from the clinical psychologists . the inclusion criteria were : 1 ) > 50 years of age ; 2 ) mild - to - moderate grade i d , according to the service admission record in their personal case files ; and 3 ) without severe psychiatric disorder or behavioral problem . it is suggested that gaining person - centered consent from such participants.29 this was achieved in this study via an ongoing evaluation of both the verbal and nonverbal cues of the participant during the research process , alongside written consent obtained from the guardian of the participant . participants were assigned to either the intervention group ( n=32 ) or the control group ( n=28 ) . the intervention group received the lswp intervention led by trained lswp instructors , in addition to their usual daily activities for approximately 6 months . the control group received their usual activities ( ie , training on self - care and daily living skills , basic work skill training , activities that developed their hobbies and interests , physical exercises to maintain or strengthen their physical fitness , vocational training in a sheltered environment ) during this period . in developing this protocol , the following recommendations were taken into consideration : 1 ) the effective features of a life story intervention design for nursing home residents ; and 2 ) suggestions on the best format for compiling life history resources for older people living in institutional settings.16,30 this life story book protocol consisted of 16 structured one - to - one or group sessions , each lasting for 1.52 hours . the entire program spanned a period of approximately 6 months . it comprised a series of activities of various psychosocial elements , such as field visits , outings , production of life story book with photos , presentation , group sharing , and collecting feedback from the caregiver . participants were guided by trained lswp instructors , who were experienced tutors for people with i d and received training and regular supervision from the clinical psychologists . , each participant will have a personalized life story book that includes the recording of significant people , places , and events for themselves ( table 1 ) . as mood , socialization , and qol were the outcomes of interest , corresponding assessment tools of mood interest and pleasure questionnaire ( mipq),31 vineland adaptive behavior scales , second edition ( vineland - ii),32 and personal well - being index intellectual disability ( cantonese ) , third edition ( pwi - id),33 were used respectively . for the purpose of this study , the analysis of vineland - ii mainly focused on the socialization domain , while the communication abilities were perceived as a background and independent variable . two - way repeated measures of analysis of variance was further used to examine the effectiveness of the lswp . a total of 60 participants with i d were recruited into the study and were assigned to either the lswp intervention group ( n=32 ) or control group ( n=28 ) . similarly , no significant differences were detected between the two groups on baseline measurements of personal well - being , mood , interest , and pleasure or adaptive behavior on socialization , as measured by the pwi , mipq , and vineland - ii scales , respectively . the lswp had the potential to prevent the deterioration of mood ( as measured by mipq ) of older adults with i d ( table 2 ) . further analysis showed that the mean mipq score declined from 67.3662.27 in the control group ; whereas , the mean mipq score for the intervention group remained almost the same from 65.6265.35 , indicating the effectiveness of lswp in preventing the negative change in mood , interest , and pleasure of the participants . independent samples t - tests were further conducted on the mean difference in the subdomains of mipq between the control and the intervention groups ( table 3 ) . the lswp showed promise in improving the socialization skills ( as measured by vineland - ii ) of older adults with i d ( table 3 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.54 ) or intervention / control group ( p=0.58 ) on the socialization skills . independent samples t - tests were also conducted on the mean difference in adaptive behaviors and other subscales of the vineland - ii scale between the control and the intervention groups , but there were no significant differences . as stated in table 4 , concerning their socialization skills , the intervention group improved a little ( m = 3.19 ; sd = 20.66 ) while their counterparts in the control group remained the same ( m = 0.07 ; sd = 19.32 ) . the lswp enhanced qol ( as measured by the pwi - id ) of older adults with i d ( table 4 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.77 ) or intervention / control group ( p=0.96 ) on the pwi score . the mean pwi score declined from 82.04 to 76.24 in the control group while that for the intervention group increased from 75.24 to 81.31 , indicating the lswp tended to enhance the qol among older adults with i d . the mean scores of both the intervention and control groups were higher than that of the norm in hong kong which is 63.99.33 independent samples t - tests were further conducted on the change of mean ( m ) in different pwi life domains between the control and the intervention groups ( table 5 ) . particularly , the personal health status of participants in the intervention group improved after receiving the lsw treatment ( m = 7.60 ) , but it deteriorated in the control group ( m = -8.57 ) . there was marked improvement concerning community - connectedness among the intervention group ( m = 6.20 ; standard deviation [ sd ] = 29.91 ) ; whereas , a decrease in community - connectedness was detected in the control group ( m = -21.43 ; sd = 34.97 ) . although there were no significant differences in the other five life domains between the control and the intervention group , the outcome of the intervention group seemed to be better than that of the control group in all of these domains , as demonstrated by the mean differences ( table 5 ) . the effectiveness of lsw tended to vary , depending on the participants communication abilities . to examine whether the effectiveness of lswp was dependent on the participants communication abilities , we divided the participants who received the intervention into two groups based on their communication abilities . a mean cut - off score of 27.7 ( according to the vineland - ii manual on adults with moderate - grade mental retardation ) was used to categorize participants into a low communication group ( mean score , 27.7 ) , and a high communication group ( mean score , > 27.7 ) . compared to the low communication score group ( m = -0.95 ; sd = 29.06 ) , the lswp seemed to be more effective in improving the pwi of the group with the higher communication score ( m = 10.60 ; sd = 15.89 ) and its subdomains except for personal safety although this did not reach statistical significance . a total of 60 participants with i d were recruited into the study and were assigned to either the lswp intervention group ( n=32 ) or control group ( n=28 ) . similarly , no significant differences were detected between the two groups on baseline measurements of personal well - being , mood , interest , and pleasure or adaptive behavior on socialization , as measured by the pwi , mipq , and vineland - ii scales , respectively . the lswp had the potential to prevent the deterioration of mood ( as measured by mipq ) of older adults with i d ( table 2 ) . further analysis showed that the mean mipq score declined from 67.3662.27 in the control group ; whereas , the mean mipq score for the intervention group remained almost the same from 65.6265.35 , indicating the effectiveness of lswp in preventing the negative change in mood , interest , and pleasure of the participants . independent samples t - tests were further conducted on the mean difference in the subdomains of mipq between the control and the intervention groups ( table 3 ) . the lswp showed promise in improving the socialization skills ( as measured by vineland - ii ) of older adults with i d ( table 3 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.54 ) or intervention / control group ( p=0.58 ) on the socialization skills . independent samples t - tests were also conducted on the mean difference in adaptive behaviors and other subscales of the vineland - ii scale between the control and the intervention groups , but there were no significant differences . as stated in table 4 , concerning their socialization skills , the intervention group improved a little ( m = 3.19 ; sd = 20.66 ) while their counterparts in the control group remained the same ( m = 0.07 ; sd = 19.32 ) . the lswp enhanced qol ( as measured by the pwi - id ) of older adults with i d ( table 4 ) . results of repeated measures analysis of variance showed no main effect of time ( p=0.77 ) or intervention / control group ( p=0.96 ) on the pwi score . the mean pwi score declined from 82.04 to 76.24 in the control group while that for the intervention group increased from 75.24 to 81.31 , indicating the lswp tended to enhance the qol among older adults with i d . the mean scores of both the intervention and control groups were higher than that of the norm in hong kong which is 63.99.33 independent samples t - tests were further conducted on the change of mean ( m ) in different pwi life domains between the control and the intervention groups ( table 5 ) . particularly , the personal health status of participants in the intervention group improved after receiving the lsw treatment ( m = 7.60 ) , but it deteriorated in the control group ( m = -8.57 ) . although there were no significant differences in the other five life domains between the control and the intervention group , the outcome of the intervention group seemed to be better than that of the control group in all of these domains , as demonstrated by the mean differences ( table 5 ) . the effectiveness of lsw tended to vary , depending on the participants communication abilities . to examine whether the effectiveness of lswp was dependent on the participants communication abilities , we divided the participants who received the intervention into two groups based on their communication abilities . a mean cut - off score of 27.7 ( according to the vineland - ii manual on adults with moderate - grade mental retardation ) was used to categorize participants into a low communication group ( mean score , 27.7 ) , and a high communication group ( mean score , > 27.7 ) . compared to the low communication score group ( m = -0.95 ; sd = 29.06 ) , the lswp seemed to be more effective in improving the pwi of the group with the higher communication score ( m = 10.60 ; sd = 15.89 ) and its subdomains except for personal safety although this did not reach statistical significance . a similar pattern was observed for the socialization skills ( low communication score group , m = 0.44 , sd = 6.30 ; high communication score group , m = 6.71 , sd = 30.69 ) . the present study represents one of the first attempts , using quantitative assessments , to evaluate the effectiveness of lswp in enhancing mood , socialization , and qol in older adults with i d . it had the potential to prevent the deterioration of mood and showed promise in improving the socialization skills in older adults with i d . in addition , we also found that the effectiveness of lsw tended to vary , depending on the participants communication abilities . first , consistent with the results of the previous studies , our lswp improved the personal well - being of the participants , and it especially enhanced participants perceived personal health and sense of community - connectedness.8,34 since there is evidence that happiness and life satisfaction prospectively predict self - rated health,35 and people s self - rated health status is significantly and positively correlated with their actual physical and mental health , which may further contribute to better qol , the improvement of the participants perceived personal health was quite a desirable outcome of lswp.36 to some extent , it indicated participants ameliorated health status and qol after receiving lswp . second , although a significant improvement in mood was not detected in the participants of lswp , the results were encouraging in that , compared to the control group who experienced a drop in the mipq score , those who participated in the lswp had a relatively stable mood reflected by the mipq score . a previous study has shown that emotional competence is positively and significantly associated with happiness and life satisfaction.37 as a matter of fact , it has been shown that people with i d are more likely to suffer from emotional disorders,1,19 and the severity of emotional problems including depressed mood and the loss of interest was positively correlated with age.5,38 these findings partly explain why a natural decline in the mipq score was detected in the control group in the present study . since previous studies have also found that emotional disorders may further affect long - term health conditions and mortality , effective interventions are needed.5 this study discovered the potential of our lswp to act as a protective factor in preventing the deterioration of mood in people with i d . third , results showed a positive trend of improvement among subjects in the intervention group in terms of their socialization skills , although this was not statistically significant . to some extent , the stronger sense of community - connectedness , the increased interest and pleasure , and the improved socialization skills can be perceived as enhanced participation , which has been found to be a valuable experience for people with different types of disabilities.39 last but not least participants with better communication skills seemed to benefit more from the program . compared to the group with a lower communication score , the lswp seemed to be more effective for the high communication group in improving their socialization skills and qol although statistical significance was not reached . although the current study represents the first attempt to comprehensively and quantitatively evaluate the effectiveness of lswp on older adults with i d , the results might be affected by the relatively small sample size . in addition , as all of the participants were recruited from the same rehabilitation organization and the intervention and control group were not equally distributed in their living conditions for the ease of administration in the delivery of lswp , this might weaken the rigor and generalizability of the findings .

polyglutamine diseases are neurodegenerative disorders caused by expansion of cag trinucleotide repeats encoding polyglutamine tracts in specific genes ( orr and zoghbi , 2007 ) . the family of polyglutamine diseases includes spinal and bulbar muscular atrophy ( sbma ) , huntington s disease ( hd ) , dentatorubral - pallidoluysian atrophy ( drpla ) , and spinocerebellar ataxia ( sca ) type 1 , 2 , 3 , 6 , 7 , and 17 . these disorders are caused by glutamine expansions in androgen receptor ( ar ) , huntingtin , atrophin-1 , ataxin-1 , ataxin-2 , ataxin-3 , cacna1a , ataxin-7 , and the tata - box binding protein ( tbp ) , respectively . one unsolved question in the field of polyglutamine diseases is why the same mutation in different genes causes the dysfunction and death of specific populations of neurons in the cns , leading to different clinical disease manifestations . the selective pattern of neuronal degeneration in the cns contrasts with the widespread distribution or housekeeping function displayed by the disease proteins . this indicates that expansion of polyglutamine tracts is necessary , but not sufficient to cause disease . evidence has been obtained that intrinsic protein features play a critical role in dictating the initiation and progression to cellular dysfunction and degeneration ( graham et al . , 2006 ; , 2002 ; klement et al . , 1998 ; tsuda et al . , 2005 ) , suggesting a mechanistic link between expanded polyglutamine - induced toxicity and protein structure / function ( parodi and pennuto , 2011 ) . sbma clinical and pathological features clearly illustrate the relevance of protein context to disease pathogenesis . sbma is an x - linked motor neuron disease characterized by selective degeneration of lower motor neurons ( kennedy et al . , 1968 ) . in the family of polyglutamine diseases , sbma is unique in that the disease fully manifests only in males . the hormone - dependent nature of sbma is well recapitulated in animal models of disease , including the fruit fly drosophila melanogaster ( pandey et al . the sex specificity of sbma and the toxicity of polyglutamine - expanded ar result from binding to its natural ligand , testosterone , or its more potent derivative , dihydrotestosterone ( dht ) . upon hormone binding , ar translocates to the nucleus , undergoes a conformational change that leads to amino / carboxy - terminal ( n / c ) interactions , binds dna at androgens response elements ( ares ) , and recruits specific transcription co - factors , including chromatin remodeling factors , to regulate the expression of androgen - responsive genes . although most of these hormone - induced post - translational events have been associated with disease pathogenesis ( katsuno et al . , 2003 , 2005 ; lieberman et al . , 2002 ; montie et al . , 2009 ; nedelsky et al . , 2010 ; orr et al . , 2010 ) , the mechanism through which hormone binding converts mutant ar into a toxic species is an important open question . in response to hormone binding , ar acquires numerous post - translational modifications ( pennuto et al . , 2009 ) , most of which play a critical role in disease pathogenesis ( parodi and pennuto , 2011 ) . we previously demonstrated that phosphorylation of polyglutamine - expanded ar by akt at serines 215 and 792 , which lie in the akt consensus site motif rxrxxs ( where r is arginine , s serine , and x any amino acid ) , reduces hormone binding and ar transactivation and protects from neurodegeneration ( palazzolo et al . similar to phosphorylation , arginine methylation is a post - translational modification with major impact on protein structure and function ( bedford and clarke , 2009 ) . arginine methylation is catalyzed by a family of enzymes known as protein arginine methyltransferases ( prmts ) , which differ in their activity , substrate specificity and subcellular localization . apart from the most recently identified prmt10 and 11 , all the other prmts have catalytic activity and are classified as type i or ii depending of the type of methylated arginine generated . type i includes prmt1 , 2 , 3 , 4 , 6 , and 8 and catalyzes the addition of two methyl groups to one of the two -guanidino nitrogen atoms of arginine , thereby generating asymmetric dimethylarginine . type ii includes prmt5 , 7 , and 9 and catalyzes the addition of one methyl group to each -guanidino nitrogen atoms to generate symmetric dimethylarginine . prmts target histones and non - histone proteins ( di lorenzo and bedford , 2011 ; wei et al . , 2014 ) and have been shown to act as co - factors of ar and other nuclear hormone receptors ( lee et al . , 2005 ; meyer et al . , 2007 ; sun et al . , 2014 ) . however , nothing is known as to whether prmts target polyglutamine proteins and play a role in polyglutamine disease pathogenesis . to address this question , we used sbma as a model of polyglutamine diseases . our results show a key role for arginine methylation in the pathogenesis of polyglutamine diseases and provide evidence that a causative link between primary structure and function of polyglutamine protein is the underlying mechanism in the pathogenesis of polyglutamine diseases . to determine whether arginine methylation contributes to polyglutamine - induced neurodegeneration , we investigated the role of prmt function on sbma pathogenesis . first , we assessed whether eight mammalian prmts ( prmt18 ) colocalize and interact with mutant ar . we analyzed the subcellular distribution of the prmts and mutant ar in cos1 cells expressing polyglutamine - expanded ar with 65 glutamine residues ( ar65q ) and the prmts tagged to enhanced gfp ( egfp ) . consistent with previous reports ( herrmann et al . , 2009 ) , prmt1 , 3 , and 4 localized predominantly to the cytosol , prmt2 and 7 localized to the nucleus and cytosol , prmt6 was present almost exclusively in the nucleus , prmt5 formed cytosolic aggregates , and prmt8 had plasma membrane localization due to myristoylation ( figure s1a ) . ar localized mostly to the cytosol in vehicle - treated cells and to the nucleus in dht - treated cells . ar subcellular localization and nuclear translocation in response to hormone treatment were not affected by the overexpression of any of these prmts . in the dht - treated cells , mutant ar co - localized with prmt2 , 6 , and 7 in the nucleus . similar results were obtained with non - expanded ar ( data not shown ) . by immunoprecipitation assay in dht - treated hek293 t cells co - expressing flag - tagged ar together with either soluble egfp or the egfp - tagged prmts , normal and polyglutamine - expanded ar specifically formed a complex with prmt2 , 6 , and 7 ( figure 1a and figure s1b ) . to test whether the prmts act as transcription co - factors of ar , we measured ar transactivation by transcriptional assay in hek293 t cells transfected with either normal ar ( ar24q ) or ar65q under the control of the cytomegalovirus promoter , and using as reporter the luciferase gene whose expression was driven by an are , as previously described ( palazzolo et al . , 2007 ) . among the prmts tested here , only prmt6 significantly increased the transcriptional activity of ar ( figure 1b ) . the effect of prmt6 was hormone dependent , indicating that prmt6 acts as a co - activator of ar rather than as a general transcription activator , and it did not result from altered ar expression ( figure s1c ) . importantly , prmt6 increased the transactivation of ar24q by 3.3-fold and that of ar65q by 5.3-fold , indicating that the effect of prmt6 on ar transactivation is enhanced by polyglutamine expansion . similar results were obtained by expressing non - expanded and polyglutamine - expanded ar under the control of an elongation factor 1 promoter ( figure s1d ) . these results show that prmt6 is a specific ar co - activator , whose function is enhanced by polyglutamine expansion . prmt6 is expressed to a similar extent in control and sbma cells , including rat pc12 cells stably expressing either normal ar with 10 glutamine residues ( ar10q ) or mutant ar with 112 glutamine residues ( ar112q ) ( walcott and merry , 2002 ) , mouse motor neuron - derived mn-1 cells stably expressing either ar24q or ar65q ( brooks et al . , 1997 ) , as well as human primary fibroblasts and induced pluripotent stem cells ( ipscs ) derived from normal subjects and sbma patients ( grunseich et al . , 2014 ) ( figure s2a ) . upon hormone treatment , normal and polyglutamine - expanded ar colocalized with endogenous prmt6 in the nucleus of control and sbma pc12 cells ( figure 1c and figure sb ) . ar112q forms intranuclear inclusions in response to hormone binding in the pc12 cells ( walcott and merry , 2002 ) , and prmt6 localized to ar - positive intranuclear inclusions in these cells ( figure 1c , arrows ) . ar and prmt6 co - localized in the nucleus of control and sbma human primary fibroblasts as well as ipscs reprogrammed to motor neurons . notably , prmt6 was expressed and co - localized with polyglutamine - expanded ar in the ventral horn motor neurons of spinal cord autopsy specimens derived from an sbma patient ( figure 1c and figure s2b ) . by immunoprecipitation assays , both normal and polyglutamine - expanded ar formed a complex with endogenous prmt6 in pc12 and mn-1 sbma cells ( figure 1d and figure s2c ) . furthermore , endogenous ar and prmt6 formed a complex in control and sbma human primary fibroblast cells and ipscs . the ar / prmt6 interaction occurred both in the absence and presence of hormone and was significantly enhanced by polyglutamine expansion in the mn-1 cells . by transcriptional assay , overexpression of prmt6 increased the transactivation of normal ar by 3.3-fold and that of polyglutamine - expanded ar by 5.7-fold in the mn-1 cells ( figure 1e ) . in mouse primary cortical neurons , prmt6 increased the transactivation of normal ar by 1.4-fold , but this difference was not significant ( figure 1f ) . rather , in primary neurons prmt6 significantly increased the transactivation of polyglutamine - expanded ar by 3.8-fold . taken together , these results indicate that polyglutamine expansion enhances the structural and functional interaction of ar with its co - activator prmt6 in neuronal and patient - derived cells . the interaction between ar and co - regulators can be inhibited by the anti - androgen 5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)-1,4,6-heptatriene-3-one ( asc - j9 ) , which has been shown to rescue the phenotype of a mouse model of sbma ( yang et al . , 2007 ) . in a transcriptional assay , asc - j9 significantly and dose dependently decreased the transcriptional activity of polyglutamine - expanded and non - expanded ar induced by prmt6 ( figure 2a and figure s3a ) . to address whether the catalytic activity of prmt6 is required to transactivate ar , we performed transcriptional assays in cells treated with the pan - prmt inhibitor adenosine dialdehyde ( adox ) . adox reduced polyglutamine - expanded ar transactivation induced by dht by 30% , and it completely abolished the effect of prmt6 on ar transactivation ( figure 2b and figure s3b ) . because adox and ami-1 are not specific inhibitors of prmt6 , we generated a prmt6 methylation - deficient mutant by substituting valine 86 with lysine and aspartate 88 with alanine ( prmt6-v86k , d88a , figure 2c ) ( boulanger et al . , 2005 ) . prmt6-v86k , d88a retained its capability to bind polyglutamine - expanded and normal ar ( figure 2b and figure s3b , insets ) , but it failed to transactivate ar ( figure 2b and figure s3b ) , indicating that prmt6 requires its catalytic activity to transactivate ar . transcriptional regulation by steroid receptors involves the interaction with co - factors bearing lxxll ( where l is leucine and x is any amino acid ) or fxxlf ( where f is phenylalanine ) motifs ( heery et al . , 1997 ) . in search for fxxlf and lxxll motifs in the prmt family , we analyzed the sequence of the 11 known prmts ( wolf , 2009 ) . none of these prmts have the fxxlf motif , while only prmt6 contains the lxxll motif lrvll ( figure 2c and figure s4 ) . to establish whether the lrvll motif of prmt6 is necessary for ar transactivation , we substituted leucines 356 and 357 with alanines , thereby generating prmt6-lxxaa mutant ( heery et al . , 1997 ) . mutation of the lxxll motif decreased the interaction with polyglutamine - expanded and normal ar ( figure 2d and figure s3c , inset ) and abolished ar transactivation ( figure 2d and figure s3c ) , indicating that the ability of prmt6 to enhance ar transcriptional activity is dependent on the integrity of the lrvll motif . steroid receptor primary structure is composed of an amino - terminal domain ( ntd ) , a dna - binding domain ( dbd ) , and the ligand - binding domain ( lbd ) ( figure 2c ) . transactivation of steroid receptors by co - factors bearing the lxxll motif occurs through interaction with the activating function 2 ( af-2 ) surface in the lbd , which provides a hydrophobic surface flanked by two conserved opposing charged amino acids , lysine 720 ( k720 ) and glutamic acid 897 ( e897 ) ( trapman and dubbink , 2007 ) . to test whether the interaction between prmt6 and ar occurs through the af-2 surface , we performed transcriptional assay in cells expressing ar variants in which either k720 was substituted with alanine ( k720a ) to reduce interaction with co - factors or e897 was substituted with lysine ( e897k ) to disrupt binding to co - factors ( figure 2e ) . as controls , we tested mutation of alanine 574 to aspartate ( a574d ) to prevent dna binding , and mutation of glycine 21 to glutamic acid ( g21e ) to abolish the n / c interactions without altering interaction with co - regulators . as expected , the a574d mutation did not respond to hormone stimulation or to prmt6 transactivation . the k720a mutation decreased the effect of prmt6 on ar transactivation by 10% , whereas the e897k mutation reduced it by 60% , indicating that the ar - prmt6 interaction requires an intact af-2 surface . next , we sought to investigate the biological significance of ar transactivation by prmt6 . to elucidate this aspect , we tested whether arginine methylation alters the expression of genes regulated by ar and prmt6 in motor neuron - derived mn-1 cells . we focused on genes that are known targets of prmt6 and ar or that have previously been implicated in sbma pathogenesis , including cyclin - dependent kinase inhibitor 1 ( p21cip / waf1 , hereafter referred to as p21),vascular endothelial growth factor receptor 2 ( vegfr2 ) , and sarco(endo)plasmic reticulum ca(2 + ) atpase 2b ( serca2b ) ( montague et al . , 2014 ; sopher et al . , we generated stable mn-1 clones expressing either ar24q or ar100q ( figures 5a and 5b ) . by real - time pcr analysis , we found that upon dht treatment , p21 and vegfr2 mrna transcript levels were downregulated in mutant cells ( figure 2f ) . we infected the cells with lentiviruses expressing either scramble or shrna against prmt6 ( phalke et al . , 2012 ) . expression of shrna against prmt6 ( # 2 ) reduced prmt6 expression by 40% ( figure 5a ) , and significantly increased the expression of p21 , vegfr2 , and serca2b specifically in the mutant cells ( figure 2f ) . on the other hand , overexpression of prmt6 had the opposite effect on p21 expression ( figure 2 g ) . these results support the idea that gene expression is altered by the interaction of polyglutamine - expanded ar and prmt6 . the observation that prmt6 catalytic activity is required to activate ar prompted us to determine whether the effect of prmt6 occurs through direct modification of ar . in an in vitro methylation assay , incubation of polyglutamine - expanded ar with prmt6 , but not prmt6-v86k , d88a , in the presence of the methyl donor [ h]s - adenosylmethionine ( [ h]-sam ) increased methylated ar by 4-fold ( figure 3a and figure s5a ) . consistent with previous findings that prmt6 undergoes auto - methylation ( frankel et al . , 2002 ) , methylated prmt6 to determine whether full - length ar is methylated , we immunoprecipitated flag - tagged ar expressed with either soluble egfp or egfp - tagged prmt6 in hek293 t cells and analyzed samples using an antibody that specifically recognizes asymmetrically dimethylated arginine ( figure 3b ) . overexpression of prmt6 did not change arginine dimethylation of both normal and mutant ar , suggesting that endogenous prmt6 is sufficient to methylate ar . on the other hand , overexpression of catalytically inactive prmt6 ( v86k , d88a ) reduced the methylation of polyglutamine - expanded ar by 40% ( figure 3c ) , probably reflecting a dominant - negative action of mutant prmt6 ( herrmann et al . , 2005 ) . next , we sought to identify the arginine residues of ar that were methylated by prmt6 . most prmts target arginine residues within gar , rxr , and rgg motifs ( feng et al . ar does not have rgg motifs , while it has one gar motif and the three rxr motifs rar , rlr , and rmr ( figure 3d ) . gar motif and r618 are part of the nuclear localization signal ( figure 3d , underlined ) . rar and rmr are part of the two akt consensus site motifs rxrxxs of ar , one located in the ntd and the other in the af-2 surface . by mass spectrometry analysis , r629 , but not r618 , was methylated by prmt6 in response to hormone treatment ( figure 3e and figure s5c ) . for technical reasons , we could not resolve the arginines at the akt consensus sites by mass spectrometry analysis . because of the relevance of these sites in sbma pathogenesis ( palazzolo et al . , 2007 , 2009 ) , we used an in vitro methylation assay to test whether prmt6 methylates the arginines of the akt consensus site motifs of ar ( figure 3f and figure s5d ) . peptides spanning these akt consensus site motifs ( wt peptides ) were incubated with purified prmt6 in the presence of [ h]-sam . as control collectively , these results suggest that ar is methylated by prmt6 at arginines 210 , 212 , 629 , 787 , and 789 . similar to ar , also forkhead box o ( foxo ) transcription factors , which play a critical role in neurodegeneration ( mojsilovic - petrovic et al . , 2009 ) , are arginine - methylated at akt consensus site motifs ( yamagata et al . importantly , arginine methylation of foxo was shown to block phosphorylation by akt , but not vice versa . to investigate the potential relationship between arginine methylation and serine phosphorylation at the ar akt consensus site motifs , we performed in vitro methylation assay using peptides in which s215 and s792 were substituted with either phospho - defective alanine ( s215a and s792a peptides ) or phospho - mimetic aspartate ( s215d and s792d peptides ) ( figure 3f and figure s5d ) , as we previously described ( palazzolo et al . , 2007 ) . s215a and s792a peptides were methylated by prmt6 , suggesting that loss of phosphorylation does not affect methylation . on the other hand , phospho - mimetic substitution of s215 and s792 abolished methylation , suggesting that phosphorylation prevents methylation . to determine whether methylation affects phosphorylation , we conducted an in vitro phosphorylation assay by incubating wt peptide spanning the akt consensus site within the af2 surface and chemically modified by arginine methylation ( me - wt peptide ) with recombinant akt in the presence of [ p]-atp ( figure 3 g ) . as negative control , we used the s792a peptide . as expected ( palazzolo et al . , 2007 ) , incubation of me - wt peptide with akt reduced the incorporation of [ p]-atp by 80% . similar results were obtained with prior incubation of wt peptide with prmt6 and [ h]-sam , and subsequent incubation with akt and [ p]-atp ( figure s5e ) . together , these results suggest that arginine methylation of the rxrxxs motif by prmt6 prevents phosphorylation by akt and vice versa . major events occurring upon hormone binding are protein stabilization , nuclear translocation , and transactivation of gene expression ( parodi and pennuto , 2011 ) . to investigate the functional significance of arginine methylation at the gar and the akt consensus site motifs , we generated the methylation - defective ar variants , ar65q - r629k and ar65q - r210k , r212k , r787k , r789k . loss of arginine methylation at these sites neither altered accumulation of monomeric ar in response to hormone binding ( figure 4a ) , nor reduced hormone - induced nuclear translocation ( figure 4b ) . in transcriptional assays , loss of methylation at r629 did not affect ar transactivation by prmt6 ( figure 4c ) . on the other hand , loss of arginine methylation at the akt consensus site motifs reduced the transactivation of normal ar by 34% and of mutant ar by 45% , respectively ( figure 4d ) . these observations indicate that prmt6 requires the arginine residues at the akt consensus site motifs in order to fully transactivate polyglutamine - expanded ar . to assess whether prmt6-induced ar transactivation is modulated by phosphorylation at the akt consensus sites , we investigated the effect of prmt6 transactivation on phosphorylation - defective ( s215a , s792a ) ar variants ( figure 4d ) . prmt6 did not alter the transactivation of phosphorylation - defective non - expanded ar , but it enhanced that of polyglutamine - expanded ar by 2.1-fold . because pharmacologic ( asc - j9 ) or genetic ( ar mutation e897k ) inhibition of the interaction between mutant ar and its co - factors suppresses toxicity ( nedelsky et al . , 2010 ; yang et al . , 2007 ) , and reduces the transactivation of ar by prmt6 , we hypothesized that ar - prmt6 interaction is pathogenetic in sbma . to test this hypothesis , we undertook both a loss- and a gain - of - function approach to target prmt6-ar interaction . the sbma mn-1 cells that we generated showed reduced cell viability compared to normal cells , although this difference was not hormone dependent ( figure 5a ) . silencing endogenous prmt6 by 30%40% with two conversely , overexpression of wild - type prmt6 as well as prmt6-v86k , d88a and -lxxaa mutants decreased cell viability by 30% in both normal and mutant cells , indicating that the effect of prmt6 overexpression on the survival of these cells was independent of its ability to transactivate ar ( figure 5b ) . notably , in the mutant cells overexpression of wild - type , but not mutant ( v86k , d88a and lxxaa ) , prmt6 reduced cell viability by 66% in a hormone - dependent manner , indicating that prmt6 gain of function in mutant cells enhances the toxicity of mutant ar in a hormone - dependent fashion . pc12 cells expressing ar112q showed reduced cell viability when treated with hormone ( figure 5c ) . overexpression of prmt6 decreased cell viability by 17% and 21% in the vehicle- and dht - treated cells , respectively . in these cells , dht treatment also reduced cell size by 15% , which was further reduced by prmt6 overexpression by 14% and 27% in the absence and presence of dht , respectively ( figure 5c ) . a hallmark of polyglutamine diseases is the accumulation of mutant proteins in forms of aggregates or micro - oligomers , which can be revealed as high molecular weight species that accumulate in the stacking portion of polyacrylamide gels ( palazzolo et al . , 2009 ) . overexpressing prmt6 significantly enhanced by 1.26-fold polyglutamine - expanded ar aggregation , further supporting a toxic gain - of - function effect of prmt6 on mutant ar ( figure 5d ) . treatment of the cells with the pi3k / akt signaling inhibitor ly294002 for 24 hr , a condition that does not elicit overt toxicity ( palazzolo et al . , 2007 ) , reduced the protective effect of prmt6 knockdown on cell viability , suggesting that prmt6 enhances polyglutamine - expanded ar toxicity by counteracting its phosphorylation by akt ( figure 5e ) . consistent with this model , prmt6 decreased neither the viability nor the size of pc12 cells expressing the phospho - mimetic ar variant ar100q - s215d , s792d ( figure 5f ) . to test whether prmt6 modifies the sbma phenotype in vivo , we used fly models of sbma . overexpression of polyglutamine - expanded ar ( ar52q ) in the eye caused degeneration of the posterior side ommatidia ( figures 6a and 6b ) ( nedelsky et al . also ar with a 12 glutamine - long tract ( ar12q ) resulted in mild neurodegeneration when overexpressed in this system , whereas ar without a polyglutamine tract ( ar0q ) was not toxic . we crossed the sbma flies with flies in which the prmt6 drosophila ortholog , dart8 , was knocked down by 31% by specific rna interference ( figure 6c ) . knocking down endogenous dart8 itself did not cause any obvious ommatidial degeneration in flies ( figures 6a , 6b , and 6d ) . however , knockdown of dart8 suppressed polyglutamine - expanded ar - induced neurodegeneration , without altering ar expression ( figure s6a ) . notably , dart8 knockdown also suppresses the phenotype caused by overexpression of non - expanded ar . knockdown of the prmt8 drosophila ortholog , dart2 , did not suppress the degenerative eye phenotype in this sbma fly model , suggesting that the effect of dart8 knockdown is specific ( figure s6b ) . consistent with the idea that silencing prmt6 ameliorates phenotype because it results in increased phosphorylation at the akt consensus site motifs , dart8 knockdown did not modify the phenotype of flies expressing phosphorylation - defective ar65q - s215a , s792a ( figures 6e and 6f ) . overexpression of prmt6 in the eye of sbma flies did not modify phenotype , suggesting that the endogenous fly ortholog of prmt6 is sufficient to cause neurodegeneration ( figures s6c and s6d ) . collectively , these results indicate that prmt6 is a modifier of mutant ar toxicity in vivo . here , we show that prmt6 co - localizes and forms a complex with ar and that the resulting transactivation of the receptor is significantly enhanced by polyglutamine expansion . we had previously shown that ar is phosphorylated by akt at the rxrxxs motifs . here , we report that prmt6 methylates the ar at arginine residues spanning the akt consensus site motifs and that arginine methylation and serine phosphorylation at these sites are mutually exclusive . importantly , inhibition of prmt6 suppressed the toxicity of mutant ar in vitro and in vivo , whereas overexpression of prmt6 enhanced toxicity . our findings establish a key role for arginine methylation and prmt6 in the pathogenesis of polyglutamine diseases . emerging evidence in the field of polyglutamine diseases supports the idea that the toxic gain of function conferred by polyglutamine expansion arises from alteration of the normal , native function(s ) of the mutant protein ( orr , 2012 ) . although the physiological function(s ) of several polyglutamine proteins is not known , there is evidence from proteins of known function that glutamine expansions act by enhancing the normal function of the disease protein , and finally cause neurodegeneration ( mcmahon et al . , 2005 ; mo et al . , 2010 ) . indeed , amplification of protein function by overexpression of ar and ataxin-1 with non - pathogenic repeat lengths leads to a neurodegenerative phenotype similar to that caused by polyglutamine expansion ( fernandez - funez et al . consistent with this concept , knockdown of prmt6 not only suppressed the toxicity of polyglutamine - expanded ar , but it also ameliorated the phenotype of flies overexpressing normal ar , further supporting that prmt6 contributes to toxicity by enhancing the native function of ar . moreover , there is evidence that polyglutamine expansion leads to amplification of interaction with native cellular partners , as reported for a variety of polyglutamine proteins , including ataxin-1 ( lim et al . , 2008 ) , ar ( nedelsky et al . , 2010 ) , and tbp ( friedman et al . , 2007 ) . expanding this idea , we here present evidence that prmt6 acts as a co - activator of ar whose function is enhanced by polyglutamine expansion . in sbma , pathogenic interactions occur through the ar co - factor interaction surface , af-2 , and an intact af-2 domain is indeed required for toxicity ( nedelsky et al . , 2010 ) . we show here that the interaction between polyglutamine - expanded ar and prmt6 is mediated by the af-2 surface of ar and the steroid receptor interaction motif , lxxll , of prmt6 . hormone binding induces a conformational change in the lbd that leads to generation of a hydrophobic pocket that initially binds to a hydrophobic helix in the ntd of ar , thereby generating intra- and inter - molecular n / c interactions . n / c interactions occur before and are lost upon dna binding ( van royen et al . , 2007 ) . subsequently , the hydrophobic pocket mediates binding to lxxll motifs in transcriptional co - factors ( heery et al . , 1997 ) . pharmacologic ( asc - j9 ) or genetic ( ar mutation e897k ) intervention to disrupt the interaction of ar with co - regulators suppresses the toxicity of mutant ar in mouse and fly models of sbma ( nedelsky et al . a mutation abolishing dna binding ( a574d ) , which occurs before binding to co - regulators , as well as a mutation that disrupts binding to co - regulators ( e897k ) suppress the eye degenerative phenotype caused by polyglutamine - expanded ar in flies . substitution of k720 with alanine ( k720a ) , which reduces interaction with co - factors , partially attenuated neurodegeneration . on the other hand , a mutation of glycine 21 to glutamic acid ( g21e ) that abolishes the n / c interactions without altering interaction with co - regulators had no effect on toxicity . consistent with these observations , asc - j9 as well as mutations k720a and e897k , but not g21e , reduced transactivation of ar by prmt6 . post - translational modifications are critical regulators of protein function and are modifiers of polyglutamine protein toxicity ( pennuto et al . , 2009 ) . methylation , together with phosphorylation , is a major post - translational modification occurring in mammalian cells , with about 2% of cellular proteins containing dimethylated arginine residues ( bedford and clarke , 2009 ) . we have previously shown that phosphorylation of polyglutamine - expanded ar by akt suppresses toxicity ( palazzolo et al . importantly , we demonstrate here that arginine methylation prevents phosphorylation by akt , and vice versa , thereby implying that arginine methylation and serine phosphorylation at these sites are mutually exclusive in ar . phosphorylation of polyglutamine - expanded huntingtin at serine 421 by akt has been shown to be protective in striatal neurons ( humbert et al . , 2002 ) , whereas phosphorylation of polyglutamine - expanded ataxin-1 at serine 776 enhances toxicity ( emamian et al . , 2003 ) . our findings indicate the existence of an additional important level of regulation of phosphorylation at the rxrxxs motif by arginine methylation , which may also play a critical role in polyglutamine diseases other than sbma . arginine methylation has been recently implicated in the pathogenesis of other neurodegenerative diseases , such as amyotrophic lateral sclerosis ( als ) . the als - linked fused in sarcoma ( fus ) undergoes extensive arginine methylation , and this modification alters its subcellular localization and toxicity ( dormann et al . , 2012 ; scaramuzzino et al . , 2013 ; tradewell et al . , moreover , arginine methylation of spliceosomal proteins affects interaction with survival of motor neuron ( smn ) , another protein involved in motor neuron disease ( brahms et al . . however , the mechanism through which arginine methylation affects neuronal survival in pathological conditions is poorly understood . given the role of the prmts as major modifiers of histones , the interaction between ar and prmt6 may alter histone epigenetic marks , thereby contributing to the transcription abnormalities that characterize sbma neuron and muscle cells ( lieberman et al . consistent with this idea , we found that polyglutamine - expanded ar and prmt6 suppress the expression of specific genes in motor neuron - derived cells , further supporting that prmt6 contributes to disease by altering ar function and transcription regulation . the enhanced interaction between polyglutamine - expanded ar and prmt6 may also lead to sequestration of prmt6 away from active chromatin sites , thereby causing a loss of prmt6 function . however , the observation that loss of prmt6 function in mice does not cause any overt phenotype ( neault et al . , 2012 ) , whereas gain of prmt6 function causes premature death ( di lorenzo et al . , 2014 ) , argues against this idea . rather , this evidence supports our hypothesis that a gain of functional interaction between prmt6 and its native partners / substrates , e.g. , polyglutamine - expanded ar as shown here , is the mechanism underlying cell dysfunction and degeneration . in conclusion , we propose a model in which polyglutamine - expanded ar is phosphorylated by akt , an event that blocks binding to hormones , protects from toxicity , and prevents methylation ( figure 7 ) ( palazzolo et al . , 2007 , 2009 ) . on the other hand , the interaction between polyglutamine - expanded ar and prmt6 leads to arginine methylation of the ar with enhancement of protein function and toxicity . mn-1 , pc12 , cos1 , hek293 t , mouse- , and patient - derived cells were cultured as previously described ( basso et al . , 2012 ; grunseich et al . , 2014 ; palazzolo et al . , 2007 ; walcott and merry , 2002 ) . human spinal cord slides were fixed with 4% pfa , then placed in blocking solution ( 10% ngs , 0.3% triton x-100 in pbs ) for 45 min at room temperature . primary antibody staining was done at 4c overnight in pbs containing 5% ngs and 0.1% triton x-100 , using ar and prmt6 antibodies . slides were then incubated with secondary antibody and then washed before drying and adding vectashield / dapi stain ( vector lab ) . for ar staining in ipsc - derived motor neurons , slides were treated with 100 mm glycine after fixation and blocked in pbs with 3% bsa . antibody staining was performed in pbs with 3% bsa and 0.1% tween overnight with 0.1% tween / pbs used for all washes . cell lysates were collected in lysis buffer ( 150 mm nacl , 6 mm na2hpo4 , 4 mm nah2po4 , 2 mm edta [ ph8 ] , 1% nadoc , 0.5% triton x-100 , sds 0.1% ) . transcriptional assays were performed using the dual - luciferase assay kit ( promega ) , according to manufacturers instructions . cell toxicity was measured by xtt assay in mn-1 cells and trypan blue assay in pc12 cells . 2 g rna were retro - transcribed using the superscript iii first - strand synthesis system kit ( invitrogen ) . gene expression was measured by quantitative real - time pcr using 7900 ht fast real - time pcr system ( applied biosystems ) . the level of each transcript was measured with the threshold cycle ( ct ) method . values were normalized to the mean of the cells expressing ar24q or ar100q , which were assigned as 100% . for in vitro methylation assays , immunoprecipitated flag - tagged ar65q was incubated with immunoprecipitated egfp , egfp - tagged prmt6 , or prmt6-kla in the presence of s - adenosyl - l-[methyl - h ] methionine ( perkin elmer ) . 20 g of indicated peptides ( united biosystem ) were incubated with immunoprecipitated egfp or egfp - tagged prmt6 and s - adenosyl - l-[methyl - h ] methionine . after washing the beads , for in vitro phosphorylation assays , the indicated peptides were incubated in phosphorylation buffer ( 20 mm naf , 20 mm mops [ ph 7.2 ] , 25 mm beta - glycerolphosphate , 10 mm mgcl2 , 25 m atp , 1 mm na3vo4 , 5 mm egta , 1 mm dtt ) , followed by the addition of 50 ng recombinant akt ( upstate ) and [ -p ] atp ( perkin elmer ) . after 30 min of incubation , samples were subjected to sds - page , transferred on pvdf membrane . the ar0q and ar65q - s215a , s792a transgenic line was generated at best gene . the dart8 ( 100228 ) and dart2 ( 26058 ) rnai lines were obtained from the vienna drosophila rnai center . quantification of the eye phenotypes was performed as previously described ( pandey et al . flies collected for scanning electron microscopy ( sem ) were processed as previously described ( lanson et al . , 2011 ) .

summarypolyglutamine expansion in androgen receptor ( ar ) is responsible for spinobulbar muscular atrophy ( sbma ) that leads to selective loss of lower motor neurons . using sbma as a model , we explored the relationship between protein structure / function and neurodegeneration in polyglutamine diseases . we show here that protein arginine methyltransferase 6 ( prmt6 ) is a specific co - activator of normal and mutant ar and that the interaction of prmt6 with ar is significantly enhanced in the ar mutant . ar and prmt6 interaction occurs through the prmt6 steroid receptor interaction motif , lxxll , and the ar activating function 2 surface . ar transactivation requires prmt6 catalytic activity and involves methylation of arginine residues at akt consensus site motifs , which is mutually exclusive with serine phosphorylation by akt . the enhanced interaction of prmt6 and mutant ar leads to neurodegeneration in cell and fly models of sbma . these findings demonstrate a direct role of arginine methylation in polyglutamine disease pathogenesis .

Introduction Results Discussion Experimental Procedures

the family of polyglutamine diseases includes spinal and bulbar muscular atrophy ( sbma ) , huntington s disease ( hd ) , dentatorubral - pallidoluysian atrophy ( drpla ) , and spinocerebellar ataxia ( sca ) type 1 , 2 , 3 , 6 , 7 , and 17 . these disorders are caused by glutamine expansions in androgen receptor ( ar ) , huntingtin , atrophin-1 , ataxin-1 , ataxin-2 , ataxin-3 , cacna1a , ataxin-7 , and the tata - box binding protein ( tbp ) , respectively . , 2005 ) , suggesting a mechanistic link between expanded polyglutamine - induced toxicity and protein structure / function ( parodi and pennuto , 2011 ) . sbma is an x - linked motor neuron disease characterized by selective degeneration of lower motor neurons ( kennedy et al . in the family of polyglutamine diseases , sbma is unique in that the disease fully manifests only in males . upon hormone binding , ar translocates to the nucleus , undergoes a conformational change that leads to amino / carboxy - terminal ( n / c ) interactions , binds dna at androgens response elements ( ares ) , and recruits specific transcription co - factors , including chromatin remodeling factors , to regulate the expression of androgen - responsive genes . we previously demonstrated that phosphorylation of polyglutamine - expanded ar by akt at serines 215 and 792 , which lie in the akt consensus site motif rxrxxs ( where r is arginine , s serine , and x any amino acid ) , reduces hormone binding and ar transactivation and protects from neurodegeneration ( palazzolo et al . similar to phosphorylation , arginine methylation is a post - translational modification with major impact on protein structure and function ( bedford and clarke , 2009 ) . arginine methylation is catalyzed by a family of enzymes known as protein arginine methyltransferases ( prmts ) , which differ in their activity , substrate specificity and subcellular localization . however , nothing is known as to whether prmts target polyglutamine proteins and play a role in polyglutamine disease pathogenesis . to address this question , we used sbma as a model of polyglutamine diseases . our results show a key role for arginine methylation in the pathogenesis of polyglutamine diseases and provide evidence that a causative link between primary structure and function of polyglutamine protein is the underlying mechanism in the pathogenesis of polyglutamine diseases . to determine whether arginine methylation contributes to polyglutamine - induced neurodegeneration , we investigated the role of prmt function on sbma pathogenesis . we analyzed the subcellular distribution of the prmts and mutant ar in cos1 cells expressing polyglutamine - expanded ar with 65 glutamine residues ( ar65q ) and the prmts tagged to enhanced gfp ( egfp ) . in the dht - treated cells , mutant ar co - localized with prmt2 , 6 , and 7 in the nucleus . by immunoprecipitation assay in dht - treated hek293 t cells co - expressing flag - tagged ar together with either soluble egfp or the egfp - tagged prmts , normal and polyglutamine - expanded ar specifically formed a complex with prmt2 , 6 , and 7 ( figure 1a and figure s1b ) . to test whether the prmts act as transcription co - factors of ar , we measured ar transactivation by transcriptional assay in hek293 t cells transfected with either normal ar ( ar24q ) or ar65q under the control of the cytomegalovirus promoter , and using as reporter the luciferase gene whose expression was driven by an are , as previously described ( palazzolo et al . the effect of prmt6 was hormone dependent , indicating that prmt6 acts as a co - activator of ar rather than as a general transcription activator , and it did not result from altered ar expression ( figure s1c ) . importantly , prmt6 increased the transactivation of ar24q by 3.3-fold and that of ar65q by 5.3-fold , indicating that the effect of prmt6 on ar transactivation is enhanced by polyglutamine expansion . these results show that prmt6 is a specific ar co - activator , whose function is enhanced by polyglutamine expansion . ar112q forms intranuclear inclusions in response to hormone binding in the pc12 cells ( walcott and merry , 2002 ) , and prmt6 localized to ar - positive intranuclear inclusions in these cells ( figure 1c , arrows ) . ar and prmt6 co - localized in the nucleus of control and sbma human primary fibroblasts as well as ipscs reprogrammed to motor neurons . notably , prmt6 was expressed and co - localized with polyglutamine - expanded ar in the ventral horn motor neurons of spinal cord autopsy specimens derived from an sbma patient ( figure 1c and figure s2b ) . the ar / prmt6 interaction occurred both in the absence and presence of hormone and was significantly enhanced by polyglutamine expansion in the mn-1 cells . by transcriptional assay , overexpression of prmt6 increased the transactivation of normal ar by 3.3-fold and that of polyglutamine - expanded ar by 5.7-fold in the mn-1 cells ( figure 1e ) . taken together , these results indicate that polyglutamine expansion enhances the structural and functional interaction of ar with its co - activator prmt6 in neuronal and patient - derived cells . the interaction between ar and co - regulators can be inhibited by the anti - androgen 5-hydroxy-1,7-bis(3,4-dimethoxyphenyl)-1,4,6-heptatriene-3-one ( asc - j9 ) , which has been shown to rescue the phenotype of a mouse model of sbma ( yang et al . to address whether the catalytic activity of prmt6 is required to transactivate ar , we performed transcriptional assays in cells treated with the pan - prmt inhibitor adenosine dialdehyde ( adox ) . adox reduced polyglutamine - expanded ar transactivation induced by dht by 30% , and it completely abolished the effect of prmt6 on ar transactivation ( figure 2b and figure s3b ) . to establish whether the lrvll motif of prmt6 is necessary for ar transactivation , we substituted leucines 356 and 357 with alanines , thereby generating prmt6-lxxaa mutant ( heery et al . mutation of the lxxll motif decreased the interaction with polyglutamine - expanded and normal ar ( figure 2d and figure s3c , inset ) and abolished ar transactivation ( figure 2d and figure s3c ) , indicating that the ability of prmt6 to enhance ar transcriptional activity is dependent on the integrity of the lrvll motif . steroid receptor primary structure is composed of an amino - terminal domain ( ntd ) , a dna - binding domain ( dbd ) , and the ligand - binding domain ( lbd ) ( figure 2c ) . transactivation of steroid receptors by co - factors bearing the lxxll motif occurs through interaction with the activating function 2 ( af-2 ) surface in the lbd , which provides a hydrophobic surface flanked by two conserved opposing charged amino acids , lysine 720 ( k720 ) and glutamic acid 897 ( e897 ) ( trapman and dubbink , 2007 ) . to test whether the interaction between prmt6 and ar occurs through the af-2 surface , we performed transcriptional assay in cells expressing ar variants in which either k720 was substituted with alanine ( k720a ) to reduce interaction with co - factors or e897 was substituted with lysine ( e897k ) to disrupt binding to co - factors ( figure 2e ) . as controls , we tested mutation of alanine 574 to aspartate ( a574d ) to prevent dna binding , and mutation of glycine 21 to glutamic acid ( g21e ) to abolish the n / c interactions without altering interaction with co - regulators . the k720a mutation decreased the effect of prmt6 on ar transactivation by 10% , whereas the e897k mutation reduced it by 60% , indicating that the ar - prmt6 interaction requires an intact af-2 surface . to elucidate this aspect , we tested whether arginine methylation alters the expression of genes regulated by ar and prmt6 in motor neuron - derived mn-1 cells . we focused on genes that are known targets of prmt6 and ar or that have previously been implicated in sbma pathogenesis , including cyclin - dependent kinase inhibitor 1 ( p21cip / waf1 , hereafter referred to as p21),vascular endothelial growth factor receptor 2 ( vegfr2 ) , and sarco(endo)plasmic reticulum ca(2 + ) atpase 2b ( serca2b ) ( montague et al . expression of shrna against prmt6 ( # 2 ) reduced prmt6 expression by 40% ( figure 5a ) , and significantly increased the expression of p21 , vegfr2 , and serca2b specifically in the mutant cells ( figure 2f ) . these results support the idea that gene expression is altered by the interaction of polyglutamine - expanded ar and prmt6 . the observation that prmt6 catalytic activity is required to activate ar prompted us to determine whether the effect of prmt6 occurs through direct modification of ar . overexpression of prmt6 did not change arginine dimethylation of both normal and mutant ar , suggesting that endogenous prmt6 is sufficient to methylate ar . ar does not have rgg motifs , while it has one gar motif and the three rxr motifs rar , rlr , and rmr ( figure 3d ) . rar and rmr are part of the two akt consensus site motifs rxrxxs of ar , one located in the ntd and the other in the af-2 surface . , 2007 , 2009 ) , we used an in vitro methylation assay to test whether prmt6 methylates the arginines of the akt consensus site motifs of ar ( figure 3f and figure s5d ) . peptides spanning these akt consensus site motifs ( wt peptides ) were incubated with purified prmt6 in the presence of [ h]-sam . , 2009 ) , are arginine - methylated at akt consensus site motifs ( yamagata et al . importantly , arginine methylation of foxo was shown to block phosphorylation by akt , but not vice versa . to investigate the potential relationship between arginine methylation and serine phosphorylation at the ar akt consensus site motifs , we performed in vitro methylation assay using peptides in which s215 and s792 were substituted with either phospho - defective alanine ( s215a and s792a peptides ) or phospho - mimetic aspartate ( s215d and s792d peptides ) ( figure 3f and figure s5d ) , as we previously described ( palazzolo et al . to determine whether methylation affects phosphorylation , we conducted an in vitro phosphorylation assay by incubating wt peptide spanning the akt consensus site within the af2 surface and chemically modified by arginine methylation ( me - wt peptide ) with recombinant akt in the presence of [ p]-atp ( figure 3 g ) . together , these results suggest that arginine methylation of the rxrxxs motif by prmt6 prevents phosphorylation by akt and vice versa . to investigate the functional significance of arginine methylation at the gar and the akt consensus site motifs , we generated the methylation - defective ar variants , ar65q - r629k and ar65q - r210k , r212k , r787k , r789k . loss of arginine methylation at these sites neither altered accumulation of monomeric ar in response to hormone binding ( figure 4a ) , nor reduced hormone - induced nuclear translocation ( figure 4b ) . on the other hand , loss of arginine methylation at the akt consensus site motifs reduced the transactivation of normal ar by 34% and of mutant ar by 45% , respectively ( figure 4d ) . these observations indicate that prmt6 requires the arginine residues at the akt consensus site motifs in order to fully transactivate polyglutamine - expanded ar . to assess whether prmt6-induced ar transactivation is modulated by phosphorylation at the akt consensus sites , we investigated the effect of prmt6 transactivation on phosphorylation - defective ( s215a , s792a ) ar variants ( figure 4d ) . because pharmacologic ( asc - j9 ) or genetic ( ar mutation e897k ) inhibition of the interaction between mutant ar and its co - factors suppresses toxicity ( nedelsky et al . , 2007 ) , and reduces the transactivation of ar by prmt6 , we hypothesized that ar - prmt6 interaction is pathogenetic in sbma . silencing endogenous prmt6 by 30%40% with two conversely , overexpression of wild - type prmt6 as well as prmt6-v86k , d88a and -lxxaa mutants decreased cell viability by 30% in both normal and mutant cells , indicating that the effect of prmt6 overexpression on the survival of these cells was independent of its ability to transactivate ar ( figure 5b ) . a hallmark of polyglutamine diseases is the accumulation of mutant proteins in forms of aggregates or micro - oligomers , which can be revealed as high molecular weight species that accumulate in the stacking portion of polyacrylamide gels ( palazzolo et al . overexpressing prmt6 significantly enhanced by 1.26-fold polyglutamine - expanded ar aggregation , further supporting a toxic gain - of - function effect of prmt6 on mutant ar ( figure 5d ) . , 2007 ) , reduced the protective effect of prmt6 knockdown on cell viability , suggesting that prmt6 enhances polyglutamine - expanded ar toxicity by counteracting its phosphorylation by akt ( figure 5e ) . to test whether prmt6 modifies the sbma phenotype in vivo , we used fly models of sbma . consistent with the idea that silencing prmt6 ameliorates phenotype because it results in increased phosphorylation at the akt consensus site motifs , dart8 knockdown did not modify the phenotype of flies expressing phosphorylation - defective ar65q - s215a , s792a ( figures 6e and 6f ) . overexpression of prmt6 in the eye of sbma flies did not modify phenotype , suggesting that the endogenous fly ortholog of prmt6 is sufficient to cause neurodegeneration ( figures s6c and s6d ) . here , we show that prmt6 co - localizes and forms a complex with ar and that the resulting transactivation of the receptor is significantly enhanced by polyglutamine expansion . here , we report that prmt6 methylates the ar at arginine residues spanning the akt consensus site motifs and that arginine methylation and serine phosphorylation at these sites are mutually exclusive . our findings establish a key role for arginine methylation and prmt6 in the pathogenesis of polyglutamine diseases . emerging evidence in the field of polyglutamine diseases supports the idea that the toxic gain of function conferred by polyglutamine expansion arises from alteration of the normal , native function(s ) of the mutant protein ( orr , 2012 ) . expanding this idea , we here present evidence that prmt6 acts as a co - activator of ar whose function is enhanced by polyglutamine expansion . in sbma , pathogenic interactions occur through the ar co - factor interaction surface , af-2 , and an intact af-2 domain is indeed required for toxicity ( nedelsky et al . we show here that the interaction between polyglutamine - expanded ar and prmt6 is mediated by the af-2 surface of ar and the steroid receptor interaction motif , lxxll , of prmt6 . hormone binding induces a conformational change in the lbd that leads to generation of a hydrophobic pocket that initially binds to a hydrophobic helix in the ntd of ar , thereby generating intra- and inter - molecular n / c interactions . pharmacologic ( asc - j9 ) or genetic ( ar mutation e897k ) intervention to disrupt the interaction of ar with co - regulators suppresses the toxicity of mutant ar in mouse and fly models of sbma ( nedelsky et al . a mutation abolishing dna binding ( a574d ) , which occurs before binding to co - regulators , as well as a mutation that disrupts binding to co - regulators ( e897k ) suppress the eye degenerative phenotype caused by polyglutamine - expanded ar in flies . importantly , we demonstrate here that arginine methylation prevents phosphorylation by akt , and vice versa , thereby implying that arginine methylation and serine phosphorylation at these sites are mutually exclusive in ar . our findings indicate the existence of an additional important level of regulation of phosphorylation at the rxrxxs motif by arginine methylation , which may also play a critical role in polyglutamine diseases other than sbma . given the role of the prmts as major modifiers of histones , the interaction between ar and prmt6 may alter histone epigenetic marks , thereby contributing to the transcription abnormalities that characterize sbma neuron and muscle cells ( lieberman et al . consistent with this idea , we found that polyglutamine - expanded ar and prmt6 suppress the expression of specific genes in motor neuron - derived cells , further supporting that prmt6 contributes to disease by altering ar function and transcription regulation . the enhanced interaction between polyglutamine - expanded ar and prmt6 may also lead to sequestration of prmt6 away from active chromatin sites , thereby causing a loss of prmt6 function . in conclusion , we propose a model in which polyglutamine - expanded ar is phosphorylated by akt , an event that blocks binding to hormones , protects from toxicity , and prevents methylation ( figure 7 ) ( palazzolo et al . on the other hand , the interaction between polyglutamine - expanded ar and prmt6 leads to arginine methylation of the ar with enhancement of protein function and toxicity .

at the end of the last century , in addition to the conventional screening of naturally occurring bioactive compounds , a strategy was developed to construct desired novel hybrid antibiotics by combinatorial biosynthesis , by specifically combining genes from various biosynthetic pathways originating from different antibiotics producing organisms . in streptomyces species , the most important producers of bioactive molecules , genetic approaches to improve secondary metabolite production are generally hampered by various phenomena such as restriction barriers , absence of an efficient gene transfer systems , and lack of suitable cloning vectors . recently , in order to circumvent these problems , there has been considerable interest in the use of intergeneric conjugation as an efficient means of gene transfer and expression in streptomyces species . this technique allows the construction and the manipulation of recombinant plasmids in e. coli and their subsequent transfer to a recipient of interest . the initial protocol for intergeneric transfer of plasmids from e. coli to streptomyces species was developed by mazodier et al . . since then , this method has been successfully applied to several streptomyces species [ 24 ] via methylation - deficient e. coli strains , such as strain et12567 , as a dna donor to avoid methylated dna restriction systems of actinomycetes . several cloning vectors that could be transferred from e. coli to streptomyces species by conjugation method have been constructed [ 7 , 8 ] . among them , pset152 is a nonreplicative plasmid in streptomyces that carries the attp site and integrase gene of c31 phage and consequently can integrate into the chromosomal attb site of the bacteriophage c31 . like integrases , c31 integrase catalyses the site - specific recombination between the chromosomal attb site and an attp site to form attl and attr . however , unlike those of integrases , the sites are both very small , with attp and attb being just 39 and 34 pb in size , respectively . the dna sequences of attb were characterized from various streptomyces species , and the analysis of these sequences revealed the presence of high conserved positions . in this genus of bacteria , the presence of pseudo - attb sites ( 50%70.3% nt identity to attb sites ) has also been reported , but with an integration frequency 300 times lower than that with the corresponding attb site . however , the absence of this attb site from some actinomycete strains , such as saccharopolyspora erythraea , has also been reported . we have previously reported the isolation from tunisian soil of two new actinomycete strains called streptomyces sp . us 24 and streptomyces sp . tn 58 , producing diverse interesting biological activities , and described the purification and structure elucidation of two and five active molecules from the streptomyces sp . the active molecules produced by streptomyces sp . us 24 are the 3-indolethanol and a diketopiperazine ( dkp ) derivative , the cyclo ( l - phe , l - pro ) diketopiperazine . dkp active molecules form a very important family because of their many potential uses as antibacterial , fungicidal , herbicidal , antiviral , immune - suppressor , and antitumor agents , and therefore they are very attractive for the production of novel hybrid active compounds by combinatorial biosynthesis . among the five active molecules characterised from the streptomyces sp . tn 58 , two belong to the rhamnopyranoside family : the 1-o-(2-aminobenzoyl)--l - rhamnoside and the 4-hydroxybenzoyl -l - rhamnopyranoside . these two active compounds possess inhibitory activity towards 3-hydroxysteroid dehydrogenase ( 3-hsd ) , a useful target for anti - inflammatory and antiphlogistic drugs . it should be noted that until now , the streptomyces sp . tn 58 is the only described strain which produces these two molecules directly without any supplement addition into the culture media . identification and expression of genes involved in biosynthetic pathways of these molecules constitute a real opportunity for the comprehension of their natural biosynthesis and subsequently the production of hybrid molecules . the aim of this work was thus to succeed in carrying out intergeneric conjugation between e. coli et12567 and streptomyces sp . us 24 and tn 58 strains , to analyse the integration sites of the integrative vector pset152 into the chromosome of these two streptomyces strains , to study whether the site - specific integration of pset152 affects essential functions such as the production of active molecules in these two studied strains , and to verify the expression of a heterologous gene in streptomyces sp . e.coli strain et12567 ( puz8002 ) [ 2 , 5 ] , used as the donor in intergeneric conjugation , is a methylation - defective strain ( dam-13 : : tn9 dcm-6 hsdm cmr ) . e. coli strain dh5 ( f80 dlaczm15 (laczya - argf ) u169 enda1 reca1hsdr17 ( rk , mk ) deor thi-1 suse44 gyra96 rela1 ) and e. coli hb101 ( fhsds2 reca13 ara14 pro a2 lacy1 galk2 rps l20 xyl 5 mtl sup e44 ) were used as host strain , and e. coli atcc 8739 and micrococcus luteus lb 14110 were used as indicator micro - organisms for the antibacterial activity assays . the two streptomyces strains us 24 and tn 58 were used as recipient for intergeneric conjugation . cbs4 strain was the glucose isomerase deficient streptomyces violaceoniger in which the xyla gene of the streptomyces sk strain , under the control of the erme - up constitutive promoter , was integrated in its chromosome via the integrative vector pts55 . pset152 is an integrative plasmid carrying c31 int and att functions and an apramycin resistance gene for selection in streptomyces and e. coli . pgem - t easy vector is a ta cloning vector amp ( promega ) used for cloning of the pcr products . ampicillin ( 50 g ml ) , chloramphenicol ( 25 g ml ) , nalidixic acid , and kanamycin ( 50 g ml ) were added to growth media when required and 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ( 40 g ml ) when appropriate . transformation of e. coli dh5 or hb101 with pgem - t pset152 derivatives was carried out according to the manufacturer 's instructions ( promega ) . 24 and tn 58 strains were grown in tryptone soya broth ( tsb : 30 g tryptic soy broth plus 5 g yeast extract per 1000 ml distilled water ) medium for the preparation of genomic dna and in r2ye plates for the preparation of spore stocks . tsb supplemented with 30 g of apramycin ml was used to grow exconjugants for the preparation of genomic dna and to maintain them . as1 medium ( 1 g yeast extract , 5 g soluble starch , 0.2 g l - alanine , 0.2 g l - arginine , 0.5 g l - asparagine , 2.5 g nacl , 10 g na2so4 and 20 g agar , ph adjusted at 7.5 with koh ) was used for conjugation experiments . for bioactive molecules production , streptomyces sp . us 24 strain and the corresponding exconjugants were grown in tsb medium , supplemented at 1% ( w / v ) with starch and with a trace element solution : ( 7.5 ml l final ; 0.4 g l zncl2 ; 2 g l feso47h2o ; 0.065 g l h3bo3 and 0.135 g l mona2o42h2o ) . streptomyces tn 58 strain and corresponding exconjugants were grown in tsb medium , supplemented with glycerol at 1% ( w / v ) and with potassium at 1 mmol l. for antibacterial activity determination , indicator microorganisms were grown overnight in lb medium at 30c for m. luteus lb14110 and at 37c for e. coli atcc 8739 , then diluted 1 : 100 in lb medium and incubated for 5 hours under constant agitation at 200 rpm at the appropriate temperature . for growth study of the two bacteria : streptomyces tn 58/xyla and cbs4 , spores at 10 ml of each strain were used to inoculate 1000 ml erlenmeyer flasks with four indents , containing 200 ml of minimum liquid medium ( [ nh4]2so4 2.5 g l , mgso4 0.3 g l , cacl2 0.2 g l , kh2po4 1 g l , yeast extract 1 g l and 1 ml of trace element solution ) in presence of xylose as solely carbon source at 10 g l. biomass of the two studied streptomyces strains was determined at different growth times by measurement by weighing to constant dry weight after drying at 105c . intergeneric conjugation between e. coli and streptomyces us 24 and tn 58 strains was performed as described previously by flett et al . , with minor modifications . e. coli et12567 ( puz8002/pset152 ) was grown to an absorbance of 0.40.6 at 600 nm . the cells were pelleted by centrifugation , washed twice in an equal volume of lb , pelleted again , and finally resuspended in 1/10 volume of lb . aliquots of the two studied streptomyces strain spores suspension stored at 20c were used as recipients . spores ( eq10 ) were washed in 2 yeast extract tryptone medium , resuspended in 500 l of 2 yeast extract tryptone medium , and incubated at 50c for 10 minutes to induce germination . donor cells ( 500 l approximately 10 cells ) were added to the treated spores , the mixture was pelleted by centrifugation , and finally the pellets were resuspended in the residual liquid , and the undiluted mixtures were plated . the mating mixtures were spread on as1 plates containing 10 mm mgcl2 and incubated for 18 hours at 37c for the streptomyces us 24 and at 30c for the streptomyces tn 58 strain . the plates were overlaid each with 1 ml of water containing 500 g of nalidixic acid and 1 mg of apramycin , incubated further for 5 days at the appropriate temperature , and the exconjugants were counted . control experiments were performed as described above but without the addition of e. coli donor cells . the viable count of the donor culture was determined by spreading the cells on lb agar plates supplemented with appropriate antibiotics . small - scale plasmid preparations from e. coli were performed as described by . digestion with restriction endonucleases , and separation of dna fragments by agarose gel electrophoresis , alkaline calf intestinal phosphatase , ligation of dna fragments , and transformation were done according to for e. coli and for streptomyces . for blot manipulations , about 1 g and 50 ng were loaded for the digested genomic dna and plasmids dna , respectively . p - labeled probes were prepared using the random prime labelling system ( amersham ) . pcr amplification of the attb sites of the two studied streptomyces strains was performed using the two primers attb1 and attb2 previously described by . approximately 200 ng genomic template dna was used with 100 pmol of each primer per 50 l reaction volume . to improve the denaturation of the dna , 5% ( v / v ) dmso amplifications were performed in a gene amp pcr system 2700 ( applied biosystems ) using 1u pfu dna polymerase ( stratagene ) and the recommended buffer system according to the following amplification profile : 94c ( 5 minutes ) followed by 45 cycles of denaturation at 94c ( 30 seconds ) , annealing at 60c ( 1 minute ) and extension at 72c ( 1 minute ) . the pcr reaction mix was analysed by agarose gel electrophoresis , and the products having the expected sizes were purified then cloned into pgem - t easy vector . reactions were performed with a thermo sequenase cycle sequencing kit ( amersham ) and specific primers . for the extraction of the active molecules from the streptomyces sp . us 24 and tn 58 strains and their corresponding exconjugants ( four exconjugants randomly chosen for each strain ) , spores at 10 ml were used to inoculate 500 ml erlenmeyer flasks with four indents containing 100 ml of culture medium . after incubation for 24 hours in an orbital incubator with shaking at 250 rpm at the appropriate temperature , the preculture was used to inoculate ( 5% v / v ) a total volume of 1000 ml culture medium having the same composition of the preculture . after three days incubation at 37c or 30c for streptomyces us 24 and tn 58 strains , respectively , and their corresponding exconjugants , in an orbital incubator with shaking at 250 rpm , the culture broths were filtered to separate mycelium and supernatant . each supernatant was extracted twice with an equal volume of ethyl acetate and then evaporated on a rotavapor ( laborata 4000 ) , and obtained crude extract was dissolved in 1 ml ethyl acetate and used for antibacterial activities as follows : a paper disk was impregnated with 80 l of the corresponding sample and then laid on the surface of an agar plates containing 3 ml of top agar inseeded by 40 l of a 5-hour old culture of m. luteus lb 14110 or e. coli atcc 8739 . after 2 hours at 4c , plates containing m. luteus were incubated at 30c and those inoculated with e. coli at 37c , all for overnight . plates were examined for evidence of antibacterial activities represented by a zone of inhibition of growth of the corresponding indicator micro - organisms around the paper disk . thin layer chromatography ( tlc ) was performed on silica gel plates sio2 ( merck ) . sk placed under the control of the erme - up constitutive promoter from pmm6 was cloned into the streptomyces integrative vector pset152 linearised by bamhi , leading to the pss6 vector construct . this plasmid was firstly obtained within e. coli hb101 strain and then transferred to e. coli et12567/puz8002 for the construction of the streptomyces tn 58/xyla strains by conjugal transfer . preparation of the cell - free lysate of the streptomyces sk , tn 58 , tn 58/pset152 , tn 58/pss6 , and cbs4 ( used as positive control ) strains was achieved as follows . the cells were grown in tsb medium for 48 hours and were harvested by centrifugation at 8000 rpm for 10 minutes , and the pellets were suspended in te - buffer with 10 mm mgcl2 and 1 mm cocl2 . after incubation for an hour on ice in the presence of 5 mgml lysozyme , 100 gml pmsf , and 1 gml pepstatin a , cells were disrupted by sonication at 4c for 6 minutes ( pulsations of 3 seconds , amplify 90 ) using a vibra cell sonicator ( fisher - bioblock scientific ) , and debris were removed by centrifugation at 20 000 rpm for 30 minutes . glucose isomerase activity was determined using fructose as substrate by quantitatively measuring the glucose production . the activity was assayed in a reaction mixture containing the enzyme ( 100 l of an appropriate diluted purified or crude extract ) with 10 mm mgcl2 , 1 mm cocl2 and 15% fructose , in a volume of 400 l . in standard condition , assays were incubated for 30 minutes at 80c , and the reaction was stopped by cooling the tubes on ice . the amount of glucose generated was determined by glucose - oxidase ( god - pap ) enzyme system , and a650 was measured after 40 minutes at room temperature . one unit of glucose isomerase activity is defined as the amount of enzyme needed to produce 1 mol of product per minute under the assay conditions . protein concentration was determined by bradford 's method using the bovine serum albumin as standard . the plasmid pset152 was mobilized from e. coli et12567 ( puz8002 ) into streptomyces sp . us 24 and tn 58 strains . exconjugants were obtained at a frequency of approximately 5 10 and 3 10 per recipient spore of streptomyces sp . us 24 and tn 58 strains , respectively , which showed a high frequency of exconjugants . indeed , nikodinovic et al . , consider that the transformation efficiency ( 5 10 exconjugants recipient ) of the streptomyces nodosus by conjugal transfer of dna from e. coli was a high transformation frequency . the integrative plasmid pset152 can not replicate in streptomyces ; so stable exconjugants should be obtained only if pset152 has been integrated into the chromosome of studied strain . in order to identify the copy number of pset152 in the recipient streptomyces sp . chromosomal dnas were extracted , digested by bamhi and probed with the whole linearised vector pset152 in a blot analysis . since pset152 had a single bamhi site , dna from exconjugants was expected to show hybridisation of two fragments to probe in the case of single copy integration . for streptomyces us24 exconjugants , obtained hybridisation patterns were identical and showed the presence of three bands of about 5.7 , 6.2 , and 7.8 kb ( figure 1(a ) ) . the same probe did not hybridise to any dna fragment of the wild type genomic dna . this result implied that the plasmid was presumably integrated at the same locus in all corresponding exconjugants . however , among the three obtained bands , the one of 5.7 kb corresponds exactly to the size of the linearized vector pset152 . this fact can be explained via the tandem integration of two copies of the pset152 vector in the chromosome of streptomyces sp . the presence of tandemly repeated copies of pset152 vector in the attb site can be explained by the property of pset152 to integrate at attr or attl sites via attp / attr or attp / attl recombination . so , in certain cases , we can assist to the multiple independent recombination events , and pset152 can integrate at attl or attr sites via attp / attl or attp / attr . , if this event occurs , this reaction must be specific to the attl and attr sites as tandem repeats of pset152 in the pseudo - attb sites were not generally observed . alternatively , the generation of tandem repeats of pset152 in the attb may be a consequence of the rate of integration . if the rate of recombination into the attb site is rapid , integration occurs early during the mating period , and when the hyphae containing integrated pset152 receives a further copy of pset152 , this last one may , generally , integrate by homologous recombination to generate a tandem repeat . concerning streptomyces tn 58 , two identical hybridisation signals were obtained for all eight analysed exconjugants corresponding to two dna fragments of about 6.8 and 8 kb ( figure 2(a ) ) . this result implied that a single copy of the pset152 plasmid was integrated at the same locus in all the exconjugants in strain streptomyces sp . tn 58 . we can suggest that the chromosome of this strain contains a single functional attb site for vectors that integrate site specifically using the bacteriophage c31 att / int system ( figure 2(b ) ) . all together , our data strongly suggest the presence of an efficiently recognised attb site in the chromosome of the two studied streptomyces strains . 24 and tn 58 strains chromosomes implies the existence of one attb site recognised by the c31 integrase . taking advantage of the two pcr primers ( attb1 and attb2 ) described by combes et al . , , we recovered a unique pcr product at the expected size of approximately 0.3 kb from the chromosomal dnas of the two studied strains . these fragments were cloned into the pgem - t easy vector yielding pss4 and pss5 plasmids for us 24 and tn 58 strains , respectively . the two inserts possess a size of 292 bp and 293 bp for us 24 and tn 58 strains , respectively , and a high nucleotide identity of 86% ( figure 3 ) . these two amplified dna fragments encode a homolog of the streptomyces coelicolor a3(2 ) sco3798 orf with a nucleotide identity of 92% and 86% for streptomyces this orf is a putative chromosome condensation protein , with sequence similarity to a mammalian - encoded protein , pirin ( an identified nuclear protein that interacts with bcl-3 and nuclear factor i ) , in which the attb site of c31 lies in various streptomyces species . for streptomyces species , all characterised c31 attb sites showed high nucleotide sequence similarity varying from 82.4% to 100% however , the presence of pseudo - attb sites ( 50%70.3% nt identity to attb sites ) into which integration occurred at a frequency 300 times lower than into the corresponding attb site has also been reported in this genus of bacteria . the multiple - sequence alignment of attb sites of streptomyces sp . us 24 and tn 58 with several other streptomyces species is shown in figure 4 . this alignment indicates that these sequences are conserved , and some positions in the minimal site can tolerate nucleotide changes . kuhstoss and rao reported , by comparing the s. ambofaciens attb site with attp , that the core sequence ( i.e. , the region at which the crossover occurs ) is 5ttg . combes et al . , had shortened this sequence to two nucleotides 5tt which was present in the attb site of both studied strains streptomyces sp . us it has been reported that integration of the integrative vectors into the c31 attb site can cause detrimental effects on antibiotic production in some strains . in fact , it should be considered that the decrease in antibiotics production for many streptomyces strains carrying integrated site specific plasmids such as s. fradiae producer of tylosin , s. kanamyceticus producer of kanamycin and others has been reported . therefore , while integrative vectors could be used for the development of stable recombinant producers of antibiotics , their possible negative effect on the level of antibiotic production should be considered . thus , in order to determine whether the site - specific integration of the plasmid pset152 affected relevant functions of streptomyces sp . us 24 and streptomyces sp . tn 58 strains , we have followed the growth of both strain and their corresponding exconjugants carrying plasmid pset152 . all exconjugants grew well in the minimal medium , and there was no difference compared to the wild type strains . the presence of pset152 had no effects on growth and sporulation of the two strains . this stability was determined as the proportion of colonies that retained resistance to apramycin ( at 50 g ml ) after several passages of the exconjugants under nonselective conditions . plasmid pset152 was inherited every time for the all studied exconjugants under nonselective conditions for both strains . meanwhile , antimicrobial activity was determined by observing bacterial growth inhibition of the wild type strains ( us 24 and tn 58 ) and corresponding exconjugants . us 24 and streptomyces sp . tn 58 and their corresponding exconjugants ( figure 5 and table 1 ) . in addition , the analysis on tlc plates of the ten studied active crude extracts revealed the presence of the expected active molecules in each strain and its corresponding exconjugants . for the streptomyces us 24 strain and its four studied exconjugants , two bands were easily detected on tlc plates under uv light at 254 nm after spraying with anisaldehyde / sulphuric acid . the first band gives an orange colouration with a retention factor , rf = 0.46 ( chcl3/meoh 10% ) and the second one a violet colouration , rf = 0.37 ( ch2cl2/9% meoh ) . according to our previously work , these two bands correspond to the 3-indolethanol and the cyclo ( l - phe , l - pro ) diketopiperazine active molecules , respectively . concerning the streptomyces sp . tn 58 strain and its studied exconjugants , the five active molecules ( m1 to m5 ) were present in all five crude extracts . m1 belongs to the acetyltryptamine family and gives a colouration orange and violet after anisaldehyde / sulphuric acid and erhlich 's reagent visualisation . m2 , rf = 0.57 ( ch2cl2/1% meoh ) , and m3 , rf = 0.47 , ( chcl3/5% meoh ) belong to the thiazole and brevinamide families , respectively . m2 gives a chestnut colouration on spraying with anisaldehyde / sulphuric acid and m3 gives a pinkish colouration after erhlich 's reagent visualisation . the two other active molecules ( m4 and m5 ) characterised from the streptomyces sp . tn 58 strain , and belonging to the rhamnopyranosides family , do not have antimicrobial activities , but they possess inhibitory activity towards 3-hydroxysteroid dehydrogenase ( 3-hsd ) . indeed , the rhamnosylated aromatic compounds were easily detected on tlc plates due to their striking yellow / greenish colouration after visualisation with anisaldehyde vapour . for all five analysed crude extracts , the wild type one ( from the streptomyces sp . tn 58 strain ) and those from the four corresponding exconjugants , we obtained similar tlc migration profiles and two yellow / greenish bands having a retention factor ( chcl3/15% meoh ) of 0.22 and 0.33 corresponding to the two molecules belonging to the rhamnopyranoside family . for the five active molecules of the streptomyces sp . tn 58 and its four studied exconjugants , tlc profiles , bands colouration , and retention factors of the different active compounds were in perfect concordance with the results of previously works . these data clearly demonstrated that the morphological differentiation and active molecule production of all studied pset152 integrated exconjugants were similar to those of wild type streptomyces sp . consequently , conjugal transfer using attp / b site integration can be concluded to be a suitable means of gene transfer and expression for both studied strains . the establishment of a gene transfer system and the stable integration of cloned genes at the specific loci in the genome of streptomyces sp . xylose isomerase ( d - xylose ketol isomerase ec 5.3.1.5 ) catalyses the reversible isomerisation of d - xylose into d - xylulose . it is also referred to as glucose isomerase ( gi ) because of its ability to convert d - glucose to d - fructose . this property is widely exploited industrially for the production of high - fructose syrup from starch . sp sk , and we attempted to examine whether this gene could express and fold effectively in strain tn 58 . it should be noted that the wild type streptomyces tn 58 strain can not assimilate the xylose as solely carbon source . based on intergeneric conjugal transfer , transformation efficiency of strain tn 58 by plasmid pss6 was achieved at high frequency ( 10 exconjugants recipient ) . the chromosomal structure of three tn 58/xyla strains , arbitrary chosen , was checked by southern blot hybridisation using the 800 bp psti dna fragment internal to pset152 as probe , which contain the orit region . as expected , a unique and identical large bamhi band was obtained for the three tn 58/xyla strains chromosomal dna , whereas wild type strain tn 58 chromosomal dna does not hybridise to the probe ( data not shown ) . one of the three recombinant strains studied was subcloned five times on a solid media in the absence of apramycin followed by cultivation for several generations in liquid media in the absence of selection pressure . the plating of this culture on solid media , on both with and without selective pressures , showed that 100% of the colonies were apramycin resistant and able to grow in the presence of xylose as solely carbon source . determination of the mycelial intracellular glucose isomerase activity of streptomyces tn 58 , streptomyces tn 58/pset152 , streptomyces sk , cbs4 , and tn 58/xyla strains shows that glucose isomerase activity of the tn 58/xyla recombinant strain is of about eighteenfold higher than that of the sk strain and comparable to that of cbs4 strain . no activity was detected for the wild type tn 58 strain and the recombinant strain tn 58/pset152 ( table 2 ) . study of the growth of the two strains , streptomyces tn 58/xyla and cbs4 , in minimum liquid media containing xylose as solely carbon source showed that the two strains grew well , and for each time , the resulting biomass was quite similar . it should be noted that glucose isomerase activity is correlated with biomass production , and optimum activity has been obtained after 48 hours of incubation . according to these results , we can deduce that site - specific recombination directed by pset152 makes it possible to stably insert heterologous dna in strain tn 58 . this should be very important for a number of cases such as genes expression , genetic complementation , and biosynthetic pathway manipulations of the active molecules of this strain especially those belonging to the rhamnopyranoside family .

streptomyces sp . us 24 and streptomyces sp . tn 58 , two strains producing interesting bioactive molecules , were successfully transformed using e. coli et12567 ( puz8002 ) , as a conjugal donor , carrying the integrative plasmid pset152 . for the streptomyces sp . us 24 strain , two copies of this plasmid were tandemly integrated in the chromosome , whereas for streptomyces sp . tn 58 , the integration was in single copy at the attb site . plasmid pset152 was inherited every time for all analysed streptomyces sp . us 24 and streptomyces sp . tn 58 exconjugants under nonselective conditions . the growth , morphological differentiation , and active molecules production of all studied pset152 integrated exconjugants were identical to those of wild type strains . consequently , conjugal transfer using pset152 integration system is a suitable means of genes transfer and expression for both studied strains . to validate the above gene transfer system , the glucose isomerase gene ( xyla ) from streptomyces sp . sk was expressed in strain streptomyces sp . tn 58 . obtained results indicated that heterologous glucose isomerase could be expressed and folded effectively . glucose isomerase activity of the constructed tn 58 recombinant strain is of about eighteenfold higher than that of the streptomyces sp . sk strain . such results are certainly of importance due to the potential use of improved strains in biotechnological process for the production of high - fructose syrup from starch .

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

at the end of the last century , in addition to the conventional screening of naturally occurring bioactive compounds , a strategy was developed to construct desired novel hybrid antibiotics by combinatorial biosynthesis , by specifically combining genes from various biosynthetic pathways originating from different antibiotics producing organisms . in streptomyces species , the most important producers of bioactive molecules , genetic approaches to improve secondary metabolite production are generally hampered by various phenomena such as restriction barriers , absence of an efficient gene transfer systems , and lack of suitable cloning vectors . recently , in order to circumvent these problems , there has been considerable interest in the use of intergeneric conjugation as an efficient means of gene transfer and expression in streptomyces species . since then , this method has been successfully applied to several streptomyces species [ 24 ] via methylation - deficient e. coli strains , such as strain et12567 , as a dna donor to avoid methylated dna restriction systems of actinomycetes . in this genus of bacteria , the presence of pseudo - attb sites ( 50%70.3% nt identity to attb sites ) has also been reported , but with an integration frequency 300 times lower than that with the corresponding attb site . however , the absence of this attb site from some actinomycete strains , such as saccharopolyspora erythraea , has also been reported . us 24 and streptomyces sp . tn 58 , producing diverse interesting biological activities , and described the purification and structure elucidation of two and five active molecules from the streptomyces sp . dkp active molecules form a very important family because of their many potential uses as antibacterial , fungicidal , herbicidal , antiviral , immune - suppressor , and antitumor agents , and therefore they are very attractive for the production of novel hybrid active compounds by combinatorial biosynthesis . among the five active molecules characterised from the streptomyces sp . tn 58 , two belong to the rhamnopyranoside family : the 1-o-(2-aminobenzoyl)--l - rhamnoside and the 4-hydroxybenzoyl -l - rhamnopyranoside . it should be noted that until now , the streptomyces sp . identification and expression of genes involved in biosynthetic pathways of these molecules constitute a real opportunity for the comprehension of their natural biosynthesis and subsequently the production of hybrid molecules . the aim of this work was thus to succeed in carrying out intergeneric conjugation between e. coli et12567 and streptomyces sp . us 24 and tn 58 strains , to analyse the integration sites of the integrative vector pset152 into the chromosome of these two streptomyces strains , to study whether the site - specific integration of pset152 affects essential functions such as the production of active molecules in these two studied strains , and to verify the expression of a heterologous gene in streptomyces sp . e.coli strain et12567 ( puz8002 ) [ 2 , 5 ] , used as the donor in intergeneric conjugation , is a methylation - defective strain ( dam-13 : : tn9 dcm-6 hsdm cmr ) . e. coli strain dh5 ( f80 dlaczm15 (laczya - argf ) u169 enda1 reca1hsdr17 ( rk , mk ) deor thi-1 suse44 gyra96 rela1 ) and e. coli hb101 ( fhsds2 reca13 ara14 pro a2 lacy1 galk2 rps l20 xyl 5 mtl sup e44 ) were used as host strain , and e. coli atcc 8739 and micrococcus luteus lb 14110 were used as indicator micro - organisms for the antibacterial activity assays . the two streptomyces strains us 24 and tn 58 were used as recipient for intergeneric conjugation . cbs4 strain was the glucose isomerase deficient streptomyces violaceoniger in which the xyla gene of the streptomyces sk strain , under the control of the erme - up constitutive promoter , was integrated in its chromosome via the integrative vector pts55 . 24 and tn 58 strains were grown in tryptone soya broth ( tsb : 30 g tryptic soy broth plus 5 g yeast extract per 1000 ml distilled water ) medium for the preparation of genomic dna and in r2ye plates for the preparation of spore stocks . for bioactive molecules production , streptomyces sp . us 24 strain and the corresponding exconjugants were grown in tsb medium , supplemented at 1% ( w / v ) with starch and with a trace element solution : ( 7.5 ml l final ; 0.4 g l zncl2 ; 2 g l feso47h2o ; 0.065 g l h3bo3 and 0.135 g l mona2o42h2o ) . streptomyces tn 58 strain and corresponding exconjugants were grown in tsb medium , supplemented with glycerol at 1% ( w / v ) and with potassium at 1 mmol l. for antibacterial activity determination , indicator microorganisms were grown overnight in lb medium at 30c for m. luteus lb14110 and at 37c for e. coli atcc 8739 , then diluted 1 : 100 in lb medium and incubated for 5 hours under constant agitation at 200 rpm at the appropriate temperature . intergeneric conjugation between e. coli and streptomyces us 24 and tn 58 strains was performed as described previously by flett et al . e. coli et12567 ( puz8002/pset152 ) was grown to an absorbance of 0.40.6 at 600 nm . donor cells ( 500 l approximately 10 cells ) were added to the treated spores , the mixture was pelleted by centrifugation , and finally the pellets were resuspended in the residual liquid , and the undiluted mixtures were plated . the mating mixtures were spread on as1 plates containing 10 mm mgcl2 and incubated for 18 hours at 37c for the streptomyces us 24 and at 30c for the streptomyces tn 58 strain . the plates were overlaid each with 1 ml of water containing 500 g of nalidixic acid and 1 mg of apramycin , incubated further for 5 days at the appropriate temperature , and the exconjugants were counted . digestion with restriction endonucleases , and separation of dna fragments by agarose gel electrophoresis , alkaline calf intestinal phosphatase , ligation of dna fragments , and transformation were done according to for e. coli and for streptomyces . for the extraction of the active molecules from the streptomyces sp . us 24 and tn 58 strains and their corresponding exconjugants ( four exconjugants randomly chosen for each strain ) , spores at 10 ml were used to inoculate 500 ml erlenmeyer flasks with four indents containing 100 ml of culture medium . after incubation for 24 hours in an orbital incubator with shaking at 250 rpm at the appropriate temperature , the preculture was used to inoculate ( 5% v / v ) a total volume of 1000 ml culture medium having the same composition of the preculture . after three days incubation at 37c or 30c for streptomyces us 24 and tn 58 strains , respectively , and their corresponding exconjugants , in an orbital incubator with shaking at 250 rpm , the culture broths were filtered to separate mycelium and supernatant . each supernatant was extracted twice with an equal volume of ethyl acetate and then evaporated on a rotavapor ( laborata 4000 ) , and obtained crude extract was dissolved in 1 ml ethyl acetate and used for antibacterial activities as follows : a paper disk was impregnated with 80 l of the corresponding sample and then laid on the surface of an agar plates containing 3 ml of top agar inseeded by 40 l of a 5-hour old culture of m. luteus lb 14110 or e. coli atcc 8739 . sk placed under the control of the erme - up constitutive promoter from pmm6 was cloned into the streptomyces integrative vector pset152 linearised by bamhi , leading to the pss6 vector construct . this plasmid was firstly obtained within e. coli hb101 strain and then transferred to e. coli et12567/puz8002 for the construction of the streptomyces tn 58/xyla strains by conjugal transfer . preparation of the cell - free lysate of the streptomyces sk , tn 58 , tn 58/pset152 , tn 58/pss6 , and cbs4 ( used as positive control ) strains was achieved as follows . after incubation for an hour on ice in the presence of 5 mgml lysozyme , 100 gml pmsf , and 1 gml pepstatin a , cells were disrupted by sonication at 4c for 6 minutes ( pulsations of 3 seconds , amplify 90 ) using a vibra cell sonicator ( fisher - bioblock scientific ) , and debris were removed by centrifugation at 20 000 rpm for 30 minutes . glucose isomerase activity was determined using fructose as substrate by quantitatively measuring the glucose production . the plasmid pset152 was mobilized from e. coli et12567 ( puz8002 ) into streptomyces sp . us 24 and tn 58 strains . us 24 and tn 58 strains , respectively , which showed a high frequency of exconjugants . , consider that the transformation efficiency ( 5 10 exconjugants recipient ) of the streptomyces nodosus by conjugal transfer of dna from e. coli was a high transformation frequency . the integrative plasmid pset152 can not replicate in streptomyces ; so stable exconjugants should be obtained only if pset152 has been integrated into the chromosome of studied strain . for streptomyces us24 exconjugants , obtained hybridisation patterns were identical and showed the presence of three bands of about 5.7 , 6.2 , and 7.8 kb ( figure 1(a ) ) . however , among the three obtained bands , the one of 5.7 kb corresponds exactly to the size of the linearized vector pset152 . this fact can be explained via the tandem integration of two copies of the pset152 vector in the chromosome of streptomyces sp . the presence of tandemly repeated copies of pset152 vector in the attb site can be explained by the property of pset152 to integrate at attr or attl sites via attp / attr or attp / attl recombination . alternatively , the generation of tandem repeats of pset152 in the attb may be a consequence of the rate of integration . if the rate of recombination into the attb site is rapid , integration occurs early during the mating period , and when the hyphae containing integrated pset152 receives a further copy of pset152 , this last one may , generally , integrate by homologous recombination to generate a tandem repeat . concerning streptomyces tn 58 , two identical hybridisation signals were obtained for all eight analysed exconjugants corresponding to two dna fragments of about 6.8 and 8 kb ( figure 2(a ) ) . this result implied that a single copy of the pset152 plasmid was integrated at the same locus in all the exconjugants in strain streptomyces sp . we can suggest that the chromosome of this strain contains a single functional attb site for vectors that integrate site specifically using the bacteriophage c31 att / int system ( figure 2(b ) ) . all together , our data strongly suggest the presence of an efficiently recognised attb site in the chromosome of the two studied streptomyces strains . 24 and tn 58 strains chromosomes implies the existence of one attb site recognised by the c31 integrase . , , we recovered a unique pcr product at the expected size of approximately 0.3 kb from the chromosomal dnas of the two studied strains . these fragments were cloned into the pgem - t easy vector yielding pss4 and pss5 plasmids for us 24 and tn 58 strains , respectively . the two inserts possess a size of 292 bp and 293 bp for us 24 and tn 58 strains , respectively , and a high nucleotide identity of 86% ( figure 3 ) . these two amplified dna fragments encode a homolog of the streptomyces coelicolor a3(2 ) sco3798 orf with a nucleotide identity of 92% and 86% for streptomyces this orf is a putative chromosome condensation protein , with sequence similarity to a mammalian - encoded protein , pirin ( an identified nuclear protein that interacts with bcl-3 and nuclear factor i ) , in which the attb site of c31 lies in various streptomyces species . for streptomyces species , all characterised c31 attb sites showed high nucleotide sequence similarity varying from 82.4% to 100% however , the presence of pseudo - attb sites ( 50%70.3% nt identity to attb sites ) into which integration occurred at a frequency 300 times lower than into the corresponding attb site has also been reported in this genus of bacteria . us 24 and tn 58 with several other streptomyces species is shown in figure 4 . , had shortened this sequence to two nucleotides 5tt which was present in the attb site of both studied strains streptomyces sp . us it has been reported that integration of the integrative vectors into the c31 attb site can cause detrimental effects on antibiotic production in some strains . thus , in order to determine whether the site - specific integration of the plasmid pset152 affected relevant functions of streptomyces sp . us 24 and streptomyces sp . tn 58 strains , we have followed the growth of both strain and their corresponding exconjugants carrying plasmid pset152 . all exconjugants grew well in the minimal medium , and there was no difference compared to the wild type strains . this stability was determined as the proportion of colonies that retained resistance to apramycin ( at 50 g ml ) after several passages of the exconjugants under nonselective conditions . plasmid pset152 was inherited every time for the all studied exconjugants under nonselective conditions for both strains . meanwhile , antimicrobial activity was determined by observing bacterial growth inhibition of the wild type strains ( us 24 and tn 58 ) and corresponding exconjugants . us 24 and streptomyces sp . in addition , the analysis on tlc plates of the ten studied active crude extracts revealed the presence of the expected active molecules in each strain and its corresponding exconjugants . for the streptomyces us 24 strain and its four studied exconjugants , two bands were easily detected on tlc plates under uv light at 254 nm after spraying with anisaldehyde / sulphuric acid . according to our previously work , these two bands correspond to the 3-indolethanol and the cyclo ( l - phe , l - pro ) diketopiperazine active molecules , respectively . tn 58 strain and its studied exconjugants , the five active molecules ( m1 to m5 ) were present in all five crude extracts . m2 , rf = 0.57 ( ch2cl2/1% meoh ) , and m3 , rf = 0.47 , ( chcl3/5% meoh ) belong to the thiazole and brevinamide families , respectively . the two other active molecules ( m4 and m5 ) characterised from the streptomyces sp . tn 58 strain , and belonging to the rhamnopyranosides family , do not have antimicrobial activities , but they possess inhibitory activity towards 3-hydroxysteroid dehydrogenase ( 3-hsd ) . for all five analysed crude extracts , the wild type one ( from the streptomyces sp . for the five active molecules of the streptomyces sp . tn 58 and its four studied exconjugants , tlc profiles , bands colouration , and retention factors of the different active compounds were in perfect concordance with the results of previously works . these data clearly demonstrated that the morphological differentiation and active molecule production of all studied pset152 integrated exconjugants were similar to those of wild type streptomyces sp . consequently , conjugal transfer using attp / b site integration can be concluded to be a suitable means of gene transfer and expression for both studied strains . the establishment of a gene transfer system and the stable integration of cloned genes at the specific loci in the genome of streptomyces sp . this property is widely exploited industrially for the production of high - fructose syrup from starch . sp sk , and we attempted to examine whether this gene could express and fold effectively in strain tn 58 . as expected , a unique and identical large bamhi band was obtained for the three tn 58/xyla strains chromosomal dna , whereas wild type strain tn 58 chromosomal dna does not hybridise to the probe ( data not shown ) . the plating of this culture on solid media , on both with and without selective pressures , showed that 100% of the colonies were apramycin resistant and able to grow in the presence of xylose as solely carbon source . determination of the mycelial intracellular glucose isomerase activity of streptomyces tn 58 , streptomyces tn 58/pset152 , streptomyces sk , cbs4 , and tn 58/xyla strains shows that glucose isomerase activity of the tn 58/xyla recombinant strain is of about eighteenfold higher than that of the sk strain and comparable to that of cbs4 strain . no activity was detected for the wild type tn 58 strain and the recombinant strain tn 58/pset152 ( table 2 ) . study of the growth of the two strains , streptomyces tn 58/xyla and cbs4 , in minimum liquid media containing xylose as solely carbon source showed that the two strains grew well , and for each time , the resulting biomass was quite similar . it should be noted that glucose isomerase activity is correlated with biomass production , and optimum activity has been obtained after 48 hours of incubation . this should be very important for a number of cases such as genes expression , genetic complementation , and biosynthetic pathway manipulations of the active molecules of this strain especially those belonging to the rhamnopyranoside family .

traditionally , the common model of oral health needs assessment depends almost entirely on professional opinions . normative need assessment refers to the impairments and diseases which expert , administrator or scientist defines as need . in spite of the usefulness of this concept in the estimation of people , procedures , and delivering services costs , it has considerable limitations . a major shortcoming of the normative approach is that it fails to take into account the broader concepts of health as it has been stated by world health organization ( who ) in 1948 , which have led to incorporate functional , psychological , and social well - being to almost all disciplines of health . more considerably , this method is criticized for unrealistic estimation of need for treatment planning , especially in developing countries with scarce oral health care recourses . demand for orthodontic treatment is mostly related to psychosocial factors like personal concern about appearance . it seems that in most patients , malocclusion is a deviation from a documented average rather than a disease , so distinction between acceptable and unacceptable occlusion is influenced by psychological and social factors . so a person 's feelings about his dental appearance should be essential in assessing the need and outcome of orthodontic treatment . on the other hand , deficiencies of normative need assessment are more serious in large - scale population studies and this method leads to a high estimation of treatment needs which is unlikely to be met by decision makers . therefore , a comprehensive system of assessing orthodontic need requires the integration of normative clinical measures with patient - based indicators of the individual 's emotion . the studies of gherunspong et al . , using oral impact on daily performance ( oidp ) and locker and jokovic using oral health impact profile ( ohip-49 ) indicated that diagnostic values of such measures are not good enough to work as screening tests . but evidently , they identified the individuals with some impacts from their conditions and consequently may conduct us to the sub - groups who could benefit more from dental treatments . perceived need to dental treatment and oral health - related quality of life ( ohrqol ) have been utilized as the subjective assessment measures in the literatures , previously . teenagers show a perception of dental beauty and orthodontic treatment need particularly attributes to their peers and environments . child oral health impact profile ( cohip ) is a psychometrically sound instrument intended to measure ohrqol among school - age children with different oral health conditions . as this instrument emphasizes on the social - emotional and school environment aspects of well - being in details , it seems that it is more appropriate to assess the quality of life in the period of adolescence . the aesthetic component of index of orthodontic treatment need ( ac - iotn ) is a standardized rating scale of dental attractiveness . it has been allocated for more than two decades as a tool in patient counseling ; assisting subjects to gain a realistic impression of their relative dental attractiveness . several studies have investigated the relationship between normative need assessors and ac - iotn for evaluating the patient 's perception . the findings support that the perception of orthodontic treatment need is a multi - factorial process and is influenced by some personal , cultural , and social factors . regarding the fact that adolescents from lower socio - economic levels usually report lower levels of self - perceived health , it seems crucial to consider the role of socio - economic status ( ses ) to correlate between professionally and self - assessed needs . in this study , we aimed to compare the diagnostic value of two instruments , cohip and ac - iotn , for self - perceived orthodontic need assessment and explore the role of some confounders in the relationship between professional and self - assessed need . the specific objectives of the study are : the prevalence of orthodontic treatment need ( otn ) in a sample of iranian adolescents by self - perceived and normative methods.the role of variables such as gender , age , ses , health behaviors , and oral health impacts in the similarity of the assessment about the treatment need by individuals and dentists.the evaluation of the diagnostic validity of cohip and self - perceived ac through the analysis of sensitivity , specificity , positive predictive value , and negative predictive value . the prevalence of orthodontic treatment need ( otn ) in a sample of iranian adolescents by self - perceived and normative methods . the role of variables such as gender , age , ses , health behaviors , and oral health impacts in the similarity of the assessment about the treatment need by individuals and dentists . the evaluation of the diagnostic validity of cohip and self - perceived ac through the analysis of sensitivity , specificity , positive predictive value , and negative predictive value . a cross - sectional survey was conducted in the 13 - 18-years - old school children in the city of isfahan , the second most populated city of iran . the sample size was calculated to be able to detect a difference of two in the score of oral health impacts by 90% power at 5% significance and considered 10% attrition rate . to obtain a representative random sample , divisions of the ministry of education and gender were determined as the variables for stratification . in each district , two boys and two girls schools were selected randomly from the electronically registered public schools list at the pertinent home page ( 20 schools ) . thereafter , 30 eligible students from each school were invited to the study by convenient sampling . the inclusion criteria were the age between 13 years and 18 years and having written parental informed consent . the students who had orthodontic appliances or reported a history of orthodontic treatment were excluded from the main study . the study was approved by the human research ethics committee of shahid beheshti university of medical sciences and isfahan university of medical sciences . also , legal process in the bureau of education , school administrations , and parent 's levels were passed . the dental health component ( dhc ) were recorded in five severity level from one no treatment need to five very great need after the assessments of all occlusion features ( overjet , overbite , reverse jet , anterior and posterior crossbite , anterior and lateral open bite , displacement of the teeth , and molar relationship ) . prior to the study , two dentists ( principle investigators ) were trained by a qualified orthodontist in the field of epidemiologic malocclusion studies and calibrated to achieve desirable agreement in the pilot study . according to the intra - class correlation coefficient ( icc ) , the intra - examiner reliability by one week interval was above 0.9 in two dentists . ac of the iotn was accounted by two schemes blindly ; self - assessed and professionally - assessed ac score . the participants and dentist , separately , evaluated overall dental attractiveness in according to 10 standard photos ranging from the most attractive ( score 1 ) to the least attractive ( score 10 ) appearance of a person . ten grade of ac was then transformed into three categories of no / slight need : ac = 1 - 4 , borderline : ac = 5 - 7 , and definite need : ac = 8 - 10 . the participants were also asked whether they thought they have bad - form or bad - sized teeth or whether the space between their teeth are irregular . the answers were coded in a 5-point likert scale as " 0 : never , 1 : almost never , 2 : sometimes , 3 : fairly often , 4 : all the time " . using the cut - off point of three , the responses then dichotomized to frequent or infrequent categories . to identify the oral health - related quality of life scores of the sample the instrument consist of 34 items in five subscales including oral health , functional well - being , social - emotional well - being , school environment and self - image which has been developed for school - aged children . participants describe their past three months experiences in the phrases ranging from 0 never to 4 almost all of the time ( e.g. in the past three months , how often have you had pain in your teeth ? ) . higher cohip scores reflect more positive ohrqol , while lower scores reflect lower ohrqol . to explore the factors determine the similar assessment of the need to orthodontic treatment by the individual and the dentist , a variable computed to detect the persons who reported the same scores on dhc grade and self - perceived ac grade . the primary grading as " no / slight need , borderline need and definite need " for each index used to create a dichotomous variable entitled similarity in the judgment . socioeconomic status was assessed based on the parents educational levels which were asked in five categories as illiterate , school without formal qualification , high school with diploma , undergraduate university level , and postgraduate university level . in further analysis , it was transformed into a binary variable as low : without academic qualification and high : with academic qualification . the statistical package for social sciences ( spss ) software version 15 was applied to analyze the data . to describe data , mean , standard deviation , proportion , and graphs were used . to investigate the relationship between background variables and the similarity of need treatment evaluation by participants and dentist , multiple logistic regressions was applied . to measure the results at the p - value level of 0.05 were considered as significant . to evaluate validity of subjective indicators in identifying those needing orthodontic treatment , sensitivity , specificity , positive predictive value , and negative predictive value were calculated . ac score equal to eight or more , cohip score below the median ( 107 ) or positive response to the teeth irregularity question were considered as patients by a self - assessment instrument . a cross - sectional survey was conducted in the 13 - 18-years - old school children in the city of isfahan , the second most populated city of iran . the sample size was calculated to be able to detect a difference of two in the score of oral health impacts by 90% power at 5% significance and considered 10% attrition rate . to obtain a representative random sample , divisions of the ministry of education and gender were determined as the variables for stratification . in each district , two boys and two girls schools were selected randomly from the electronically registered public schools list at the pertinent home page ( 20 schools ) . thereafter , 30 eligible students from each school were invited to the study by convenient sampling . the inclusion criteria were the age between 13 years and 18 years and having written parental informed consent . the students who had orthodontic appliances or reported a history of orthodontic treatment were excluded from the main study . the study was approved by the human research ethics committee of shahid beheshti university of medical sciences and isfahan university of medical sciences . also , legal process in the bureau of education , school administrations , and parent 's levels were passed . the dental health component ( dhc ) were recorded in five severity level from one no treatment need to five very great need after the assessments of all occlusion features ( overjet , overbite , reverse jet , anterior and posterior crossbite , anterior and lateral open bite , displacement of the teeth , and molar relationship ) . prior to the study , two dentists ( principle investigators ) were trained by a qualified orthodontist in the field of epidemiologic malocclusion studies and calibrated to achieve desirable agreement in the pilot study . according to the intra - class correlation coefficient ( icc ) , the intra - examiner reliability by one week interval was above 0.9 in two dentists . ac of the iotn was accounted by two schemes blindly ; self - assessed and professionally - assessed ac score . the participants and dentist , separately , evaluated overall dental attractiveness in according to 10 standard photos ranging from the most attractive ( score 1 ) to the least attractive ( score 10 ) appearance of a person . ten grade of ac was then transformed into three categories of no / slight need : ac = 1 - 4 , the participants were also asked whether they thought they have bad - form or bad - sized teeth or whether the space between their teeth are irregular . the answers were coded in a 5-point likert scale as " 0 : never , 1 : almost never , 2 : sometimes , 3 : fairly often , 4 : all the time " . using the cut - off point of three , the responses then dichotomized to frequent or infrequent categories . to identify the oral health - related quality of life scores of the sample , the instrument consist of 34 items in five subscales including oral health , functional well - being , social - emotional well - being , school environment and self - image which has been developed for school - aged children . participants describe their past three months experiences in the phrases ranging from 0 never to 4 almost all of the time ( e.g. in the past three months , how often have you had pain in your teeth ? ) . higher cohip scores reflect more positive ohrqol , while lower scores reflect lower ohrqol . to explore the factors determine the similar assessment of the need to orthodontic treatment by the individual and the dentist , a variable computed to detect the persons who reported the same scores on dhc grade and self - perceived ac grade . the primary grading as " no / slight need , borderline need and definite need " for each index used to create a dichotomous variable entitled similarity in the judgment . socioeconomic status was assessed based on the parents educational levels which were asked in five categories as illiterate , school without formal qualification , high school with diploma , undergraduate university level , and postgraduate university level . in further analysis , it was transformed into a binary variable as low : without academic qualification and high : with academic qualification . the statistical package for social sciences ( spss ) software version 15 was applied to analyze the data . to describe data , mean , standard deviation , proportion , and graphs were used . to investigate the relationship between background variables and the similarity of need treatment evaluation by participants and dentist , multiple logistic regressions was applied . to measure the results at the p - value level of 0.05 were considered as significant . to evaluate validity of subjective indicators in identifying those needing orthodontic treatment , sensitivity , specificity , ac score equal to eight or more , cohip score below the median ( 107 ) or positive response to the teeth irregularity question were considered as patients by a self - assessment instrument . twenty seven ( 4.5% ) of the students who had a history of previous orthodontic treatment were excluded , so complete data was gained from 570 ( 95% ) students . the mean age was 14.9 1.2 and 52.8% of them were female . according to the dhc grade 4 or 5 as the criteria ( gold standard ) for need to orthodontic treatment , 16.4% ( n = 92 ) of examined children had definite treatment need . a considerable number of children ( 90% ) perceived to have acceptable anterior teeth or ac grade1 - 4 . distribution of iotn scores in 13 - 18-year - old school children regarding patient 's and dentist 's perceptions ( n= 570 ) the mean ( sd ) cohip score of the population was 103.6 ( 18 ) . while it showed a range of 15 - 135 the prevalence of participants with at least one frequent impact was 66% , 34% , and 32% in the oral health , social - emotional , and self - image subscales , respectively . there were not any association between gender and age variables with the qol score ( p < 0.05 ) , but a gradient was seen in this score with the worsening malocclusion disorders . the cohip scores in different categories of the study population are brought in table 1 . mean and standard deviation ( sd ) of cohip scores in 13 - 18-year - old school children regarding the degree of need to orthodontic treatment and the history of orthodontic treatment ( n=570 ) twelve persons thought that they are in ac grade of eight and above , while only seven of them had definite normative need . half of the students did not need orthodontics by perceptional and normative assessment as well . however , 27% with borderline and 13% with definite need to treatment perceived well about themselves . as it is presented in tables 2 and 3 , the distribution of individuals in different categories has significant differences ( p > 0.001 ) by changing the indicators of need assessment . need to orthodontic treatment on the basis of dhc - iotn ( gold standard ) and subjective ( self - perceived ac - iotn ) instrument in 13 - 18-year - old school children ( n=562 ) in isfahan frequency of persons with different grades of orthodontic treatment need on the basis of professionally - assessed ac - iotn and subjective ( self - perceived ac - iotn ) instrument in 13 - 18-year - old school children ( n=562 ) in isfahan regarding the ac - iotn , a comparison between the persons and dentists evaluation of need showed poor / slight agreement level . table 4 illustrates the percentage of agreement and the kappa coefficient ( ) with considering the agreement occurring by chance . the agreements between the patients ( self - perceived ac ) and dentists ( dhc / normative ac ) regarding the judgments about the needs to orthodontic treatment logistic regression analysis revealed that students with no caries experience , better quality of life , higher father 's educational level , and brushing habits are more likely to have judgment comparable with dentists . however , gender , age , and mother 's education did not seem to have any role in this decision [ table 5 ] . multiple logistic regression to investigate the influences of the independent variables on the similar judgment in the participant and dentist about treatment need in 13 - 18-year - old school children in isfahan the diagnostic power of three subjective indicators with respect to normative need to orthodontics treatment was summarized in table 6 . in this sample , 61% ( 95% ci = 0.50 ; 0.71 ) of all the patients with definite need had a positive impact - related test ( sensitivity ) and the probability of negative cohip test result given no disease ( specificity ) was 50% ( 95% ci = 0.45 ; 0.55 ) . positive predictive value of 20% means that only one - fifth of those who had reported impact on life had need to treatment . it revealed that in our sample the probability of not having the condition is around 85% if the test is negative ; either the cohip or ac or single question is allocated . regarding a question on the irregular teeth , the strength of ac to detect the healthy ones showed to be excellent , but its low sensitivity disabled the recognition of the persons with serious need . diagnostic validity of subjective indicators in identifying those needing orthodontic treatment in 13 - 18-year - old school children in isfahan ( n=570 ) the findings of the present study clearly revealed a considerable difference between subjectively and normatively assessed needs relating to treatment of malocclusions in 13 - 18-year - old iranian adolescents . more than half of the students under assessment were defined as healthy by an expert while this rate was 90% when reported by the students themselves . it was in concordance with the previous studies which highlighted disagreement between two kinds of assessment on dento - facial irregularities . there was no significant difference in two genders either in their normative need to treatment or in the perception of need to treatment . it was in contrast with some studies which had shown subjective needs for aesthetic - related treatment were more frequent in the females . it is well approved that great concerns about appearance is the main factor for motivation to seek orthodontic treatment rather than function or health . this is a major worry in the adolescents because of the influences on their communications , and it is suggested that individuals satisfied with their body image tend to have more successful social contacts . but according to our findings , a considerable proportion of the students with definite need did not expressed any deficiency in their dental appearance . there may be two explanations ; the first one is that the appropriateness of ac index to detect the felt need in this age group is questionable . teenagers tend to show themselves in a perfect manner and do not tend to be in a flawed state . de oliveira and sheiham and mandall et al . also had concluded that ac was not sufficiently reliable to be used as a consumer measure in child and adolescents . second is that the dhc takes into account possible occlusal findings that could be functionally detrimental , but are not aesthetically important like posterior crossbite , posterior displacements , or increased overbite . also , it is good to mention that there are cultural and religious norms that play an important role in the mode of self - display in the youth in our country ; it could lead to fade the importance of other factors . while the utilization of quality of life measures has been encouraged in the oral health studies , it is emphasized particularly in the orthodontic treatment need assessments , recently . however , it has been shown that although the clinical condition have a distinct impact on individual 's life and the association between malocclusion and qol was confirmed , there are crucial problems to conjoining the results of these two perspectives . in this study , only 10% of all examined students had criteria of both affected qol and normative need together . similar to the finding of the study on brazilian adolescents,39% of the patients with grade 4 and 46% of whom with grade 3 of dhc - iotn did not report considerable impact on their quality of life . regarding administering a valid questionnaire to quantify the impacts of oral and dental problems on adolescent 's daily living , our assumption was that all domains of this instrument ( oral health , function , social - emotional , schooling , and self - image ) could be affected by malocclusion . moreover , study of dunlow indicated that cohip is sensitive to malocclusion and its subscales have an acceptable concurrency with dento - facial image , social anxiety , and self concept measures . but the present results revealed that this instrument is neither sensitive nor specific enough to detect the patients with malocclusions as a screening tool . this issue confirmed the previous reports which have mentioned that subjective instruments are not reliable in orthodontic need assessment because of their poor performance to screen clinical diseases . regarding the existing discrepancy between two methods of need evaluation , we tried to uncover the variables that played role in the process of judgment by patient 's perspective . while some factors such as ohrqol , dental caries history and father 's education increased the chance of same judgment on need , the coefficient of determination which was gained in the fitting models was 0.07 . it means that more than 90% of variability of judgment is accounted for the factors other than our tested variables which should be discovered by a qualitative research . in line with the multi - dimensional identity of health , individual 's perceptions are the outcomes of complex bio - psychosocial processes and they are affected by various moderating factors such as the person 's overall characteristics , living environment , health literacy , and so on . the main limitation of this study is the conceptual inflictions about the use of generic ohrqol instrument as measuring tool in otn . to remove all doubts , in addition , it should be thought that the implication of such measures in clinical or patient seeking settings is completely different from public setting . definition of the minimal clinically importance and cut - off point determination of subjective instruments was another issue in the procedure of need assessment . despite the advantages of integration of patient - centered outcome measuring , this approach in oral health need assessment needs to further research yet . this study demonstrated that regarding ac as a patient - based outcome , about 50% of the participants had no need on the dentist 's and individual 's belief . while in the other 50% with borderline need , the appearance was acceptable . using cohip , a high proportion of false positive cases has weakened any decision foundation for treatment ; however , individuals with an impact - related need can be prioritized for treatment in program planning . the frequency of definite need to orthodontics has decreased from 16.4% to 10% by application of impact - related need assessment .

background : a comprehensive system of assessing orthodontic need requires the integration of normative clinical measures with patient - based indicators . this study sought to discover weather an oral health - related quality of life measure or aesthetic component of index of orthodontic treatment need ( ac - iotn ) could be used as a predictor of orthodontic treatment need . factors affecting the judgment of patient and dentist about this need are discussed.materials and methods : oral examination on 597 iranian students between 13 years and 18 years was done to reach the grade of dental health component ( dhc ) . the child oral health impact profile ( cohip ) and ac - iotn were recorded . the diagnostic values of subjective tests were assessed . multiple logistic regressions were applied to investigate the role of variables in the persons perceptions.results:half of the 570 eligible students did not need orthodontic treatment either on professional or self - assessment ; 60% of patients with definite need had a distinct impact on their quality of life . the specificity of ac to detect the healthy persons was excellent ( 0.99 ) but its sensitivity was low ( 0.08 ) . cohip score gave a better sensitivity but its specificity was 50% . caries experience , quality of life , father 's education , and brushing habits were the factors relating to the same judgment of persons and dentists ( p > 0.02).conclusion : regarding the discrepancies between two assessment methods , present instruments did not meet the predictor 's competencies . the patient - based methods could not substitute the professional assessment , but by identification , the persons with higher impacts would benefit the prioritization process .

INTRODUCTION MATERIALS AND METHODS Study population Data collection Statistical analysis RESULTS DISCUSSION CONCLUSION

traditionally , the common model of oral health needs assessment depends almost entirely on professional opinions . normative need assessment refers to the impairments and diseases which expert , administrator or scientist defines as need . in spite of the usefulness of this concept in the estimation of people , procedures , and delivering services costs , it has considerable limitations . a major shortcoming of the normative approach is that it fails to take into account the broader concepts of health as it has been stated by world health organization ( who ) in 1948 , which have led to incorporate functional , psychological , and social well - being to almost all disciplines of health . demand for orthodontic treatment is mostly related to psychosocial factors like personal concern about appearance . so a person 's feelings about his dental appearance should be essential in assessing the need and outcome of orthodontic treatment . therefore , a comprehensive system of assessing orthodontic need requires the integration of normative clinical measures with patient - based indicators of the individual 's emotion . , using oral impact on daily performance ( oidp ) and locker and jokovic using oral health impact profile ( ohip-49 ) indicated that diagnostic values of such measures are not good enough to work as screening tests . but evidently , they identified the individuals with some impacts from their conditions and consequently may conduct us to the sub - groups who could benefit more from dental treatments . perceived need to dental treatment and oral health - related quality of life ( ohrqol ) have been utilized as the subjective assessment measures in the literatures , previously . teenagers show a perception of dental beauty and orthodontic treatment need particularly attributes to their peers and environments . child oral health impact profile ( cohip ) is a psychometrically sound instrument intended to measure ohrqol among school - age children with different oral health conditions . as this instrument emphasizes on the social - emotional and school environment aspects of well - being in details , it seems that it is more appropriate to assess the quality of life in the period of adolescence . the aesthetic component of index of orthodontic treatment need ( ac - iotn ) is a standardized rating scale of dental attractiveness . it has been allocated for more than two decades as a tool in patient counseling ; assisting subjects to gain a realistic impression of their relative dental attractiveness . several studies have investigated the relationship between normative need assessors and ac - iotn for evaluating the patient 's perception . the findings support that the perception of orthodontic treatment need is a multi - factorial process and is influenced by some personal , cultural , and social factors . regarding the fact that adolescents from lower socio - economic levels usually report lower levels of self - perceived health , it seems crucial to consider the role of socio - economic status ( ses ) to correlate between professionally and self - assessed needs . in this study , we aimed to compare the diagnostic value of two instruments , cohip and ac - iotn , for self - perceived orthodontic need assessment and explore the role of some confounders in the relationship between professional and self - assessed need . the specific objectives of the study are : the prevalence of orthodontic treatment need ( otn ) in a sample of iranian adolescents by self - perceived and normative methods.the role of variables such as gender , age , ses , health behaviors , and oral health impacts in the similarity of the assessment about the treatment need by individuals and dentists.the evaluation of the diagnostic validity of cohip and self - perceived ac through the analysis of sensitivity , specificity , positive predictive value , and negative predictive value . the prevalence of orthodontic treatment need ( otn ) in a sample of iranian adolescents by self - perceived and normative methods . the role of variables such as gender , age , ses , health behaviors , and oral health impacts in the similarity of the assessment about the treatment need by individuals and dentists . the evaluation of the diagnostic validity of cohip and self - perceived ac through the analysis of sensitivity , specificity , positive predictive value , and negative predictive value . a cross - sectional survey was conducted in the 13 - 18-years - old school children in the city of isfahan , the second most populated city of iran . the sample size was calculated to be able to detect a difference of two in the score of oral health impacts by 90% power at 5% significance and considered 10% attrition rate . to obtain a representative random sample , divisions of the ministry of education and gender were determined as the variables for stratification . thereafter , 30 eligible students from each school were invited to the study by convenient sampling . the inclusion criteria were the age between 13 years and 18 years and having written parental informed consent . the students who had orthodontic appliances or reported a history of orthodontic treatment were excluded from the main study . also , legal process in the bureau of education , school administrations , and parent 's levels were passed . the dental health component ( dhc ) were recorded in five severity level from one no treatment need to five very great need after the assessments of all occlusion features ( overjet , overbite , reverse jet , anterior and posterior crossbite , anterior and lateral open bite , displacement of the teeth , and molar relationship ) . prior to the study , two dentists ( principle investigators ) were trained by a qualified orthodontist in the field of epidemiologic malocclusion studies and calibrated to achieve desirable agreement in the pilot study . according to the intra - class correlation coefficient ( icc ) , the intra - examiner reliability by one week interval was above 0.9 in two dentists . ac of the iotn was accounted by two schemes blindly ; self - assessed and professionally - assessed ac score . the participants and dentist , separately , evaluated overall dental attractiveness in according to 10 standard photos ranging from the most attractive ( score 1 ) to the least attractive ( score 10 ) appearance of a person . ten grade of ac was then transformed into three categories of no / slight need : ac = 1 - 4 , borderline : ac = 5 - 7 , and definite need : ac = 8 - 10 . to identify the oral health - related quality of life scores of the sample the instrument consist of 34 items in five subscales including oral health , functional well - being , social - emotional well - being , school environment and self - image which has been developed for school - aged children . participants describe their past three months experiences in the phrases ranging from 0 never to 4 almost all of the time ( e.g. in the past three months , how often have you had pain in your teeth ? ) to explore the factors determine the similar assessment of the need to orthodontic treatment by the individual and the dentist , a variable computed to detect the persons who reported the same scores on dhc grade and self - perceived ac grade . the primary grading as " no / slight need , borderline need and definite need " for each index used to create a dichotomous variable entitled similarity in the judgment . socioeconomic status was assessed based on the parents educational levels which were asked in five categories as illiterate , school without formal qualification , high school with diploma , undergraduate university level , and postgraduate university level . to describe data , mean , standard deviation , proportion , and graphs were used . to investigate the relationship between background variables and the similarity of need treatment evaluation by participants and dentist , multiple logistic regressions was applied . to evaluate validity of subjective indicators in identifying those needing orthodontic treatment , sensitivity , specificity , positive predictive value , and negative predictive value were calculated . ac score equal to eight or more , cohip score below the median ( 107 ) or positive response to the teeth irregularity question were considered as patients by a self - assessment instrument . a cross - sectional survey was conducted in the 13 - 18-years - old school children in the city of isfahan , the second most populated city of iran . the sample size was calculated to be able to detect a difference of two in the score of oral health impacts by 90% power at 5% significance and considered 10% attrition rate . to obtain a representative random sample , divisions of the ministry of education and gender were determined as the variables for stratification . thereafter , 30 eligible students from each school were invited to the study by convenient sampling . the inclusion criteria were the age between 13 years and 18 years and having written parental informed consent . the students who had orthodontic appliances or reported a history of orthodontic treatment were excluded from the main study . also , legal process in the bureau of education , school administrations , and parent 's levels were passed . the dental health component ( dhc ) were recorded in five severity level from one no treatment need to five very great need after the assessments of all occlusion features ( overjet , overbite , reverse jet , anterior and posterior crossbite , anterior and lateral open bite , displacement of the teeth , and molar relationship ) . prior to the study , two dentists ( principle investigators ) were trained by a qualified orthodontist in the field of epidemiologic malocclusion studies and calibrated to achieve desirable agreement in the pilot study . according to the intra - class correlation coefficient ( icc ) , the intra - examiner reliability by one week interval was above 0.9 in two dentists . ac of the iotn was accounted by two schemes blindly ; self - assessed and professionally - assessed ac score . the participants and dentist , separately , evaluated overall dental attractiveness in according to 10 standard photos ranging from the most attractive ( score 1 ) to the least attractive ( score 10 ) appearance of a person . ten grade of ac was then transformed into three categories of no / slight need : ac = 1 - 4 , the participants were also asked whether they thought they have bad - form or bad - sized teeth or whether the space between their teeth are irregular . using the cut - off point of three , the responses then dichotomized to frequent or infrequent categories . to identify the oral health - related quality of life scores of the sample , the instrument consist of 34 items in five subscales including oral health , functional well - being , social - emotional well - being , school environment and self - image which has been developed for school - aged children . participants describe their past three months experiences in the phrases ranging from 0 never to 4 almost all of the time ( e.g. in the past three months , how often have you had pain in your teeth ? ) to explore the factors determine the similar assessment of the need to orthodontic treatment by the individual and the dentist , a variable computed to detect the persons who reported the same scores on dhc grade and self - perceived ac grade . the primary grading as " no / slight need , borderline need and definite need " for each index used to create a dichotomous variable entitled similarity in the judgment . socioeconomic status was assessed based on the parents educational levels which were asked in five categories as illiterate , school without formal qualification , high school with diploma , undergraduate university level , and postgraduate university level . the statistical package for social sciences ( spss ) software version 15 was applied to analyze the data . to describe data , mean , standard deviation , proportion , and graphs were used . to investigate the relationship between background variables and the similarity of need treatment evaluation by participants and dentist , multiple logistic regressions was applied . to evaluate validity of subjective indicators in identifying those needing orthodontic treatment , sensitivity , specificity , ac score equal to eight or more , cohip score below the median ( 107 ) or positive response to the teeth irregularity question were considered as patients by a self - assessment instrument . twenty seven ( 4.5% ) of the students who had a history of previous orthodontic treatment were excluded , so complete data was gained from 570 ( 95% ) students . according to the dhc grade 4 or 5 as the criteria ( gold standard ) for need to orthodontic treatment , 16.4% ( n = 92 ) of examined children had definite treatment need . distribution of iotn scores in 13 - 18-year - old school children regarding patient 's and dentist 's perceptions ( n= 570 ) the mean ( sd ) cohip score of the population was 103.6 ( 18 ) . while it showed a range of 15 - 135 the prevalence of participants with at least one frequent impact was 66% , 34% , and 32% in the oral health , social - emotional , and self - image subscales , respectively . there were not any association between gender and age variables with the qol score ( p < 0.05 ) , but a gradient was seen in this score with the worsening malocclusion disorders . the cohip scores in different categories of the study population are brought in table 1 . mean and standard deviation ( sd ) of cohip scores in 13 - 18-year - old school children regarding the degree of need to orthodontic treatment and the history of orthodontic treatment ( n=570 ) twelve persons thought that they are in ac grade of eight and above , while only seven of them had definite normative need . half of the students did not need orthodontics by perceptional and normative assessment as well . however , 27% with borderline and 13% with definite need to treatment perceived well about themselves . as it is presented in tables 2 and 3 , the distribution of individuals in different categories has significant differences ( p > 0.001 ) by changing the indicators of need assessment . need to orthodontic treatment on the basis of dhc - iotn ( gold standard ) and subjective ( self - perceived ac - iotn ) instrument in 13 - 18-year - old school children ( n=562 ) in isfahan frequency of persons with different grades of orthodontic treatment need on the basis of professionally - assessed ac - iotn and subjective ( self - perceived ac - iotn ) instrument in 13 - 18-year - old school children ( n=562 ) in isfahan regarding the ac - iotn , a comparison between the persons and dentists evaluation of need showed poor / slight agreement level . the agreements between the patients ( self - perceived ac ) and dentists ( dhc / normative ac ) regarding the judgments about the needs to orthodontic treatment logistic regression analysis revealed that students with no caries experience , better quality of life , higher father 's educational level , and brushing habits are more likely to have judgment comparable with dentists . however , gender , age , and mother 's education did not seem to have any role in this decision [ table 5 ] . multiple logistic regression to investigate the influences of the independent variables on the similar judgment in the participant and dentist about treatment need in 13 - 18-year - old school children in isfahan the diagnostic power of three subjective indicators with respect to normative need to orthodontics treatment was summarized in table 6 . in this sample , 61% ( 95% ci = 0.50 ; 0.71 ) of all the patients with definite need had a positive impact - related test ( sensitivity ) and the probability of negative cohip test result given no disease ( specificity ) was 50% ( 95% ci = 0.45 ; 0.55 ) . positive predictive value of 20% means that only one - fifth of those who had reported impact on life had need to treatment . regarding a question on the irregular teeth , the strength of ac to detect the healthy ones showed to be excellent , but its low sensitivity disabled the recognition of the persons with serious need . diagnostic validity of subjective indicators in identifying those needing orthodontic treatment in 13 - 18-year - old school children in isfahan ( n=570 ) the findings of the present study clearly revealed a considerable difference between subjectively and normatively assessed needs relating to treatment of malocclusions in 13 - 18-year - old iranian adolescents . more than half of the students under assessment were defined as healthy by an expert while this rate was 90% when reported by the students themselves . it was in contrast with some studies which had shown subjective needs for aesthetic - related treatment were more frequent in the females . this is a major worry in the adolescents because of the influences on their communications , and it is suggested that individuals satisfied with their body image tend to have more successful social contacts . but according to our findings , a considerable proportion of the students with definite need did not expressed any deficiency in their dental appearance . there may be two explanations ; the first one is that the appropriateness of ac index to detect the felt need in this age group is questionable . also had concluded that ac was not sufficiently reliable to be used as a consumer measure in child and adolescents . second is that the dhc takes into account possible occlusal findings that could be functionally detrimental , but are not aesthetically important like posterior crossbite , posterior displacements , or increased overbite . also , it is good to mention that there are cultural and religious norms that play an important role in the mode of self - display in the youth in our country ; it could lead to fade the importance of other factors . while the utilization of quality of life measures has been encouraged in the oral health studies , it is emphasized particularly in the orthodontic treatment need assessments , recently . however , it has been shown that although the clinical condition have a distinct impact on individual 's life and the association between malocclusion and qol was confirmed , there are crucial problems to conjoining the results of these two perspectives . in this study , only 10% of all examined students had criteria of both affected qol and normative need together . similar to the finding of the study on brazilian adolescents,39% of the patients with grade 4 and 46% of whom with grade 3 of dhc - iotn did not report considerable impact on their quality of life . regarding administering a valid questionnaire to quantify the impacts of oral and dental problems on adolescent 's daily living , our assumption was that all domains of this instrument ( oral health , function , social - emotional , schooling , and self - image ) could be affected by malocclusion . but the present results revealed that this instrument is neither sensitive nor specific enough to detect the patients with malocclusions as a screening tool . this issue confirmed the previous reports which have mentioned that subjective instruments are not reliable in orthodontic need assessment because of their poor performance to screen clinical diseases . regarding the existing discrepancy between two methods of need evaluation , we tried to uncover the variables that played role in the process of judgment by patient 's perspective . while some factors such as ohrqol , dental caries history and father 's education increased the chance of same judgment on need , the coefficient of determination which was gained in the fitting models was 0.07 . in line with the multi - dimensional identity of health , individual 's perceptions are the outcomes of complex bio - psychosocial processes and they are affected by various moderating factors such as the person 's overall characteristics , living environment , health literacy , and so on . the main limitation of this study is the conceptual inflictions about the use of generic ohrqol instrument as measuring tool in otn . definition of the minimal clinically importance and cut - off point determination of subjective instruments was another issue in the procedure of need assessment . despite the advantages of integration of patient - centered outcome measuring , this approach in oral health need assessment needs to further research yet . this study demonstrated that regarding ac as a patient - based outcome , about 50% of the participants had no need on the dentist 's and individual 's belief . while in the other 50% with borderline need , the appearance was acceptable . the frequency of definite need to orthodontics has decreased from 16.4% to 10% by application of impact - related need assessment .

besides lysine acetylation , we recently identified a variety of short - chain lysine acylations in core histones , including lysine propionylation , butyrylation , 2-hydroxyisobutyrylation , crotonylation , malonylation , succinylation , and glutarylation ( chen et al . , 2007 , dai et al . , 2014 , tan et al . , 2011 , tan et al . , 2014 , xie et al . , 2012 ) . emerging data suggest that these new histone lysine acylations may have unique functions that depend not only on cell metabolism , but also on their ability to be deposited or removed by specific enzymes ( dai et al . , 2014 , , 2012 , rousseaux and khochbin , 2015 , sabari et al . , 2015 , sin et al . , 2012 , tan et al . , 2011 ) . nevertheless , the functional impact of differential histone acylation on chromatin recognition by specific factors has remained unexplored . , we decided to investigate histone butyrylation , because , in contrast to the acetyl ( 2-carbon ) and propionyl ( 3-carbon ) groups , the butyryl ( 4-carbon ) group restricts the binding of bromodomains ( flynn et al . , 2015 ) . more specifically , we focused our attention on histone h4 at k5 and k8 , whose acetylation is required to bind the first bromodomain of brdt , a testis - specific member of the bet protein family ( morinire et al . , 2009 ) . our previous work showed that brdt stimulates the transcription of certain spermatogenic - specific genes by recruiting the p - tefb complex and by directly binding to their transcriptional start sites ( tsss ) . additionally , during late spermatogenesis , brdt s first bromodomain is necessary for the replacement of histones by non - histone sperm - specific transition proteins ( tps ) and protamines ( prms ) ( gaucher et al . , 2012 ) . given the critical role of h4k5 and h4k8 acetylation in brdt - driven activities , we hypothesized that other mutually exclusive histone marks at these two residues might have key regulatory roles in sperm cell genome programming . here , we identify major histone lysine butyrylation sites in cells from different species , including mouse spermatogenic cells . using spermatogenesis as an integrated biological model system , in addition to in vitro experiments and targeted proteomic approaches our data indicate that interchangeable acetylation and butyrylation at h4k5 and h4k8 not only stimulates transcription , but could also underlie a highly dynamic interaction of histone post - translational modification ( ptm)-binding factors such as brdt . additional data further show that stable differential use of acetylation and butyrylation could also durably affect genome organization in the maturing sperm . altogether , these findings indicate how competition between histone acylation states could be an important epigenetic regulatory mechanism . to identify histone butyryllysine ( kbu ) sites and study their function , we first confirmed the presence of histone kbu by western blotting . our data suggest that histone kbu is an evolutionarily conserved ptm in eukaryotic cells ( figure 1a ) . we then used mass spectrometry to identify possible kbu sites in core histones from three species ( chen et al . kbu sites were detected in the n - terminal tails of h3 ( k9 , k14 , k18 , k23 , k27 , k36 , k37 , k79 , and k122 ) , h4 ( k5 , k8 , k12 , and k16 ) , and h2b ( k5 and k20 ) ( figure 1b ; data s1 ) . to examine the function of kbu in chromatin biology , we used mammalian spermatogenesis as a model system . spermatogenic cells can be roughly classified into three major types : proliferative progenitor cells ( spermatogonia ) , meiotic cells ( spermatocytes ) , and post - meiotic haploid cells ( spermatids ) . during differentiation , highly specific gene expression programs are activated in both meiotic and early post - meiotic cells . large - scale genome reorganization also takes place in spermatids , where a genome - wide replacement of histones by tps and prms occurs in post - meiotic cells known as elongating and condensing spermatids ( gaucher et al . , 2010 , goudarzi et al . 2004 ) . to confirm the existence of histone butyrylation in spermatogenic cells , we identified histone kbu sites by mass spectrometry analysis of histones from mouse testis . we detected ten butyrylation sites including h4k5bu and h4k8bu ( occurring separately or in combination ) , supporting the presence of these two histone marks in spermatogenic cells ( figure 1b ; data s1 ; see also figure 7b ) . we then used highly specific anti - h4k5bu and anti - h4k8bu antibodies , along with the anti - h4k5ac and anti - h4k8ac antibodies , to investigate the stage - specific presence of these marks in spermatogenic cells . immunohistochemistry analysis showed that h4k5 and k8 butyrylation is enhanced in elongating spermatids ( figure 2a ) , similar to earlier observations for histone h4k5 and h4k8 acetylation ( hazzouri et al . , 2000 ) . to study the dynamic changes of butyrylation versus acetylation at both h4k5 and h4k8 sites in spermatogenic cells , we examined the co - existence of h4k5ac and h4k5bu as well as that of h4k8ac and h4k8bu . in elongating spermatids , h4k5ac and h4k8ac are widely distributed , but their localization becomes biased toward the sub - acrosomal regions in later stages . in contrast , h4k5bu- and h4k8bu - containing nucleosomes are homogenously distributed in the same cells ( figure 2b ) . in late elongating spermatids , while acetylated histones are removed and degraded ( gaucher et al . , 2012 , qian et al . , 2013 ) , butyrylated h4 species escape this wave of acetylation - dependent histone removal , to finally disappear in condensing spermatids ( figure 2b ) . interestingly , immunoblotting experiments , using fractionated spermatogenic cells , confirmed that butyrylation persists longer than acetylation on h4k5 and k8 sites ( figure 2c ) . these results highlight a bimodal histone removal process , whereby the removal of h4 k5/k8 butyrylated nucleosome occurs after that of h4 k5/k8 acetylated nucleosomes . our immunofluorescence analysis showed distinctive patterns of acetylation and butyrylation at h4k5 and h4k8 , with both marks showing enhanced labeling in elongating spermatids , when cellular transcription dramatically decreases ( gaucher et al . , 2010 ) . this observation motivated us to study their genomic distributions and potential functions at earlier stages of spermatogenesis , when specific spermatogenic gene expression programs are activated ( dai et al . , 2014 , gaucher et al . , 2012 , tan et al . , 2011 ) . toward this goal , mouse spermatogenic cells were fractionated into spermatocytes and post - meiotic round spermatids ( dai et al . , 2014 , gaucher et al . , 2012 , tan et al the two pools of cells were subjected to chromatin immunoprecipitation followed by deep sequencing ( chip - seq ) using four anti - ptm - specific antibodies ( anti - h4k5ac , -h4k8ac , -h4k5bu , and -h4k8bu ) . analysis of the genomic distribution of these marks revealed that regions surrounding gene tsss ( located upstream of tsss and at the 5utr ) exhibit the highest coverage by the four ptms in both spermatocytes and spermatids ( figure 3a ) . further analyses showed that the four h4 ptms are enriched at tsss in a manner dependent on the transcriptional activity of the corresponding genes ( figure 3b ) . a critical concern in the above experiments is the possibility that histone sites subject to acetylation may be butyrylated at only low background levels , which would be misleadingly overestimated by the chip - seq analysis due to vastly different affinities of the antibodies used . to address this issue , we used surface plasmon resonance ( spr ) to measure the affinities of antibodies for their respective targets . these measurements showed that all four antibodies have similar ranges of affinity ( figure s1a ) . additionally , a chip - qpcr approach demonstrated that the four histone marks are significantly detected at selected genomic regions ( figures s1b and s1c ) . together , these experiments confirmed that h4k5k8 butyrylation occurs at levels that largely exceed background noise . following these control experiments , we investigated in more detail the relationship between gene expression and the co - occurrence of the four tss - associated histone marks . remarkably , the most active genes were found associated with all four marks at their tss regions ( figures 3b and 4a ) . in contrast , genes lacking any one of these marks at their tss showed significantly reduced expression ( figure 4a ) . other genomic elements did not show such a direct relationship between the co - existence of the four histone marks and gene expression ( figure s2 ) . to further analyze the relationship between the four acylation histone marks and gene activity , we took advantage of the differences in gene expression programs between spermatocytes ( meiotic cells ) and the transcriptionally active haploid round spermatids ( generated after meiosis ) . using our chip - seq data from these two cell types , we divided genes into four categories according to the intensity of tss labeling by the four h4 acylation marks , namely : ( 1 ) genes bearing none of the four marks ( labeled no ) and ( 24 ) genes bearing all four marks , either with ( 2 ) comparable ( = ) , ( 3 ) higher ( > ) , or ( 4 ) lower ( < ) peak intensities in spermatocytes compared to round spermatids ( figure 4b ) . genes in category ( 1 ) are largely unexpressed , while those in category ( 2 ) exhibited no change in expression level between meiotic and post - meiotic cells . in striking contrast , genes in categories ( 3 ) and ( 4 ) exhibited differential expression in the two cell types , which was positively associated with the change in intensity of tss labeling by the four histone marks ( figure 4b ) . additional support for a positive correlation between the presence of the four ptms in the tsss and gene expression is the observation that all four marks are depleted on the sex chromosomes compared to autosomes , consistent with the chromosome - wide meiotic transcriptional inactivation known to characterize sex chromosomes ( figure 4c ) . to further investigate this observation , we specifically considered the fraction of sex chromosome - linked genes that escapes meiotic sex chromosome inactivation ( namekawa et al . , 2006 ) . while all four ptms were identified on a majority ( 73% ) of tsss associated with active autosomal genes , co - occurrence of the four ptms was observed in only a minority ( 23% ) of tsss associated with sex - linked genes that are reactivated in post - meiotic cells . hence , over three - quarters of this latter category of tsss bear between zero and three of the four histone marks . interestingly , most of the tsss bearing 13 of the ptms were depleted of h4k8ac , but not of h4k8bu ( figure 4c ) . this is consistent with other studies showing depletion of acetylation on the tsss of sex - linked post - meiotic genes and their labeling with other acyl groups such as crotonyl ( sin et al . , 2012 , tan et al . , 2011 our chip - seq data showed that , like acetylation , histone butyrylation is associated with high levels of gene expression , suggesting the possibility that histone butyrylation directly stimulates gene expression . to test this hypothesis , we exploited a reconstituted activator - dependent in vitro transcription system . our early studies had shown that in vitro , p300 and creb binding protein ( cbp ) can catalyze lysine butyrylation by transferring the butyryl group from [ c ] butyryl - coa to core histone proteins ( chen et al . this activity was also observed in ex vivo transfection experiments ( chen et al . , 2007 ) . moreover , in vitro butyrylation activity was confirmed both on a reconstituted chromatin template and on histone octamers by mass spectrometry analysis . further analysis of this in vitro activity revealed that p300 efficiently butyrylates the sites of interest , h4k5 and h4k8 , in histone octamers as well as in chromatin ( figures 5a and 5b ; data s1 ) . after confirming that p300 is a histone butyryltransferase , we used a p300- and p53-dependent in vitro transcription system ( figure 5c ) to test if histone butyrylation could stimulate transcription . we observed that p300-catalyzed histone butyrylation indeed directly stimulates transcription ( figure 5d ) . the mutation of lysine residues to arginine either on h3 or h4 tails eliminated acyl - coa - stimulated transcription , indicating that acetyl / butyryl - coa activates transcription through p300-catalyzed histone lysine acylation ( figure 5e ) . this experiment clearly demonstrates that , like acetylation , histone butyrylation can also directly stimulate gene transcriptional activity . association of the four histone acylation marks with the tsss of most of the highly active genes in spermatogenic cells raises the possibility that the high transcriptional activity of these genes is mediated by the binding of brdt to the acylation marks on their tsss . to test this hypothesis , we compared previously identified brdt - bound tsss ( gaucher et al . , 2012 ) with tsss labeled with the four histone marks . this analysis showed that most of the brdt - bound gene tsss also bore high levels of acetylation and butyrylation marks at h4k5 and h4k8 ( figures 6a and 6b ) . to study if brdt s first bromodomain ( bd1 ) mediates this interaction , we used spermatogenic cells from mice expressing a mutated form of brdt lacking bd1 ( gaucher et al . the chip - seq analysis showed that , in both spermatocytes and round spermatids , the deletion of bd1 considerably weakens brdt binding to tsss bearing the four acylation marks ( figure 6a , brdt bd1 ) . this result indicates that bd1 has a major role in targeting gene tsss bearing h4k5k8 acetylation / butyrylation . to further characterize histone acylation at gene tsss bound by brdt , we considered the occurrence of the four studied h4 ptms at tsss as a function of brdt - binding . remarkably , the vast majority of brdt - associated tsss bore all four acylation marks ( figure 6b ) . these observations therefore led us to question the ability of brdt to bind h4 bearing butyrylation at either k5 or k8 or both . to test the ability of brdt s bromodomains to bind a butyrylated h4 tail , we first carried out an in vitro binding assay using biotinylated h4 tail peptides bearing all four possible combinations of the k5ac , k5bu , k8ac , and k8bu acylation marks . we incubated the peptides with extracts from transfected brdt - expressing cos7 cells and performed pull - down experiments . as expected , brdt efficiently interacts with h4 peptides that are either fully acetylated ( i.e. , on k5 , k8 , k12 , and k16 ) or diacetylated on k5 and k8 ( figure s3a , upper ) . strikingly , whereas brdt binding was only slightly affected by the replacement of h4k8ac by h4k8bu , it was completely abolished by the substitution of h4k5ac by h4k5bu . pull - down experiments using brdt mutants bearing inactive bromodomains bd1 or bd2 demonstrated that brdt binding to all the tested peptides depends on the integrity of its first bromodomain ( figure s3a , lower ) . these data clearly imply that the butyrylation of h4k5 inhibits the binding of brdt to histone h4 . to validate this result our results confirmed that butyrylation at h4k5 abolishes the binding of brdt to h4 tails ( figure 6c , upper ) . as further confirmation , we repeated the pull - down assay on protein extracts from wild - type mouse testis using either fully acetylated or fully butyrylated immobilized h4 tail peptides and analyzed the bound fractions by mass spectrometry . brdt was easily identified among the proteins affinity - isolated by the h4ac - containing peptide , but not by the h4bu - containing peptide ( figure s3b ; data s1 ) . the use of extracts from mice testes expressing the brdt bd1 mutant confirmed that the brdt - h4 tail interactions described above are primarily mediated by the bd1 domain ( figure 6c , lower right ) . this result was further corroborated by an experiment with jq1 , a bet bromodomain inhibitor , which abolished the binding of testis - derived brdt to acetylated h4 and h4k5ack8bu peptides ( figure 6c , lower left ) . taken together , these findings establish that h4k5bu , but not h4k8bu , abolishes the interaction between brdt and the histone h4 tail . to understand the molecular basis of the inhibitory effect of h4k5 butyrylation on brdt binding in the crystal structure of brdt - bd1 bound to h4k5ack8ac , residue k5ac is intimately recognized by bd1 , whereas k8ac makes fewer contacts ( morinire et al . , 2009 ) . modeling shows that replacing the k8 acetyl group with a butyryl group allows the additional atoms to be accommodated without compromising any of the interactions between bd1 and the peptide ( figure 6d ) . in contrast , replacing the k5 acetyl group by a butyryl group results in a steric clash with residues in the domain s za loop , implying that some structural adjustments to the model are required to accommodate the butyryl group . comparison with the published co - crystal structure of brd4-bd1 bound to h3k14bu indicates the type of adjustments required to accommodate h4k5bu ( vollmuth and geyer , 2010 ) . aligning the latter structure with that of the brdt - bd1/h4k5ack8ac complex shows that h3k14bu occupies approximately the same position as k5ac ( figure 6e ) . however , the bulkier butyryl group results in the displacement of a water molecule within the ligand - binding pocket and causes the k14bu main - chain and side - chain atoms to be shifted relative to those of k5ac . in the context of an h4k5buk8ac peptide , such shifts would be predicted to disrupt several direct and indirect hydrogen bonds between bd1 and the peptide ( see figure legend for details ) . thus , the modeling approach provides a plausible structural basis for the poor affinity observed for the binding of brdt to the h4k5buk8ac peptide compared with that to the h4k5ack8ac and h4k5ack8bu peptides . our pull - down and structural modeling data strongly suggest that brdt is inhibited from binding tss regions where histone h4 is modified by lysine butyrylation at the k5 position . this conclusion appears contradictory to the chip - seq data , where h4k5bu and brdt were both associated with the same tss regions . a hypothesis that would reconcile these observations is that acetylation and butyrylation of h4k5 exhibit a rapid turnover . indeed , acyltransferases associated with highly active gene tsss might feasibly drain cellular acetyl - coa as well as butyryl - coa toward these sites , leading to a mixture of histone h4 acetylation and butyrylation marks . to test this hypothesis , we performed in vitro assays by incubating reconstituted histone octamers with purified p300 and an equimolar mixture of acetyl - coa and butyryl - coa . our results show that p300 can use acyl - coas to catalyze acetylation and butyrylation at both h4k5 and h4k8 sites ( figure 7a ) , as we detected h4 peptides bearing various combinations of acetylated or butyrylated h4k5 and h4k8 ( figure 7a ; data s1 ) . encouraged by this in vitro result , we then investigated whether histone h4 isolated from spermatogenic cells also contains diverse lysine acylation marks . mass spectrometry analysis of these samples detected h4 peptides with various combinations of acetylation and butyrylation at h4k5 and h4k8 . in addition , depending on cell type , the stoichiometry of h4k5bu and h4k8bu could be higher than some of the widely studied histone marks such as h3k4me3 ( kulej et al . , 2015 ) ( figures 7b and s4a ; table s2 ) , but lower than those of h4 tail acetylation , such as that of k16 , which can be as high as 20% of h4 species ( kulej et al . , 2015 ) . acetylation is known to have a high turnover rate at gene tsss ( crump et al . , 2011 ) . therefore , due to continuous acylation by histone acetyltransferase ( hats ) such as p300 and the rapid turnover of these ptms , it is feasible that h4k5 and k8 alternate between acetylated and butyrylated states . this model is supported by the detection of various combinations of h4k5k8 acetylation and butyrylation either in in vitro hat assays ( figure 7a ) or in vivo in spermatogenic cells ( figure 7b ) . a direct consequence of such alternating histone acetylation / butyrylation would be the dynamic binding of brdt , which would oscillate between high- and low - affinity states depending on the acylation status of h4k5 ( figure 7c ) . such dynamic histone h4 acylation could be facilitated by open nucleosomes on the corresponding gene tsss by specific histone variants such as h2a.lap1 , a histone h2a variant capable of inducing unstable and open nucleosomes and known to associate with active gene tsss in spermatogenic cells ( nekrasov et al . indeed , analysis of chip - seq data for h2a.lap1 revealed that the four h4 ptms are particularly enriched on h2a.lap1-associated tsss ( figure s4b ) . taken together , the above results support a model whereby alternating competing histone acetylation and butyrylation underlie a dynamic interaction between the histone modifications and the cognate bromodomain . the present study reports our findings regarding the interplay between histone acetylation and butyrylation on the histone h4 tail during sperm cell differentiation . we found that in vitro , p300 uses available acetyl - coa and butyryl - coa sources to acylate the h4 tail at all the acceptor lysines in an indiscriminate manner . a proteomic approach also revealed the co - existence of the same combinations of h4 k5k8 acetyl and butyryl marks in different spermatogenic cell types . chip - seq analysis on fractionated spermatogenic cells further demonstrated that h4k5/k8 acetyl / butyryl are particularly enriched at the tsss of the most active genes . strikingly , however , functional and structural analysis revealed that the acylation state of h4k5 is a critical determinant of brdt binding affinity , as brdt binds the acetylated , but not the butyrylated state . in this context , a stable maintenance of differential acetylation and butyrylation could have important functional consequences for the genome reorganization observed during spermatogenesis . we found that , in contrast to earlier stages , in elongating spermatids , h4k5 and h4k8 acetylation and butyrylation become more markedly associated with specific regions of the genome . indeed , we previously showed that in elongating spermatids , when histone hyperacetylation and a general transcriptional shut - down occur , bd1 is indispensable for the replacement of acetylated histones by tps ( gaucher et al . , 2012 ) . additionally , the hyperacetylated histones are known to be directly targeted for degradation by a pa200-containing specialized proteasome ( qian et al . , 2013 ) , suggesting that the brdt - bound histone population enters this pathway . here , we show that in elongating spermatids , butyrylated histones survive this wave of acetylation - dependent histone removal and degradation . these observations suggest that a stable differential labeling of h4 by acetylation and butyrylation may control the timing of histone removal . in this context , it is also possible to speculate that nucleosomes bearing butyrylated h4 could undergo a direct histone - to - prm replacement due to the inability of brdt to mediate the exchange of histones by tps . indeed , tp1-tp2 double ko cells can undergo direct histone - to - prm replacement but , in this case , prms are unable to tightly compact the genome ( for review , see gaucher et al . , 2010 ) ) . the prediction is therefore that regions bearing butyrylated histones would evolve to a less compact structure in mature sperms than regions that are marked by acetylated histones before their removal . this regulatory mechanism could be an elegant way to introduce differences in genome compaction by prms . differential histone tail acylation might also play important roles in the control of somatic cell gene expression , differentiation , and genome programming . indeed , a recent study of p300-mediated histone h3k18 crotonylation revealed that an increase in cellular levels of crotonyl - coa favors a more efficient de novo gene activation ( sabari et al . , 2015 ) . although the precise mechanism underlying the role of h3k18cr in transcription is unknown , the differential affinity of a bromodomain protein toward crotonyl versus acetyl marks could conceivably account for the observed phenomenon . previous reports demonstrated a rapid turnover of histone acetylation on a sub - fraction of nucleosomes ( clayton et al . , 2006 , waterborg , 2002 ) , overlapping with active gene tsss ( crump et al . , however , the functional implications of the short half - life of histone acetylation on active chromatin regions , especially on highly transcribed gene tsss , have remained elusive . the results described in our present study are consistent with a rapid turnover of both acetyl and butyryl marks on the h4 tail . given the differential affinity of brdt bd1 for the acetylated and butyrylated forms of h4k5 , a rapid alternation of acylation states at h4k5 would result in a highly dynamic interaction between brdt and chromatin . thus , it is tempting to speculate that the generally observed rapid turnover of acetyl marks may be functionally significant because it enables rapid transitions between alternative states of lysine acylation . such a mechanism , while maintaining histones permanently modified , would allow for a dynamic association with specific bromodomains , which might be important for sustaining successive cycles of transcription . thus , either a change in the ratio of cellular acyl - coas ( for instance caused by metabolic disorders ; pougovkina et al . , 2014 ) or differential activities of histone deacetylase ( hdacs ) in removing acyl - groups ( rousseaux and khochbin , 2015 ) could reprogram gene expression profiles . in conclusion , we provide here the first demonstration that the interchangeable use of two closely related histone acylation marks at a specific site , h4k5 , has important functional consequences by modulating the ability of a transcriptional regulator , brdt , to recognize chromatin . this finding should improve our understanding of gene transcriptional regulation and its link to cell metabolism . pan anti - kbu antibody , anti - histone site - specific kbu , and acetyllysine antibodies used were purchased from ptm biolabs and anti - histone h3 and anti - histone h4 antibodies were from abcam . mouse monoclonal antibodies against h4k5ac and h4k8ac were a generous gift from dr . h. kimura . anti - brdt is a homemade antibody previously described ( gaucher et al . , 2012 ) . peptides bearing one or a few acetyl and kbu residues were custom synthesized and were verified by hplc and mass spectrometry . jq1 was synthesized by charles mckenna and elena ferri ( university of southern california ) ( emadali et al . , 2013 ) and used as described in this reference . the antigen recognition capacities of the antibodies used in chip - seq and chip - qpcr were determined using spr as described in the supplemental information . acetylation and butyrylation of histones were determined on 200 g of core histones extracted from the different studied cell types . histone from spermatogenic cells underwent chemical butyrylation with deuterated ( d5 ) butyryl anhydride and processed as described in the supplemental information . all the experimental procedures , including immunostaining , cell fractionation , and chip - seq were carried out exactly as described previously ( dai et al . , 2014 , gaucher et al . , 2012 , tan et al . , 2011 ) . chip - qpcr was performed following our chip - seq protocol , but the recovered dna was amplified using quantitative pcr and specific primers corresponding to regions indicated in figure s1 . table s1 indicates the sequence of these primers and details of the chip experiments are described in supplemental information . brdt pull - down assays were performed as previously described ( huang et al . , 2010 , pivot - pajot et al . , 2003 ) . the in vitro transcription assay was also previously described ( an et al . , 2002 , an and roeder , 2004 , sabari et al . the bioinformatic analyses followed a pipeline similar to the one previously described ( dai et al . montellier et al . , 2013 , tan et al . , 2011 ) and are detailed in the supplemental information . in vitro acetyl / butyryl - transferase assays on histone octamer used recombinant p300 expressed and purified from baculovirus infected in sf21 insect cells . protein purification and the details of the hat assays are described in the supplemental information . a .- l.v . designed appropriate primers and performed qpcr after chip from total germ cells . d.z . performed the ip / pull - down / ms / ms experiments , ptm quantifications , antibody characterization ( spr ) , and coordinated the ms analyses . h.h . and o.k . performed the hplc - ms / ms experiments and data analysis .

summaryrecently discovered histone lysine acylation marks increase the functional diversity of nucleosomes well beyond acetylation . here , we focus on histone butyrylation in the context of sperm cell differentiation . specifically , we investigate the butyrylation of histone h4 lysine 5 and 8 at gene promoters where acetylation guides the binding of brdt , a bromodomain - containing protein , thereby mediating stage - specific gene expression programs and post - meiotic chromatin reorganization . genome - wide mapping data show that highly active brdt - bound gene promoters systematically harbor competing histone acetylation and butyrylation marks at h4 k5 and h4 k8 . despite acting as a direct stimulator of transcription , histone butyrylation competes with acetylation , especially at h4 k5 , to prevent brdt binding . additionally , h4 k5k8 butyrylation also marks retarded histone removal during late spermatogenesis . hence , alternating h4 acetylation and butyrylation , while sustaining direct gene activation and dynamic bromodomain binding , could impact the final male epigenome features .

Introduction Results Discussion Experimental Procedures Author Contributions

besides lysine acetylation , we recently identified a variety of short - chain lysine acylations in core histones , including lysine propionylation , butyrylation , 2-hydroxyisobutyrylation , crotonylation , malonylation , succinylation , and glutarylation ( chen et al . emerging data suggest that these new histone lysine acylations may have unique functions that depend not only on cell metabolism , but also on their ability to be deposited or removed by specific enzymes ( dai et al . nevertheless , the functional impact of differential histone acylation on chromatin recognition by specific factors has remained unexplored . , we decided to investigate histone butyrylation , because , in contrast to the acetyl ( 2-carbon ) and propionyl ( 3-carbon ) groups , the butyryl ( 4-carbon ) group restricts the binding of bromodomains ( flynn et al . more specifically , we focused our attention on histone h4 at k5 and k8 , whose acetylation is required to bind the first bromodomain of brdt , a testis - specific member of the bet protein family ( morinire et al . additionally , during late spermatogenesis , brdt s first bromodomain is necessary for the replacement of histones by non - histone sperm - specific transition proteins ( tps ) and protamines ( prms ) ( gaucher et al . given the critical role of h4k5 and h4k8 acetylation in brdt - driven activities , we hypothesized that other mutually exclusive histone marks at these two residues might have key regulatory roles in sperm cell genome programming . here , we identify major histone lysine butyrylation sites in cells from different species , including mouse spermatogenic cells . using spermatogenesis as an integrated biological model system , in addition to in vitro experiments and targeted proteomic approaches our data indicate that interchangeable acetylation and butyrylation at h4k5 and h4k8 not only stimulates transcription , but could also underlie a highly dynamic interaction of histone post - translational modification ( ptm)-binding factors such as brdt . additional data further show that stable differential use of acetylation and butyrylation could also durably affect genome organization in the maturing sperm . to identify histone butyryllysine ( kbu ) sites and study their function , we first confirmed the presence of histone kbu by western blotting . kbu sites were detected in the n - terminal tails of h3 ( k9 , k14 , k18 , k23 , k27 , k36 , k37 , k79 , and k122 ) , h4 ( k5 , k8 , k12 , and k16 ) , and h2b ( k5 and k20 ) ( figure 1b ; data s1 ) . to examine the function of kbu in chromatin biology , we used mammalian spermatogenesis as a model system . spermatogenic cells can be roughly classified into three major types : proliferative progenitor cells ( spermatogonia ) , meiotic cells ( spermatocytes ) , and post - meiotic haploid cells ( spermatids ) . during differentiation , highly specific gene expression programs are activated in both meiotic and early post - meiotic cells . large - scale genome reorganization also takes place in spermatids , where a genome - wide replacement of histones by tps and prms occurs in post - meiotic cells known as elongating and condensing spermatids ( gaucher et al . to confirm the existence of histone butyrylation in spermatogenic cells , we identified histone kbu sites by mass spectrometry analysis of histones from mouse testis . we then used highly specific anti - h4k5bu and anti - h4k8bu antibodies , along with the anti - h4k5ac and anti - h4k8ac antibodies , to investigate the stage - specific presence of these marks in spermatogenic cells . in contrast , h4k5bu- and h4k8bu - containing nucleosomes are homogenously distributed in the same cells ( figure 2b ) . , 2013 ) , butyrylated h4 species escape this wave of acetylation - dependent histone removal , to finally disappear in condensing spermatids ( figure 2b ) . these results highlight a bimodal histone removal process , whereby the removal of h4 k5/k8 butyrylated nucleosome occurs after that of h4 k5/k8 acetylated nucleosomes . our immunofluorescence analysis showed distinctive patterns of acetylation and butyrylation at h4k5 and h4k8 , with both marks showing enhanced labeling in elongating spermatids , when cellular transcription dramatically decreases ( gaucher et al . this observation motivated us to study their genomic distributions and potential functions at earlier stages of spermatogenesis , when specific spermatogenic gene expression programs are activated ( dai et al . toward this goal , mouse spermatogenic cells were fractionated into spermatocytes and post - meiotic round spermatids ( dai et al . additionally , a chip - qpcr approach demonstrated that the four histone marks are significantly detected at selected genomic regions ( figures s1b and s1c ) . following these control experiments , we investigated in more detail the relationship between gene expression and the co - occurrence of the four tss - associated histone marks . in contrast , genes lacking any one of these marks at their tss showed significantly reduced expression ( figure 4a ) . other genomic elements did not show such a direct relationship between the co - existence of the four histone marks and gene expression ( figure s2 ) . to further analyze the relationship between the four acylation histone marks and gene activity , we took advantage of the differences in gene expression programs between spermatocytes ( meiotic cells ) and the transcriptionally active haploid round spermatids ( generated after meiosis ) . using our chip - seq data from these two cell types , we divided genes into four categories according to the intensity of tss labeling by the four h4 acylation marks , namely : ( 1 ) genes bearing none of the four marks ( labeled no ) and ( 24 ) genes bearing all four marks , either with ( 2 ) comparable ( = ) , ( 3 ) higher ( > ) , or ( 4 ) lower ( < ) peak intensities in spermatocytes compared to round spermatids ( figure 4b ) . genes in category ( 1 ) are largely unexpressed , while those in category ( 2 ) exhibited no change in expression level between meiotic and post - meiotic cells . in striking contrast , genes in categories ( 3 ) and ( 4 ) exhibited differential expression in the two cell types , which was positively associated with the change in intensity of tss labeling by the four histone marks ( figure 4b ) . additional support for a positive correlation between the presence of the four ptms in the tsss and gene expression is the observation that all four marks are depleted on the sex chromosomes compared to autosomes , consistent with the chromosome - wide meiotic transcriptional inactivation known to characterize sex chromosomes ( figure 4c ) . while all four ptms were identified on a majority ( 73% ) of tsss associated with active autosomal genes , co - occurrence of the four ptms was observed in only a minority ( 23% ) of tsss associated with sex - linked genes that are reactivated in post - meiotic cells . this is consistent with other studies showing depletion of acetylation on the tsss of sex - linked post - meiotic genes and their labeling with other acyl groups such as crotonyl ( sin et al . , 2011 our chip - seq data showed that , like acetylation , histone butyrylation is associated with high levels of gene expression , suggesting the possibility that histone butyrylation directly stimulates gene expression . after confirming that p300 is a histone butyryltransferase , we used a p300- and p53-dependent in vitro transcription system ( figure 5c ) to test if histone butyrylation could stimulate transcription . the mutation of lysine residues to arginine either on h3 or h4 tails eliminated acyl - coa - stimulated transcription , indicating that acetyl / butyryl - coa activates transcription through p300-catalyzed histone lysine acylation ( figure 5e ) . this experiment clearly demonstrates that , like acetylation , histone butyrylation can also directly stimulate gene transcriptional activity . association of the four histone acylation marks with the tsss of most of the highly active genes in spermatogenic cells raises the possibility that the high transcriptional activity of these genes is mediated by the binding of brdt to the acylation marks on their tsss . to test this hypothesis , we compared previously identified brdt - bound tsss ( gaucher et al . this analysis showed that most of the brdt - bound gene tsss also bore high levels of acetylation and butyrylation marks at h4k5 and h4k8 ( figures 6a and 6b ) . to study if brdt s first bromodomain ( bd1 ) mediates this interaction , we used spermatogenic cells from mice expressing a mutated form of brdt lacking bd1 ( gaucher et al . the chip - seq analysis showed that , in both spermatocytes and round spermatids , the deletion of bd1 considerably weakens brdt binding to tsss bearing the four acylation marks ( figure 6a , brdt bd1 ) . to further characterize histone acylation at gene tsss bound by brdt , we considered the occurrence of the four studied h4 ptms at tsss as a function of brdt - binding . remarkably , the vast majority of brdt - associated tsss bore all four acylation marks ( figure 6b ) . these observations therefore led us to question the ability of brdt to bind h4 bearing butyrylation at either k5 or k8 or both . to test the ability of brdt s bromodomains to bind a butyrylated h4 tail , we first carried out an in vitro binding assay using biotinylated h4 tail peptides bearing all four possible combinations of the k5ac , k5bu , k8ac , and k8bu acylation marks . , on k5 , k8 , k12 , and k16 ) or diacetylated on k5 and k8 ( figure s3a , upper ) . strikingly , whereas brdt binding was only slightly affected by the replacement of h4k8ac by h4k8bu , it was completely abolished by the substitution of h4k5ac by h4k5bu . these data clearly imply that the butyrylation of h4k5 inhibits the binding of brdt to histone h4 . to validate this result our results confirmed that butyrylation at h4k5 abolishes the binding of brdt to h4 tails ( figure 6c , upper ) . as further confirmation , we repeated the pull - down assay on protein extracts from wild - type mouse testis using either fully acetylated or fully butyrylated immobilized h4 tail peptides and analyzed the bound fractions by mass spectrometry . brdt was easily identified among the proteins affinity - isolated by the h4ac - containing peptide , but not by the h4bu - containing peptide ( figure s3b ; data s1 ) . this result was further corroborated by an experiment with jq1 , a bet bromodomain inhibitor , which abolished the binding of testis - derived brdt to acetylated h4 and h4k5ack8bu peptides ( figure 6c , lower left ) . to understand the molecular basis of the inhibitory effect of h4k5 butyrylation on brdt binding in the crystal structure of brdt - bd1 bound to h4k5ack8ac , residue k5ac is intimately recognized by bd1 , whereas k8ac makes fewer contacts ( morinire et al . in the context of an h4k5buk8ac peptide , such shifts would be predicted to disrupt several direct and indirect hydrogen bonds between bd1 and the peptide ( see figure legend for details ) . thus , the modeling approach provides a plausible structural basis for the poor affinity observed for the binding of brdt to the h4k5buk8ac peptide compared with that to the h4k5ack8ac and h4k5ack8bu peptides . our pull - down and structural modeling data strongly suggest that brdt is inhibited from binding tss regions where histone h4 is modified by lysine butyrylation at the k5 position . a hypothesis that would reconcile these observations is that acetylation and butyrylation of h4k5 exhibit a rapid turnover . indeed , acyltransferases associated with highly active gene tsss might feasibly drain cellular acetyl - coa as well as butyryl - coa toward these sites , leading to a mixture of histone h4 acetylation and butyrylation marks . our results show that p300 can use acyl - coas to catalyze acetylation and butyrylation at both h4k5 and h4k8 sites ( figure 7a ) , as we detected h4 peptides bearing various combinations of acetylated or butyrylated h4k5 and h4k8 ( figure 7a ; data s1 ) . encouraged by this in vitro result , we then investigated whether histone h4 isolated from spermatogenic cells also contains diverse lysine acylation marks . mass spectrometry analysis of these samples detected h4 peptides with various combinations of acetylation and butyrylation at h4k5 and h4k8 . this model is supported by the detection of various combinations of h4k5k8 acetylation and butyrylation either in in vitro hat assays ( figure 7a ) or in vivo in spermatogenic cells ( figure 7b ) . a direct consequence of such alternating histone acetylation / butyrylation would be the dynamic binding of brdt , which would oscillate between high- and low - affinity states depending on the acylation status of h4k5 ( figure 7c ) . such dynamic histone h4 acylation could be facilitated by open nucleosomes on the corresponding gene tsss by specific histone variants such as h2a.lap1 , a histone h2a variant capable of inducing unstable and open nucleosomes and known to associate with active gene tsss in spermatogenic cells ( nekrasov et al . taken together , the above results support a model whereby alternating competing histone acetylation and butyrylation underlie a dynamic interaction between the histone modifications and the cognate bromodomain . the present study reports our findings regarding the interplay between histone acetylation and butyrylation on the histone h4 tail during sperm cell differentiation . a proteomic approach also revealed the co - existence of the same combinations of h4 k5k8 acetyl and butyryl marks in different spermatogenic cell types . strikingly , however , functional and structural analysis revealed that the acylation state of h4k5 is a critical determinant of brdt binding affinity , as brdt binds the acetylated , but not the butyrylated state . in this context , a stable maintenance of differential acetylation and butyrylation could have important functional consequences for the genome reorganization observed during spermatogenesis . we found that , in contrast to earlier stages , in elongating spermatids , h4k5 and h4k8 acetylation and butyrylation become more markedly associated with specific regions of the genome . , 2013 ) , suggesting that the brdt - bound histone population enters this pathway . here , we show that in elongating spermatids , butyrylated histones survive this wave of acetylation - dependent histone removal and degradation . these observations suggest that a stable differential labeling of h4 by acetylation and butyrylation may control the timing of histone removal . in this context , it is also possible to speculate that nucleosomes bearing butyrylated h4 could undergo a direct histone - to - prm replacement due to the inability of brdt to mediate the exchange of histones by tps . differential histone tail acylation might also play important roles in the control of somatic cell gene expression , differentiation , and genome programming . indeed , a recent study of p300-mediated histone h3k18 crotonylation revealed that an increase in cellular levels of crotonyl - coa favors a more efficient de novo gene activation ( sabari et al . although the precise mechanism underlying the role of h3k18cr in transcription is unknown , the differential affinity of a bromodomain protein toward crotonyl versus acetyl marks could conceivably account for the observed phenomenon . previous reports demonstrated a rapid turnover of histone acetylation on a sub - fraction of nucleosomes ( clayton et al . , however , the functional implications of the short half - life of histone acetylation on active chromatin regions , especially on highly transcribed gene tsss , have remained elusive . given the differential affinity of brdt bd1 for the acetylated and butyrylated forms of h4k5 , a rapid alternation of acylation states at h4k5 would result in a highly dynamic interaction between brdt and chromatin . such a mechanism , while maintaining histones permanently modified , would allow for a dynamic association with specific bromodomains , which might be important for sustaining successive cycles of transcription . , 2014 ) or differential activities of histone deacetylase ( hdacs ) in removing acyl - groups ( rousseaux and khochbin , 2015 ) could reprogram gene expression profiles . in conclusion , we provide here the first demonstration that the interchangeable use of two closely related histone acylation marks at a specific site , h4k5 , has important functional consequences by modulating the ability of a transcriptional regulator , brdt , to recognize chromatin . pan anti - kbu antibody , anti - histone site - specific kbu , and acetyllysine antibodies used were purchased from ptm biolabs and anti - histone h3 and anti - histone h4 antibodies were from abcam . acetylation and butyrylation of histones were determined on 200 g of core histones extracted from the different studied cell types . , 2011 ) and are detailed in the supplemental information .

physical inactivity is a modifiable risk factor for the development of numerous chronic diseases including obesity , type 2 diabetes , hypertension , cardiovascular disease , and certain types of cancers ( 4 , 36 ) . studies have demonstrated that chronic mild - to - moderate intensity aerobic exercise elicits improvements in multiple traditional cardiovascular risk factors ( e.g. , fasting glucose , blood lipids , blood pressure ) ( 5 , 19 , 34 ) , as well as vascular health biomarkers ( 20 ) , and endothelial function ( 2 , 17 , 21 ) . these documented improvements of indices pertaining to vascular health and endothelial function have included increased flow - mediated dilation ( fmd ) , nitric oxide ( no ) , and interleukin-10 ( il-10 ) , as well as decreased endothelial microparticles ( emp ) , c - reactive protein ( crp ) , and interleukin-6 ( il-6 ) . these improvements are particularly significant because endothelial dysfunction has been considered to be an initial step in the pathogenic process underlying atherosclerosis development and has been associated with hypertension , a risk factor for cardiovascular disease ( e.g. , myocardial infarction , stroke , and heart failure ) , renal failure , and mortality ( 7 , 28 ) . aerobic exercise training ( aext ) has been associated with favorable endothelial adaptations due to increased blood flow from shear stress ( 9 , 22 , 25 , 31 ) . in addition , the subsequent improvements in clinical health values and vascular health biomarkers may lead to the development of preventive measures to reduce hypertension and the risk of cardiovascular disease . according to the u.s . department of health and human services ( dhhs ) , african american adults are 40% more likely to have hypertension than their caucasian counterparts ( 27 ) . given the disparate prevalence of the condition among african americans , as well as the well - documented influence of aerobic exercise on vascular health , particular attention should be given to the implementation and evaluation of exercise programs as a clinical intervention for this population . exercise adherence , the degree to which an individual complies with his or her program , is central to evaluating the efficacy of exercise interventions and ultimately addressing health disparities ( 23 ) . adherence may be influenced by a range of factors , including those related to the individual ( e.g. , demographic and psychological characteristics ) , the environment ( e.g. , social support and accessibility ) , as well as the behavior itself ( e.g. , intensity , duration , and perceived effort ) ( 33 ) . studies indicate that greater levels of adherence yield favorable outcomes ( 12 , 13 , 15 ) ; likewise , without adherence to exercise protocols , physiological improvements may be attenuated or may not occur ( 23 , 26 , 36 ) . adherence to exercise prescriptions is often mentioned , yet seldom reported in clinical interventions . therefore , exercise prescriptions and subsequent improvements can not be sufficiently evaluated unless adherence is accurately measured . in clinical interventions , exercise adherence is often measured as a dichotomous variable , where one either completes the treatment ( i.e. , adheres ) , or does not complete the treatment ( i.e. , does not adhere ) . studies have investigated the effects of aext on improvements in clinical and vascular health outcomes ( 4 , 16 ) ; however , few have examined the role that varied levels of adherence may play in this process , particularly among at - risk populations . the purpose of this study was to evaluate whether measures of exercise adherence were related to clinical outcome measures and indices of vascular health subsequent to a 6-month aext intervention in a middle - to - older aged african american cohort . participants included in the study were middle - to - older aged ( 40 71 y / o ) african americans at low - to - moderate risk for cardiovascular disease as previously described ( 2 ) and otherwise apparently healthy as defined by the american college of sports medicine ( acsm ) ( 1 ) . baseline testing included office blood pressure measurements , fmd studies , fasting blood sampling , and graded exercise testing . clinical laboratory values measured included body mass index ( bmi ) , maximal oxygen uptake ( vo2max ) , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , total cholesterol , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides , and fasting blood glucose . emp , crp , il-10 , il-6 , and no were measured in the plasma samples , and fmd was measured in the brachial artery as previously described ( 2 , 14 , 35 ) . participants engaged in a 6-month ( 24-week ) aerobic exercise program under direct supervision of lab personnel , 3x / week , starting with 20 minutes of exercise per session at an intensity equivalent to 50% of vo2max . training duration was increased 5 minutes each week until 40 minutes of exercise at 50% of vo2max was achieved . upon achieving 40 minutes of exercise , training intensity was increased 5% each week until 65% of vo2max was achieved . by week eight , participants reached the desired exercise duration of 40 minutes and an intensity at 65% of vo2max , which they maintained as their exercise prescription for the remainder of the study . exercise modes included treadmill walking / jogging , stair stepping , stationary cycling , elliptical cross - training , and rowing and arm ergometry . the participants used heart rate monitors during exercise in order to monitor exercise intensity , and study personnel recorded heart rates every 10 minutes to ensure adherence to the prescribed training program . cardiovascular fitness was determined by a submaximal treadmill test and individualized exercise prescriptions were developed for the aext intervention as described previously ( 2 ) . measures of adherence were calculated to account for physiological adaptations that may occur during exercise training as well as detraining effect . three different adherence measures were calculated including exercise percentage , exercise volume , and exercise score . exercise percentage was calculated by taking the 24-week program divided by the actual number of weeks that it took each participant to complete the program up to a maximum of 52 weeks ( 1 year ) . participants were required to repeat a week s exercise prescription if they did not attend at least two sessions in a given week and maintain a 60% minimum exercise percentage . in order to demonstrate whether a higher volume of exercise elicited greater physiological adaptations , exercise volume was calculated by adding the total number of minutes that the subject completed during the training program . lastly , exercise score was calculated by taking the exercise volume divided by the number of weeks it took each participant to complete the program . this adherence measure considered current guidelines for exercise including frequency , intensity , and duration ( 1 ) , and when compared to adherence volume , this measurement specifically considered whether frequency of exercise influenced physiological response . among the 42 participants who completed the 6-month aext intervention , the data used in the statistical analysis for each clinical laboratory value were bmi ( n=42 ) , vo2max ( n=41 ) , sbp ( n=41 ) , dbp ( n=41 ) , total cholesterol ( n=36 ) , ldl - c ( n=36 ) , hdl - c ( n=36 ) , triglycerides ( n=36 ) , and fasting blood glucose ( n=34 ) . the data used for each vascular health variable were fmd testing ( n=26 ) , no ( n=24 ) , cd62e+ emps ( n=28 ) , cd31 + 42- emps ( n=28 ) , crp ( n=37 ) , il-6 ( n=32 ) , and il-10 ( n=26 ) . the differences in sample sizes of each variable are related to issues such as inability to schedule subjects , failure to acquire blood samples , or faulty assay procedures . data are expressed as mean the standard error of the mean ( sem ) . the distribution of all variables was examined using the shapiro - wilk test of normality . pre- and post - aext were compared using the paired samples wilcoxon signed - rank test . spearman s rho was used to calculate correlations between the adherence measures and the changes in clinical values as well as changes in vascular health biomarkers . all statistical analyses were performed using spss version 21.0 ( spss inc . , chicago , il ) . participants included in the study were middle - to - older aged ( 40 71 y / o ) african americans at low - to - moderate risk for cardiovascular disease as previously described ( 2 ) and otherwise apparently healthy as defined by the american college of sports medicine ( acsm ) ( 1 ) . baseline testing included office blood pressure measurements , fmd studies , fasting blood sampling , and graded exercise testing . clinical laboratory values measured included body mass index ( bmi ) , maximal oxygen uptake ( vo2max ) , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , total cholesterol , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides , and fasting blood glucose . emp , crp , il-10 , il-6 , and no were measured in the plasma samples , and fmd was measured in the brachial artery as previously described ( 2 , 14 , 35 ) . participants engaged in a 6-month ( 24-week ) aerobic exercise program under direct supervision of lab personnel , 3x / week , starting with 20 minutes of exercise per session at an intensity equivalent to 50% of vo2max . training duration was increased 5 minutes each week until 40 minutes of exercise at 50% of vo2max was achieved . upon achieving 40 minutes of exercise , training intensity was increased 5% each week until 65% of vo2max was achieved . by week eight , participants reached the desired exercise duration of 40 minutes and an intensity at 65% of vo2max , which they maintained as their exercise prescription for the remainder of the study . exercise modes included treadmill walking / jogging , stair stepping , stationary cycling , elliptical cross - training , and rowing and arm ergometry . the participants used heart rate monitors during exercise in order to monitor exercise intensity , and study personnel recorded heart rates every 10 minutes to ensure adherence to the prescribed training program . cardiovascular fitness was determined by a submaximal treadmill test and individualized exercise prescriptions were developed for the aext intervention as described previously ( 2 ) . measures of adherence were calculated to account for physiological adaptations that may occur during exercise training as well as detraining effect . three different adherence measures were calculated including exercise percentage , exercise volume , and exercise score . exercise percentage was calculated by taking the 24-week program divided by the actual number of weeks that it took each participant to complete the program up to a maximum of 52 weeks ( 1 year ) . participants were required to repeat a week s exercise prescription if they did not attend at least two sessions in a given week and maintain a 60% minimum exercise percentage . in order to demonstrate whether a higher volume of exercise elicited greater physiological adaptations , exercise volume was calculated by adding the total number of minutes that the subject completed during the training program . lastly , exercise score was calculated by taking the exercise volume divided by the number of weeks it took each participant to complete the program . this adherence measure considered current guidelines for exercise including frequency , intensity , and duration ( 1 ) , and when compared to adherence volume , this measurement specifically considered whether frequency of exercise influenced physiological response . among the 42 participants who completed the 6-month aext intervention , the data used in the statistical analysis for each clinical laboratory value were bmi ( n=42 ) , vo2max ( n=41 ) , sbp ( n=41 ) , dbp ( n=41 ) , total cholesterol ( n=36 ) , ldl - c ( n=36 ) , hdl - c ( n=36 ) , triglycerides ( n=36 ) , and fasting blood glucose ( n=34 ) . the data used for each vascular health variable were fmd testing ( n=26 ) , no ( n=24 ) , cd62e+ emps ( n=28 ) , cd31 + 42- emps ( n=28 ) , crp ( n=37 ) , il-6 ( n=32 ) , and il-10 ( n=26 ) . the differences in sample sizes of each variable are related to issues such as inability to schedule subjects , failure to acquire blood samples , or faulty assay procedures . data are expressed as mean the standard error of the mean ( sem ) . the distribution of all variables was examined using the shapiro - wilk test of normality . pre- and post - aext were compared using the paired samples wilcoxon signed - rank test . spearman s rho was used to calculate correlations between the adherence measures and the changes in clinical values as well as changes in vascular health biomarkers . all statistical analyses were performed using spss version 21.0 ( spss inc . , chicago , il ) . the study group consisted of 42 african american men ( n=6 ; 14.3% ) and women ( n=36 ; 85.7% ) . ideal adherence would have been defined as a subject achieving an exercise percentage of 100% , resulting in an exercise volume of 2730.0 minutes and an exercise score of 113.8 . the calculated adherence variables included the following : exercise percentage ( 81.6 2.0% ; range : 61.0 100.0% ) , exercise volume ( 2920.8 86.8 min ; range : 2330.0 4650.0 min ) , and exercise score ( 98.0 2.8 ; range : 69.3 166.1 ) . the clinical laboratory values of the participants measured prior and subsequent to the aext intervention are presented in table 1 . the 6-month aext intervention significantly increased vo2max and significantly decreased bmi , plasma triglycerides , and fasting blood glucose . total cholesterol , ldl cholesterol , hdl cholesterol , and mean systolic and diastolic blood pressure were not significantly changed following the aext intervention . the vascular health biomarkers : fmd , no , cd62e+ emp , cd31 + 42- emp , crp , il-6 , and il-10 , considered indices of vascular health , were measured prior and subsequent to the aext intervention and are presented in table 2 . the 6-month aext intervention significantly increased fmd and no and significantly decreased cd62e+ emps , cd31+cd42- emps , crp , and il-6 . there was an increase in il-10 following the aext intervention ; however , the increase was not significant . there was a significant positive correlation between exercise volume and the change in systolic blood pressure , and a significant positive correlation between exercise score and the change in hdl cholesterol subsequent to the aext intervention . there were no other significant correlations between any of the adherence measures and the changes in clinical values of the participants . changes in vascular health biomarkers were not significantly correlated with any of the adherence measures . research data have demonstrated that significant physiological adaptations may occur subsequent to aext interventions ( 3 , 5 , 9 , 22 , 25 , 29 , 30 ) . the results from the present study expand upon our previous findings that aext is a nonpharmacologic treatment modality that may improve health and reduce cardiovascular risk in middle - to - older aged african american adults free from overt cardiovascular disease . specifically , we have reported that aext demonstrated improvements in both clinical outcome measures as well as vascular health markers ( 2 , 14 , 35 ) . as a complement to this previous work , the present study was designed to elucidate whether levels of exercise adherence played a significant role in the favorable physiological adaptations in this african american cohort . the primary findings of this study revealed that the significant improvements in clinical and vascular health measures pre - post aext in this cohort were not associated with any of the measured levels of exercise adherence . the minimal significant associations between the adherence measures and clinical health measures did not include any of the clinical health measures that were significantly changed pre - post aext . to our knowledge , this study is novel because it is the first study that has evaluated relationships between measured indices of exercise adherence and improvements in physiological markers subsequent to aext in a middle - to - older aged african american cohort . exercise adherence is not uniformly quantified in clinical research ; therefore , this should be taken into account when interpreting and comparing study findings . currently , there is no gold standard in adherence measurement ( 6 , 32 ) ; however , miller and colleagues suggest that quantifying adherence as cumulative exercise exposure , rather than simply sessions attended , may yield more accurate outcome data ( 24 ) . adherence in clinical trials has been measured a number of ways , including pedometry ( 13 ) , accelerometry ( 11 ) , self - monitoring through exercise diaries ( 23 ) , dividing kilocalories expended by kilocalories prescribed ( 8 , 11 ) , and calculating minutes completed within prescribed target heart rate range divided by total minutes prescribed ( 18 ) . the results of the aforementioned studies collectively have demonstrated favorable outcomes in body composition , clinical markers , and aerobic fitness levels . lack of adherence to clinical exercise interventions is another factor contributing to the difficulty in assessing adherence . a number of demographic , physiological , and psychological factors are associated with one s adherence to an exercise program , including cardiovascular disease risk , body mass index , exercise enjoyment , past exercise behavior , social support , and self - efficacy for exercise ( 10 , 33 , 36 ) . several strategies for enhancing adherence have been identified , including cognitive behavioral , social support , and programming approaches . one of the strategies utilized in this intervention was to gradually increase the duration and intensity of exercise throughout the program in order to minimize pain and fatigue , and to maximize feelings of exercise self - efficacy . additionally , tokens for public transit , as well as complimentary parking , were provided in order to enhance convenience . in the present study , the amount of exercise each subject completed was objectively quantified by recording frequency and duration , and heart rate monitoring was used to assess intensity . furthermore , exercise adherence was calculated three ways in order to comprehensively measure and assess the relationship between exercise adherence and physiological adaptations to exercise . the first method of measuring adherence , exercise percentage , was calculated by dividing 24 ( the number of weeks comprising the exercise prescription ) by the number of weeks it took each subject to complete the 24-week program . if participants did not attend at least two sessions in a given week , they would be required to repeat that week s exercise prescription . this method took into account the adaptations that occur from regular exercise training , as well as the detraining effects that may occur from interrupted participation . the second method of measuring adherence , exercise volume , was measured by calculating the total minutes of exercise each participant completed while enrolled in the study . participants who took longer to complete the program typically obtained higher exercise volume scores due to repeating the exercise prescription for specific weeks where they may not have met the requirements for 23 sessions in a given week . according to the acsm , it is recommended that most adults engage in moderate - intensity cardiorespiratory exercise training for a total of 150 minutes per week ; however , the exercise program should be modified according to an individual s health status , physical function , habitual physical activity , exercise responses , and stated goals ( 15 ) . in addition , adults who are unwilling or unable to meet the exercise targets outlined in acsm s position stand may still benefit from engaging in amounts of exercise less than recommended ( 15 ) . therefore , this measure of adherence considered whether the total number of minutes achieved by this african american cohort was adequate to elicit favorable physiological adaptations . in order to account for the variable amount of time it took the participants to complete the 24-week equivalent exercise training program , a third method of measuring adherence this measure was calculated by dividing each participant s total exercise minutes ( exercise volume ) by the actual number of weeks it took the participant to complete the program . this final method of calculating adherence took into account the frequency of exercise participation , given that participants worked at uniform intensities ( beginning at 50% vo2max and working up to 65% vo2max ) . exercise intensity was closely regulated with study personnel recording participants exercise heart rates every 10 minutes to ensure they were working at their prescribed intensities . this measurement permitted exercise dose ( i.e. , intensity , duration , and frequency ) to be closely monitored , which is necessary in order to amply interpret outcome variables ( 26 ) . as previously noted , the exercise scores of participants who were required to repeat weeks of exercise may have been impacted by their higher exercise volumes . while statistically significant improvements following aext in both clinical and vascular health measures were observed in this african american cohort , these improvements were not related to the participants levels of adherence to the exercise prescription . only two clinical variables were significantly correlated with exercise adherence in the present study : systolic blood pressure and hdl cholesterol . systolic blood pressure was positively correlated with exercise volume ( total minutes ) ; however , there was no relationship between systolic blood pressure and exercise percentage or exercise score . hdl cholesterol was positively correlated with exercise score , but not exercise percentage or exercise volume . the findings demonstrated that there were no statistically significant differences in systolic blood pressure and hdl cholesterol pre - post aext . further analysis revealed cohen s effect size value for the change in systolic blood pressure ( d=0.06 ) and the change in hdl cholesterol ( d=0.14 ) , and also suggests low practical significance . in addition , none of the vascular health markers were significantly correlated with any exercise adherence measure . exercise adherence in this population at levels lower than recommended in acsm s position stand may prove to have clinical and vascular health benefits . results from the present study indicate that participants benefited from aext regardless of their individual level of exercise adherence as defined in this study . exercise adherence is often difficult to quantify ; thus adherence was conceptualized three separate ways in order to comprehensively measure the construct . participants in this study improved their vascular health and decreased their risk factors for cardiovascular disease , and these favorable alterations were not associated with the measured indices of adherence to the exercise prescription . this suggests that the axiom , some exercise is better than none , may be particularly true when working with members of this at - risk population . future studies should continue to accurately quantify adherence in order to assess the exercise dose for improvement in vascular and clinical health . as previously mentioned , findings from this study demonstrate little significance between levels of exercise adherence and changes in physiological outcomes ; however , the current study had a number of limitations that should be addressed . first , the study sample size was relatively small , but this was due to the exclusions to create a more homogenous group in order to eliminate confounding variables that may influence clinical or vascular health markers . despite the small sample size , significant changes were observed in four clinical outcomes and all but one vascular health marker . second , it is important to consider that while levels of adherence varied among participants in the current study , participants who were unable to make satisfactory progress toward completion of the program ( i.e. , not attending at least 60% of sessions ) were asked to discontinue participation in the study . based on the literature , it is reasonable to deduce that excluded participants that fell below this study s minimum adherence requirements would not have experienced similar improvements in clinical and vascular health measures ( 23 , 26 , 36 ) . finally , the sample population was predominantly female african americans ; therefore , our findings may not be generalizable to african american males or other ethnic populations . accurately measuring adherence is essential because one must engage in ample aext ( e.g. , frequency , intensity , duration ) in order for physiological adaptations to occur , and ultimately , to receive health benefits . in the present study , improvements in clinical and vascular health as a result of aext were observed regardless of the variation in the exercise adherence indices measured ; however , all of the participants in this cohort met the minimum requirements for adherence as outlined in the study . given the lack of correlations between levels of exercise adherence and significant improvements in clinical and vascular health measures , achieving high levels of adherence may have less merit ; however , this warrants further investigation . future studies should further examine the role of adherence in the dose - response relationship between aext and clinical and vascular health outcomes . based on these findings , participating in a regular aext program below published guidelines for healthy populations in order to achieve minimum physiological benefits may result in unforeseen clinical and/or physiological beneficial effects in certain populations .

improvements in indices of vascular health and endothelial function have been inversely associated with hypertension , a risk factor for cardiovascular disease ( e.g. , myocardial infarction , stroke , and heart failure ) , renal failure , and mortality . aerobic exercise training ( aext ) has been positively associated with improvements in clinical health values , as well as vascular health biomarkers , and endothelial function . the purpose of this study was to evaluate whether measures of exercise adherence were related to clinical outcome measures and indices of vascular health subsequent to a 6-month aext intervention in a middle - to - older aged african american cohort . following dietary stabilization , sedentary , apparently healthy , african american adults ( 40 71 y / o ) underwent baseline testing including blood pressure , flow - mediated dilation ( fmd ) studies , fasting blood sampling , and graded exercise testing . upon completion of a supervised 6-month aext intervention , participants repeated all baseline tests . exercise adherence was measured three ways : exercise percentage , exercise volume , and exercise score . there were no significant correlations between the changes in the vascular health biomarkers of the participants and any of the adherence measures . in addition , there were no significant correlations between any of the adherence measures and the clinical values of the participants that had been significantly changed pre - post - aext . participants improved their clinical and vascular health and decreased risk factors for hypertension and cardiovascular disease regardless of their level of adherence to aext . future studies should continue to accurately quantify adherence in order to assess the exercise dose for improvements in vascular and clinical health .

INTRODUCTION METHODS Participants Protocol Statistical Analysis RESULTS DISCUSSION

physical inactivity is a modifiable risk factor for the development of numerous chronic diseases including obesity , type 2 diabetes , hypertension , cardiovascular disease , and certain types of cancers ( 4 , 36 ) . studies have demonstrated that chronic mild - to - moderate intensity aerobic exercise elicits improvements in multiple traditional cardiovascular risk factors ( e.g. , fasting glucose , blood lipids , blood pressure ) ( 5 , 19 , 34 ) , as well as vascular health biomarkers ( 20 ) , and endothelial function ( 2 , 17 , 21 ) . these documented improvements of indices pertaining to vascular health and endothelial function have included increased flow - mediated dilation ( fmd ) , nitric oxide ( no ) , and interleukin-10 ( il-10 ) , as well as decreased endothelial microparticles ( emp ) , c - reactive protein ( crp ) , and interleukin-6 ( il-6 ) . these improvements are particularly significant because endothelial dysfunction has been considered to be an initial step in the pathogenic process underlying atherosclerosis development and has been associated with hypertension , a risk factor for cardiovascular disease ( e.g. , myocardial infarction , stroke , and heart failure ) , renal failure , and mortality ( 7 , 28 ) . aerobic exercise training ( aext ) has been associated with favorable endothelial adaptations due to increased blood flow from shear stress ( 9 , 22 , 25 , 31 ) . in addition , the subsequent improvements in clinical health values and vascular health biomarkers may lead to the development of preventive measures to reduce hypertension and the risk of cardiovascular disease . department of health and human services ( dhhs ) , african american adults are 40% more likely to have hypertension than their caucasian counterparts ( 27 ) . given the disparate prevalence of the condition among african americans , as well as the well - documented influence of aerobic exercise on vascular health , particular attention should be given to the implementation and evaluation of exercise programs as a clinical intervention for this population . adherence may be influenced by a range of factors , including those related to the individual ( e.g. , demographic and psychological characteristics ) , the environment ( e.g. , social support and accessibility ) , as well as the behavior itself ( e.g. studies indicate that greater levels of adherence yield favorable outcomes ( 12 , 13 , 15 ) ; likewise , without adherence to exercise protocols , physiological improvements may be attenuated or may not occur ( 23 , 26 , 36 ) . in clinical interventions , exercise adherence is often measured as a dichotomous variable , where one either completes the treatment ( i.e. studies have investigated the effects of aext on improvements in clinical and vascular health outcomes ( 4 , 16 ) ; however , few have examined the role that varied levels of adherence may play in this process , particularly among at - risk populations . the purpose of this study was to evaluate whether measures of exercise adherence were related to clinical outcome measures and indices of vascular health subsequent to a 6-month aext intervention in a middle - to - older aged african american cohort . participants included in the study were middle - to - older aged ( 40 71 y / o ) african americans at low - to - moderate risk for cardiovascular disease as previously described ( 2 ) and otherwise apparently healthy as defined by the american college of sports medicine ( acsm ) ( 1 ) . baseline testing included office blood pressure measurements , fmd studies , fasting blood sampling , and graded exercise testing . clinical laboratory values measured included body mass index ( bmi ) , maximal oxygen uptake ( vo2max ) , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , total cholesterol , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides , and fasting blood glucose . emp , crp , il-10 , il-6 , and no were measured in the plasma samples , and fmd was measured in the brachial artery as previously described ( 2 , 14 , 35 ) . participants engaged in a 6-month ( 24-week ) aerobic exercise program under direct supervision of lab personnel , 3x / week , starting with 20 minutes of exercise per session at an intensity equivalent to 50% of vo2max . by week eight , participants reached the desired exercise duration of 40 minutes and an intensity at 65% of vo2max , which they maintained as their exercise prescription for the remainder of the study . the participants used heart rate monitors during exercise in order to monitor exercise intensity , and study personnel recorded heart rates every 10 minutes to ensure adherence to the prescribed training program . measures of adherence were calculated to account for physiological adaptations that may occur during exercise training as well as detraining effect . three different adherence measures were calculated including exercise percentage , exercise volume , and exercise score . exercise percentage was calculated by taking the 24-week program divided by the actual number of weeks that it took each participant to complete the program up to a maximum of 52 weeks ( 1 year ) . in order to demonstrate whether a higher volume of exercise elicited greater physiological adaptations , exercise volume was calculated by adding the total number of minutes that the subject completed during the training program . lastly , exercise score was calculated by taking the exercise volume divided by the number of weeks it took each participant to complete the program . this adherence measure considered current guidelines for exercise including frequency , intensity , and duration ( 1 ) , and when compared to adherence volume , this measurement specifically considered whether frequency of exercise influenced physiological response . among the 42 participants who completed the 6-month aext intervention , the data used in the statistical analysis for each clinical laboratory value were bmi ( n=42 ) , vo2max ( n=41 ) , sbp ( n=41 ) , dbp ( n=41 ) , total cholesterol ( n=36 ) , ldl - c ( n=36 ) , hdl - c ( n=36 ) , triglycerides ( n=36 ) , and fasting blood glucose ( n=34 ) . the data used for each vascular health variable were fmd testing ( n=26 ) , no ( n=24 ) , cd62e+ emps ( n=28 ) , cd31 + 42- emps ( n=28 ) , crp ( n=37 ) , il-6 ( n=32 ) , and il-10 ( n=26 ) . pre- and post - aext were compared using the paired samples wilcoxon signed - rank test . spearman s rho was used to calculate correlations between the adherence measures and the changes in clinical values as well as changes in vascular health biomarkers . participants included in the study were middle - to - older aged ( 40 71 y / o ) african americans at low - to - moderate risk for cardiovascular disease as previously described ( 2 ) and otherwise apparently healthy as defined by the american college of sports medicine ( acsm ) ( 1 ) . baseline testing included office blood pressure measurements , fmd studies , fasting blood sampling , and graded exercise testing . clinical laboratory values measured included body mass index ( bmi ) , maximal oxygen uptake ( vo2max ) , systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , total cholesterol , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides , and fasting blood glucose . emp , crp , il-10 , il-6 , and no were measured in the plasma samples , and fmd was measured in the brachial artery as previously described ( 2 , 14 , 35 ) . participants engaged in a 6-month ( 24-week ) aerobic exercise program under direct supervision of lab personnel , 3x / week , starting with 20 minutes of exercise per session at an intensity equivalent to 50% of vo2max . by week eight , participants reached the desired exercise duration of 40 minutes and an intensity at 65% of vo2max , which they maintained as their exercise prescription for the remainder of the study . the participants used heart rate monitors during exercise in order to monitor exercise intensity , and study personnel recorded heart rates every 10 minutes to ensure adherence to the prescribed training program . measures of adherence were calculated to account for physiological adaptations that may occur during exercise training as well as detraining effect . three different adherence measures were calculated including exercise percentage , exercise volume , and exercise score . in order to demonstrate whether a higher volume of exercise elicited greater physiological adaptations , exercise volume was calculated by adding the total number of minutes that the subject completed during the training program . lastly , exercise score was calculated by taking the exercise volume divided by the number of weeks it took each participant to complete the program . this adherence measure considered current guidelines for exercise including frequency , intensity , and duration ( 1 ) , and when compared to adherence volume , this measurement specifically considered whether frequency of exercise influenced physiological response . among the 42 participants who completed the 6-month aext intervention , the data used in the statistical analysis for each clinical laboratory value were bmi ( n=42 ) , vo2max ( n=41 ) , sbp ( n=41 ) , dbp ( n=41 ) , total cholesterol ( n=36 ) , ldl - c ( n=36 ) , hdl - c ( n=36 ) , triglycerides ( n=36 ) , and fasting blood glucose ( n=34 ) . the data used for each vascular health variable were fmd testing ( n=26 ) , no ( n=24 ) , cd62e+ emps ( n=28 ) , cd31 + 42- emps ( n=28 ) , crp ( n=37 ) , il-6 ( n=32 ) , and il-10 ( n=26 ) . pre- and post - aext were compared using the paired samples wilcoxon signed - rank test . spearman s rho was used to calculate correlations between the adherence measures and the changes in clinical values as well as changes in vascular health biomarkers . ideal adherence would have been defined as a subject achieving an exercise percentage of 100% , resulting in an exercise volume of 2730.0 minutes and an exercise score of 113.8 . the calculated adherence variables included the following : exercise percentage ( 81.6 2.0% ; range : 61.0 100.0% ) , exercise volume ( 2920.8 86.8 min ; range : 2330.0 4650.0 min ) , and exercise score ( 98.0 2.8 ; range : 69.3 166.1 ) . the clinical laboratory values of the participants measured prior and subsequent to the aext intervention are presented in table 1 . the 6-month aext intervention significantly increased vo2max and significantly decreased bmi , plasma triglycerides , and fasting blood glucose . total cholesterol , ldl cholesterol , hdl cholesterol , and mean systolic and diastolic blood pressure were not significantly changed following the aext intervention . the vascular health biomarkers : fmd , no , cd62e+ emp , cd31 + 42- emp , crp , il-6 , and il-10 , considered indices of vascular health , were measured prior and subsequent to the aext intervention and are presented in table 2 . the 6-month aext intervention significantly increased fmd and no and significantly decreased cd62e+ emps , cd31+cd42- emps , crp , and il-6 . there was a significant positive correlation between exercise volume and the change in systolic blood pressure , and a significant positive correlation between exercise score and the change in hdl cholesterol subsequent to the aext intervention . there were no other significant correlations between any of the adherence measures and the changes in clinical values of the participants . changes in vascular health biomarkers were not significantly correlated with any of the adherence measures . research data have demonstrated that significant physiological adaptations may occur subsequent to aext interventions ( 3 , 5 , 9 , 22 , 25 , 29 , 30 ) . the results from the present study expand upon our previous findings that aext is a nonpharmacologic treatment modality that may improve health and reduce cardiovascular risk in middle - to - older aged african american adults free from overt cardiovascular disease . specifically , we have reported that aext demonstrated improvements in both clinical outcome measures as well as vascular health markers ( 2 , 14 , 35 ) . as a complement to this previous work , the present study was designed to elucidate whether levels of exercise adherence played a significant role in the favorable physiological adaptations in this african american cohort . the primary findings of this study revealed that the significant improvements in clinical and vascular health measures pre - post aext in this cohort were not associated with any of the measured levels of exercise adherence . the minimal significant associations between the adherence measures and clinical health measures did not include any of the clinical health measures that were significantly changed pre - post aext . to our knowledge , this study is novel because it is the first study that has evaluated relationships between measured indices of exercise adherence and improvements in physiological markers subsequent to aext in a middle - to - older aged african american cohort . exercise adherence is not uniformly quantified in clinical research ; therefore , this should be taken into account when interpreting and comparing study findings . adherence in clinical trials has been measured a number of ways , including pedometry ( 13 ) , accelerometry ( 11 ) , self - monitoring through exercise diaries ( 23 ) , dividing kilocalories expended by kilocalories prescribed ( 8 , 11 ) , and calculating minutes completed within prescribed target heart rate range divided by total minutes prescribed ( 18 ) . lack of adherence to clinical exercise interventions is another factor contributing to the difficulty in assessing adherence . a number of demographic , physiological , and psychological factors are associated with one s adherence to an exercise program , including cardiovascular disease risk , body mass index , exercise enjoyment , past exercise behavior , social support , and self - efficacy for exercise ( 10 , 33 , 36 ) . several strategies for enhancing adherence have been identified , including cognitive behavioral , social support , and programming approaches . one of the strategies utilized in this intervention was to gradually increase the duration and intensity of exercise throughout the program in order to minimize pain and fatigue , and to maximize feelings of exercise self - efficacy . additionally , tokens for public transit , as well as complimentary parking , were provided in order to enhance convenience . in the present study , the amount of exercise each subject completed was objectively quantified by recording frequency and duration , and heart rate monitoring was used to assess intensity . furthermore , exercise adherence was calculated three ways in order to comprehensively measure and assess the relationship between exercise adherence and physiological adaptations to exercise . the first method of measuring adherence , exercise percentage , was calculated by dividing 24 ( the number of weeks comprising the exercise prescription ) by the number of weeks it took each subject to complete the 24-week program . this method took into account the adaptations that occur from regular exercise training , as well as the detraining effects that may occur from interrupted participation . the second method of measuring adherence , exercise volume , was measured by calculating the total minutes of exercise each participant completed while enrolled in the study . participants who took longer to complete the program typically obtained higher exercise volume scores due to repeating the exercise prescription for specific weeks where they may not have met the requirements for 23 sessions in a given week . according to the acsm , it is recommended that most adults engage in moderate - intensity cardiorespiratory exercise training for a total of 150 minutes per week ; however , the exercise program should be modified according to an individual s health status , physical function , habitual physical activity , exercise responses , and stated goals ( 15 ) . in addition , adults who are unwilling or unable to meet the exercise targets outlined in acsm s position stand may still benefit from engaging in amounts of exercise less than recommended ( 15 ) . therefore , this measure of adherence considered whether the total number of minutes achieved by this african american cohort was adequate to elicit favorable physiological adaptations . in order to account for the variable amount of time it took the participants to complete the 24-week equivalent exercise training program , a third method of measuring adherence this measure was calculated by dividing each participant s total exercise minutes ( exercise volume ) by the actual number of weeks it took the participant to complete the program . , intensity , duration , and frequency ) to be closely monitored , which is necessary in order to amply interpret outcome variables ( 26 ) . as previously noted , the exercise scores of participants who were required to repeat weeks of exercise may have been impacted by their higher exercise volumes . while statistically significant improvements following aext in both clinical and vascular health measures were observed in this african american cohort , these improvements were not related to the participants levels of adherence to the exercise prescription . only two clinical variables were significantly correlated with exercise adherence in the present study : systolic blood pressure and hdl cholesterol . systolic blood pressure was positively correlated with exercise volume ( total minutes ) ; however , there was no relationship between systolic blood pressure and exercise percentage or exercise score . hdl cholesterol was positively correlated with exercise score , but not exercise percentage or exercise volume . the findings demonstrated that there were no statistically significant differences in systolic blood pressure and hdl cholesterol pre - post aext . further analysis revealed cohen s effect size value for the change in systolic blood pressure ( d=0.06 ) and the change in hdl cholesterol ( d=0.14 ) , and also suggests low practical significance . in addition , none of the vascular health markers were significantly correlated with any exercise adherence measure . exercise adherence in this population at levels lower than recommended in acsm s position stand may prove to have clinical and vascular health benefits . results from the present study indicate that participants benefited from aext regardless of their individual level of exercise adherence as defined in this study . exercise adherence is often difficult to quantify ; thus adherence was conceptualized three separate ways in order to comprehensively measure the construct . participants in this study improved their vascular health and decreased their risk factors for cardiovascular disease , and these favorable alterations were not associated with the measured indices of adherence to the exercise prescription . future studies should continue to accurately quantify adherence in order to assess the exercise dose for improvement in vascular and clinical health . as previously mentioned , findings from this study demonstrate little significance between levels of exercise adherence and changes in physiological outcomes ; however , the current study had a number of limitations that should be addressed . first , the study sample size was relatively small , but this was due to the exclusions to create a more homogenous group in order to eliminate confounding variables that may influence clinical or vascular health markers . second , it is important to consider that while levels of adherence varied among participants in the current study , participants who were unable to make satisfactory progress toward completion of the program ( i.e. based on the literature , it is reasonable to deduce that excluded participants that fell below this study s minimum adherence requirements would not have experienced similar improvements in clinical and vascular health measures ( 23 , 26 , 36 ) . accurately measuring adherence is essential because one must engage in ample aext ( e.g. in the present study , improvements in clinical and vascular health as a result of aext were observed regardless of the variation in the exercise adherence indices measured ; however , all of the participants in this cohort met the minimum requirements for adherence as outlined in the study . given the lack of correlations between levels of exercise adherence and significant improvements in clinical and vascular health measures , achieving high levels of adherence may have less merit ; however , this warrants further investigation . future studies should further examine the role of adherence in the dose - response relationship between aext and clinical and vascular health outcomes . based on these findings , participating in a regular aext program below published guidelines for healthy populations in order to achieve minimum physiological benefits may result in unforeseen clinical and/or physiological beneficial effects in certain populations .

the human hand is an effective and efficient manipulator of the environment with which it interacts . next to the face , the hand is the most familiar region of the body as it is frequently on display , rarely covered and is a source of familiarity through interpersonal communication and physical interaction . within the biometrics and forensic science communities , the hand has been widely accepted for its role in the verification of identity , particularly in relation to variation recognised in palmar and digital epidermal prints [ 4 , 5 ] . however , the dorsum of the hand has also proved to be of value for such purposes , and features that have been investigated include variation in knuckle skin crease patterns , pigmentation distribution and superficial vein patterns [ 69 ] . the basic function of this appendage is to interact with the immediate environment , and , as a result , it is vulnerable to physical insults that may alter or modify its appearance . over 20 % of patients attending accident and emergency ( a&e ) departments in the uk each year have a hand injury , which equates to over 1.36 million attendances each year , of which 5 % will require surgery . isolated hand and wrist injuries accounted for 6.6 % of all new a&e admissions in northern ireland with a consensus that probably between 10 % and 20 % of all admissions are related to hand injuries [ 13 , 14 ] . this general incidence rate appears to hold true in other countries with the research by rosberg and dahlin in malmo , sweden , concurring that 12 % of a&e admissions relate to hand injuries . rosberg and dahlin and hill et al . suggested that the demographic most at risk from hand injuries are young males between the ages of 11 and 25 years . rosberg and dahlin also reported that injuries occur more frequently to the border regions of the hand and specifically to the thumb or small finger . it is reported that around 75 % of the population is right - handed and 15 % left - handed with the ability to be truly ambidextrous being relatively rare [ 16 , 17 ] . the dominance of the hand is reported to have a significant influence of the incidence of injury with the dominant hand more likely to show injury than the non - dominant hand . hill et al . agreed with this proposal but found that the non - dominant hand was at increased risk of injury when a knife was involved with a ratio of 1:3.4 for dominant to non - dominant hands . other common causes of injury ( broken glass , opening a tin , fall on hand , hand caught between objects , hand through window / door , etc . ) showed a higher ratio of injuries to the dominant over the non - dominant hand . sorock et al . found that right - handed people injured their left hand in 55.4 % of cases and left - handed people injured their right hand in 57.7 % of cases . it is generally held that scars resulting from medical intervention are of lesser value for verification purposes than scars that originate from an accidental aetiology as , for the former , there are common factors whereas accidental scars offer more random characteristics such as location , size , healing pattern , etc . whilst most surgical scars can be readily differentiated from accidental scars mainly due to their appearance and position in relation to langer s lines , in some instances , especially if the scars are small , it may be difficult to diagnose aetiology with accuracy . however , in reality , the incidence of surgical scars is considered to be very small compared with accidental scars , the majority of which will never result in a visit to a&e , let alone an operating theatre , and therefore , for the purposes of verification of identity , they may in fact be misrepresented in terms of their discriminatory value for the purposes of identification . within the biometrics and forensic science communities , the hand is viewed as being sufficiently individuating to fulfil the requirements for verification of identity but is considered to be of lesser value in the establishment of identity . for the purposes of verification , two data sets are required that can be compared for features of similarity and difference . the strength of that relationship can then be expressed on the basis of likelihood ratios that the two sets either arise from the same individual or from a different individual [ 2426 ] . guidelines for the forensic comparison of images within the uk were published by the national policing improvement agency on behalf of the association of chief police officers , and the document laid out several important considerations in relation to image comparison for the purpose of verification of identity in a legal setting . although aimed at comparison of the face , this document applies equally well to comparison of other regions of the body including the hand . image comparison depends strongly on the quality of the images , and the npia document stated that identification through comparison of images does not have evidential value unless it demonstrates morphologically comparable features. it also highlighted the importance that such comparisons illustrate the significance of points of similarity and difference and that it must highlight presence and absence of features . also highlighted in this report was the importance of probability factors and likelihood of features being present or absent in comparative images . the presence of scarring on the dorsum of the hand has been utilised as a feature for consideration within the multifactorial process of hand image comparison . within the centre for anatomy and human identification at the university of dundee , image comparisons are undertaken of the anatomical features of the dorsum of the hand primarily for the purposes of comparing accused to offender images . in the majority of cases , these involve images of child sexual abuse where the dorsum of the hand of the perpetrator is present . the requirement for this form of analysis is on the increase with casework showing a tenfold rise in the past 5 years . the sharing of images of child pornography is recognised as one of the fastest - growing crimes of modern times [ 28 , 29 ] with close to a million new photographs being posted on the internet daily . ease of access to the world wide web for the purposes of sharing images and the ease of availability of self - operated recording devices to perpetrators of sexual abuse is of concern to the law enforcement profession , as the sharing of images and the demand that is generated has added a new dimension to the social problem of child protection and the investigation and prosecution of child sexual offenses [ 3033 ] . as scars are a common feature in the comparison of these images , it is essential that an understanding of their incidence is more fully understood . the aim of this research was to assess the incidence and position of scars on the dorsum of the hand to facilitate an understanding of the value of hand scars in the process of verification of identity . a subsequent publication will utilise this information and use it to construct likelihood ratios for current casework analysis . the centre for anatomy and human identification at the university of dundee holds an active database of hand images using both visual and infrared light . at the time of this research , the database consisted of images of pairs of hands from 260 individuals of both sexes drawn from a cadre of disaster victim identification specialists undertaking training at the university of dundee . two hundred thirty - eight participants permitted personal information to be recorded , including sex and hand dominance , resulting in 177 males and 61 females ( 476 hands ) being available for this research ( table 1 ) . the age range for the sample was 2162 years with a mean age of 43 years , and all participants were of european ancestry . as per the expected incidence , approximately 10 % were left - handed and close to 90 % were right - handed . any individual with single or multiple digit amputations was not included nor were any individuals with developmental or congenital abnormalities.table 1sample separated according to hand dominance and sexright - handedleft - handedambidextroustotalmale156210177female555161total211261238 sample separated according to hand dominance and sex the dorsal surface of each hand was divided into 24 cells by way of a deformation grid , with each cell assigned an identification number ( fig . 1 and table 2 ) . twenty - seven landmarks were selected to permit construction of this grid , and they were chosen because of their anatomical stability and visibility in all images . once each hand was grid - mapped , then the presence of scarring was recorded ( fig . 2 ) . for each hand , the number , type and approximate size of a scar was recorded for each of the 24 cells , for both the right and left hand of each individual.fig . 1location of the 27 hand landmarks and the formation of the deformation grid that arises from these to result in 24 grid cells for recording purposestable 2definition of the points that permit construction of the hand gridlandmark1most medial point on wrist constriction2most lateral point on wrist constriction3deepest point in the interdigital webbing between digits 1 and 24most lateral and prominent point over the 1st metacarpophalangeal ( mcp ) joint5deepest point in the interdigital webbing between digits 2 and 36deepest point in the interdigital webbing between digits 3 and 47deepest point in the interdigital webbing between digits 4 and 58medial border of the base of digit 5 parallel to the mcp joint9lateral border of base of digit 2 parallel to the mcp joint10medial border of thumb parallel to the interphalangeal ( ip ) joint11lateral border of the thumb parallel to the ip joint12medial border of digit 5 parallel to the proximal interphalangeal ( pip ) joint13lateral border of digit 5 parallel to the pip joint14medial border of digit 4 parallel to the pip joint15lateral border of digit 4 parallel to the pip joint16medial border of digit 3 parallel to the pip joint17lateral border of digit 3 parallel to the pip joint18medial border of digit 2 parallel to the pip joint19lateral border of digit 2 parallel to the pip joint20medial border of digit 5 parallel to the distal interphalangeal ( dip ) joint21lateral border of digit 5 parallel to the dip joint22medial border of digit 4 parallel to the dip joint23lateral border of digit 4 parallel to the dip joint24medial border of digit 3 parallel to the dip joint25lateral border of digit 3 parallel to the dip joint26medial border of digit 2 parallel to the dip joint27lateral border of digit 2 parallel to the dip jointfig . the deformation grid has been placed according to the landmark points ( straight lines ) and the positions of scars are outlined by black circles location of the 27 hand landmarks and the formation of the deformation grid that arises from these to result in 24 grid cells for recording purposes definition of the points that permit construction of the hand grid cropped image from the database of the right hand of a female participant . the deformation grid has been placed according to the landmark points ( straight lines ) and the positions of scars are outlined by black circles the number of scars was a simple count of the scars present within a grid . if a scar crossed a grid line , it was recorded within the grid where the majority of the scar resided . scar type referred to whether the scar was linear or non - linear in its presentation . size of the scar was assessed using the photographic scale marker included in each image . scars 5 mm were classified as small , scars between 6 and 9 mm were medium and scars 10 mm were recorded as being large . only inactive scars were counted , and recent injuries to a hand that still showed inflammatory reactions were not included . table 3 shows the distribution of scar incidence across the 24 grid cells in the right and left hands of the males and females from the sample . linear and non - linear scars have been combined in this table so that a total of 251 scars were found in the male right sample ( average of 1.42 scars per hand ) , 348 in the male left sample ( average of 1.97 per hand ) , 95 in the female right sample ( average of 1.56 per hand ) and 51 in the female left sample ( average of 0.84 per hand ) . combining right and left hands resulted in an average of 3.38 scars for each male and 2.39 scars for each female . in reality , 49 % of male right hands did not show any scarring nor did 40 % of male left hands , 44 % of female right hands or 57 % of female left hands . forty - five males were scar - free ( 25 % of male sample ) showing scarring on neither their right nor their left hands whilst 16 females ( 26 % of female sample ) were devoid of scarring on both hands . the largest number of scars seen in any one hand was 14 on a male right , 16 on a male left , 10 on a female right and 6 on a female left . the largest number of scars seen in the combined right and left hands of any individual was 23 on a male and 14 on a female . mann whitney u testing showed that scars were more common in males than females ( p < 0.001 ) , more common in male rights than male lefts ( p < 0.001 ) and more common in female lefts than in female rights ( p < 0.001).table 3number of scars found in each cell separated for sex and sidecell no.male rightmale leftfemale rightfemale left142151241343313136844202531006240071431133869419261876100401111019521217257413171373143231158163216492117273413318233273191282020710002122183222111845231213002421500total2513489551 number of scars found in each cell separated for sex and side using the data in table 3 , fig . 3 was constructed to show where approximately 50 % of the scarring in each hand occurred . for male right hands , the highest incidence of scarring was found in grid cells 9 , 17 , 18 and 21 which equated to 39 % of scarring . to raise the total to 53 % scarring , grid cells 12 and 13 had to be added . for male left hands , the highest incidence of scarring was found in grid cells 7 , 17 and 18 which equated to 28 % of all scarring . to raise the total to 56 % of all scarring , grid cells 1 , 9 , 12 for female right hands , the highest incidence of scarring was found in grid cells 7 and 17 which equated to 28 % of all scarring . to raise the total to 55 % of all scarring , grid cells 9 , 12 , 13 and 18 had to be added . for female left hands , the highest incidence of scarring occurred in grid cells 3 , 9 and 22 which equated to 38 % of all scarring . to raise the total to 46 % required cells 2 , 7 , 12 , 13 , 17 and 18 to be added . when cells were examined for a common pattern in relation to where approximately 50 % of scarring occurs , grid cells 9 , 12 , 17 and 18 were common to all four groups ( side and sex ) and grid cells 7 and 13 were common to three of the four groups . figure 3 shows the common positions within the hand for the incidence of scarring , so that the distal region of the hand appears to be more commonly scarred , particularly in relation to the index and middle fingers.fig . 3location of approximately 50 % of scarring on the dorsum of the hand in right and left hands of males and females location of approximately 50 % of scarring on the dorsum of the hand in right and left hands of males and females figure 4 shows the location of what can be described as the medial border ( grid cells 5 , 10 , 15 , 20 and 24 ) , the lateral border ( grid cells 1 , 6 , 11 and 16 ) , the index finger corridor ( grid cells 7 , 12 , 17 and 21 ) and the middle finger corridor ( grid cells 9 , 13 , 18 and 22 ) . table 4 shows the incidence and location of scars in these regions separated by sex and side . an average of 7 % of all scars occur along the medial border , 8.5 % along the lateral border , 31 % in the index finger corridor and 33 % in the middle finger corridor.fig . 4location of the medial and lateral border segments and the index and middle finger corridor regionstable 4percentage incidence of scarring in different regions of the hand for rights and lefts in both sexesmalefemaleaveragerightleftrightleftindex corridor cells 7 , 12 , 17 , 213231382431middle corridor cells 9 , 13 , 18 , 223123453333lateral border cells 1 , 6 , 11 , 16815388.5medial border cells 5 , 10 , 15 , 20 , 24813347 location of the medial and lateral border segments and the index and middle finger corridor regions percentage incidence of scarring in different regions of the hand for rights and lefts in both sexes tables 5 and 6 show the incidence of scars in each of the 24 grid cells separated by scar type and size for males and females , respectively . table 5 shows that , in the male right hand , linear scars are more than twice as common as non - linear scars and mann whitney u tests show this to be highly significantly different ( p < 0.001 ) . linear scars are most commonly found in grid cells 9 , 12 , 17 , 18 and 21 whereas non - linear scars are most frequently found in grid cells 9 and 17 . small scars are the most common size ( 66 % of all scars in the male right hand ) followed by medium scars ( 26 % of all scars ) , and large scars were least common ( 8 % of all scars ) . small scars were most frequently found in grid cells 9 , 17 and 21 , with medium scars most frequently found in grid cells 3 , 9 and 18 . if large scars did occur , then they were most frequently found in grid cells 3 and 7 . in the male left hand , linear scars were more common than non - linear scars by a factor of over 4 , and mann whitney u tests showed this to be highly significantly different ( p < 0.001 ) . linear scars were found most frequently in grid cells 7 , 12 , 17 and 18 whereas non - linear scars were most frequently found in grid cells 1 and 18 . small scars were the most common ( 65 % of all scars ) and were most frequently located in grid cells 12 , 17 and 18 . medium scars were less common ( 24 % of all scars in the male left hand ) and were most commonly found in grid cells 7 and 11 whereas large scars were the least common ( 11 % of all scars ) and occurred most frequently in grid cell 1.table 5number of scars ( linear and non - linear ) in male right and left hands , in each cell , separated according to scar sizecell no.male rightmale leftlinearnon - linearsmallmediumlargelinearnon - linearsmallmediumlarge1400401389572312111036343121373948324223012002052121010100620200404007131455265151248331325461291610178112613501000000403011182451154710212152125023218431312514211218321430120202001553611142853163130172810171611242132226441816714812482642191029218062020703406472121175175014414402256110015311612357111013085024202001141230total181701656620282662268438table 6number of scars ( linear and non - linear ) in female right and left hands , separate according to scar sizecell no.female rightfemale leftlinearlon - linearsmallmediumlargelinearnon - linearsmallmediumlarge1501311010022231021300351141534314000002001150000000000600000000007941021301208313011000196160115600100000001100115050020110125252022220135252030300143030010100153011120110161111010100179493121210185261030300192011000000200000000000212130020200223122050140230000000000240000000000total732265237381333153 number of scars ( linear and non - linear ) in male right and left hands , in each cell , separated according to scar size number of scars ( linear and non - linear ) in female right and left hands , separate according to scar size table 6 shows that , in the female right hand , linear scars are more than three times as common as non - linear scars , and mann whitney u tests showed this to be highly significant ( p < 0.001 ) . linear scars are most commonly found in grid cells 7 and 17 , and although non - linear do not have a high occurrence , they tend to be found in the same grid cells as linear scars . small scars are the most common size ( 68 % of all scars in the female right hand ) followed by medium scars ( 24 % of all scars ) , and then large scars were least common ( 7 % of all scars ) . small scars were most frequently found in grid cells 7 and 17 with medium and large scars not showing a particularly high incidence in any grid cell . in the female left hand , linear scars were more common than non - linear scars by a factor of almost 3 , and again this reached statistical significance ( p < 0.01 ) . linear scars were found most frequently in grid cells 3 and 22 whilst non - linear scars were most frequently found in grid cell 9 . small scars were the most common ( 65 % of all scars in the female left hand ) and were most frequently located in grid cell 9 . medium scars were less common ( 29 % of all scars ) , and large scars were the least common ( 6 % of all scars ) . the incidence of medium and large scars did not permit identification of a most frequent grid cell . only three individuals presented with identifiable surgical scars ( 1.3 % of the total sample ) , and all were found in males and classified as large scars . one was located on the left hand in grid cell 3 , and the other two were located on the right and in grid cells 3 and 4 . the phrase the hand is the window onto the mind has been attributed to the german philosopher immanuel kant from around 1798 [ 34 , 35 ] , and certainly , the analysis of the hand has intrigued science and the public alike through their portrayal in both art and literature . that the intimate anatomy of the hand can be of investigative value was exploited by conan doyle in his novel a study in scarlet where holmes pontificated that by a man 's finger - nails , by his coat - sleeve , by his boots , by his trouser - knees , by the callosities of his forefinger and thumb , by his expression , by his shirt - cuff by each of these things a man 's calling is plainly revealed.. whilst neither finger callosities nor nails form the core of this investigation , it is evident that the persistent scars on the back of the hand paint an interesting picture in terms of marks that can be utilised as present or absent morphological features in the comparison of images for the purposes of verification of identity . previous research has indicated that males are more likely to present a larger number of scars than females , and their location will be influenced by the dominance of the hand [ 12 , 15 , 19 ] . the results of this study agree with this in part . approximately 50 % more scars occurred in male hands than in female hands . interestingly , although males showed a greater incidence on their left hands , females showed a greater incidence on their right hands . this is unlikely to be explained by hand dominance as it was shown in table 1 that the percentage of hand dominance was similar in the two groups and agreed with the general literature . therefore , there is a suggestion that the male left hand which is usually the non - dominant hand is more prone to injury than the dominant hand ( right ) whilst the reverse is the situation for females with the dominant hand being more susceptible to injury . it is possible that this result reflects differences in the type of injuries sustained by males and females [ 12 , 18 ] , and although this seems like a very stereotypical explanation , it is difficult to find a reasonable alternative other than males are more likely to scar their non - dominant hand as a result of perhaps injury caused by an implement in the dominant hand , whereas female scarring is less implement - based and more accidental in aetiology as described by hill et al . . it would appear from this research that , perhaps as expected , surgical scars are rare within the data base sample , and when they do occur , they are most commonly represented in cells 3 and 4 . the most common hand surgery is largely concerned with degenerative change including median nerve compression and arthritis , and this is more common in an elderly sample than is perhaps represented by the database cohort . on the dorsum of the hand , surgical intervention scars occur most frequently over the joint regions to address arthroplasty , but in this research , the most common surgical scars were in the region over the intercarpal joints . the database is not necessarily representative of the general population , and it is important that the database be increased significantly so that comparisons may be made within appropriate populational sectors . for the purposes of verification of identity , it has been shown that non - linear scars are less common as are scars in excess of 6 mm . therefore , these categories may initially be thought of as being of greater discriminatory value , but in reality the multiplicity of small scars ( such as seen in fig . 2 ) and their random placing due to their accidental nature resulted in no multiple scar patterning within the database being identical to any other . therefore , whilst in isolation , the larger and non - linear scars may prove to be of greater value ; the pattern of location of two or more small scars has proved to be of considerable importance in the formation of a two - dimensional locational map . contrary to the findings of rosberg and dahlin , the medial and lateral borders of the hand are not the most common location for the persistence of scarring in this sample . only 78 % of all scarring occurred along a hand border , and this was equally true for males and females and for rights and lefts . however , the highest incidence of scarring occurred along the index and middle corridor regions , where 64 % of all scarring in the hand was located . this is a clear indication that a large proportion of the scarring found in this sample has resulted from interaction injury with both dominant and non - dominant fingers being susceptible to small scale traumas . for the purposes of image comparison , scars are a useful trait to examine for presence or absence , similarity or differences in shape , size and position . they are most commonly accidental in nature and therefore not predictable per se , although the incidence of scarring along the index and middle fingers is likely to be higher than in any other part of the hand . in males , scars are more likely to occur in the index and middle finger corridors of the non - dominant hand , and in the majority of casework undertaken , we have identified that it is the left non - dominant hand of the perpetrator that tends to be captured in images , and in the majority of cases , it is the thumb , index and middle finger regions that are most frequently observed . therefore , it is exceptionally important to be aware of the incidence of scarring in this region in the general population and how that might relate to the profile of the offender hand [ 37 , 38 ] . it is accepted that the occupational origin of the individuals within a database will be important as it is likely that the more manual work that is undertaken then the greater will be the incidence of scarring . the database utilised in this communication is of considerable value , but ongoing additions to the resource from different societal sectors will allow future inferences to be made regarding the incidence of scarring in different groups . the subsequent paper will examine current casework and the probability that the perpetrator and suspect was the same person , based solely on scar - related information sourced from this communication . it is essential that the anatomical interpretation of features of the hand is fully documented if expert opinions are to be proffered on the likelihood of two individuals showing matching features .

when undertaking image comparison of the hand between accused and perpetrator , it is not unusual for scars to be identified on the back of the hand . to investigate the occurrence of scarring in a discreet sample , a database of 238 individuals was examined , and the dorsum of the right and left hands was gridded for each individual . the position , size and type of scar were recorded within each grid . it was found that , in general , males exhibited a higher incidence of scarring than females . however , males were more likely to show scarring on their left hand whereas females were more likely to exhibit scarring on their right hand . contrary to the literature , scarring was not most prevalent along the borders of the hand but occurred more frequently in association with the index and middle finger corridor regions . surgical scars were rare as were large scars whereas linear scars smaller than 6 mm were the most frequently identified . close to half of the sample did not exhibit scarring on one hand . the importance of understanding the pattern of scarring on the back of the hand is discussed in the light of forensic image comparison analysis .

Introduction Material and methods Results Discussion

next to the face , the hand is the most familiar region of the body as it is frequently on display , rarely covered and is a source of familiarity through interpersonal communication and physical interaction . however , the dorsum of the hand has also proved to be of value for such purposes , and features that have been investigated include variation in knuckle skin crease patterns , pigmentation distribution and superficial vein patterns [ 69 ] . the basic function of this appendage is to interact with the immediate environment , and , as a result , it is vulnerable to physical insults that may alter or modify its appearance . rosberg and dahlin also reported that injuries occur more frequently to the border regions of the hand and specifically to the thumb or small finger . it is reported that around 75 % of the population is right - handed and 15 % left - handed with the ability to be truly ambidextrous being relatively rare [ 16 , 17 ] . the dominance of the hand is reported to have a significant influence of the incidence of injury with the dominant hand more likely to show injury than the non - dominant hand . found that right - handed people injured their left hand in 55.4 % of cases and left - handed people injured their right hand in 57.7 % of cases . however , in reality , the incidence of surgical scars is considered to be very small compared with accidental scars , the majority of which will never result in a visit to a&e , let alone an operating theatre , and therefore , for the purposes of verification of identity , they may in fact be misrepresented in terms of their discriminatory value for the purposes of identification . within the biometrics and forensic science communities , the hand is viewed as being sufficiently individuating to fulfil the requirements for verification of identity but is considered to be of lesser value in the establishment of identity . guidelines for the forensic comparison of images within the uk were published by the national policing improvement agency on behalf of the association of chief police officers , and the document laid out several important considerations in relation to image comparison for the purpose of verification of identity in a legal setting . although aimed at comparison of the face , this document applies equally well to comparison of other regions of the body including the hand . image comparison depends strongly on the quality of the images , and the npia document stated that identification through comparison of images does not have evidential value unless it demonstrates morphologically comparable features. the presence of scarring on the dorsum of the hand has been utilised as a feature for consideration within the multifactorial process of hand image comparison . within the centre for anatomy and human identification at the university of dundee , image comparisons are undertaken of the anatomical features of the dorsum of the hand primarily for the purposes of comparing accused to offender images . in the majority of cases , these involve images of child sexual abuse where the dorsum of the hand of the perpetrator is present . as scars are a common feature in the comparison of these images , it is essential that an understanding of their incidence is more fully understood . the aim of this research was to assess the incidence and position of scars on the dorsum of the hand to facilitate an understanding of the value of hand scars in the process of verification of identity . for each hand , the number , type and approximate size of a scar was recorded for each of the 24 cells , for both the right and left hand of each individual.fig . 1location of the 27 hand landmarks and the formation of the deformation grid that arises from these to result in 24 grid cells for recording purposestable 2definition of the points that permit construction of the hand gridlandmark1most medial point on wrist constriction2most lateral point on wrist constriction3deepest point in the interdigital webbing between digits 1 and 24most lateral and prominent point over the 1st metacarpophalangeal ( mcp ) joint5deepest point in the interdigital webbing between digits 2 and 36deepest point in the interdigital webbing between digits 3 and 47deepest point in the interdigital webbing between digits 4 and 58medial border of the base of digit 5 parallel to the mcp joint9lateral border of base of digit 2 parallel to the mcp joint10medial border of thumb parallel to the interphalangeal ( ip ) joint11lateral border of the thumb parallel to the ip joint12medial border of digit 5 parallel to the proximal interphalangeal ( pip ) joint13lateral border of digit 5 parallel to the pip joint14medial border of digit 4 parallel to the pip joint15lateral border of digit 4 parallel to the pip joint16medial border of digit 3 parallel to the pip joint17lateral border of digit 3 parallel to the pip joint18medial border of digit 2 parallel to the pip joint19lateral border of digit 2 parallel to the pip joint20medial border of digit 5 parallel to the distal interphalangeal ( dip ) joint21lateral border of digit 5 parallel to the dip joint22medial border of digit 4 parallel to the dip joint23lateral border of digit 4 parallel to the dip joint24medial border of digit 3 parallel to the dip joint25lateral border of digit 3 parallel to the dip joint26medial border of digit 2 parallel to the dip joint27lateral border of digit 2 parallel to the dip jointfig . the deformation grid has been placed according to the landmark points ( straight lines ) and the positions of scars are outlined by black circles location of the 27 hand landmarks and the formation of the deformation grid that arises from these to result in 24 grid cells for recording purposes definition of the points that permit construction of the hand grid cropped image from the database of the right hand of a female participant . if a scar crossed a grid line , it was recorded within the grid where the majority of the scar resided . table 3 shows the distribution of scar incidence across the 24 grid cells in the right and left hands of the males and females from the sample . linear and non - linear scars have been combined in this table so that a total of 251 scars were found in the male right sample ( average of 1.42 scars per hand ) , 348 in the male left sample ( average of 1.97 per hand ) , 95 in the female right sample ( average of 1.56 per hand ) and 51 in the female left sample ( average of 0.84 per hand ) . combining right and left hands resulted in an average of 3.38 scars for each male and 2.39 scars for each female . forty - five males were scar - free ( 25 % of male sample ) showing scarring on neither their right nor their left hands whilst 16 females ( 26 % of female sample ) were devoid of scarring on both hands . the largest number of scars seen in the combined right and left hands of any individual was 23 on a male and 14 on a female . mann whitney u testing showed that scars were more common in males than females ( p < 0.001 ) , more common in male rights than male lefts ( p < 0.001 ) and more common in female lefts than in female rights ( p < 0.001).table 3number of scars found in each cell separated for sex and sidecell no.male rightmale leftfemale rightfemale left142151241343313136844202531006240071431133869419261876100401111019521217257413171373143231158163216492117273413318233273191282020710002122183222111845231213002421500total2513489551 number of scars found in each cell separated for sex and side using the data in table 3 , fig . for male right hands , the highest incidence of scarring was found in grid cells 9 , 17 , 18 and 21 which equated to 39 % of scarring . for male left hands , the highest incidence of scarring was found in grid cells 7 , 17 and 18 which equated to 28 % of all scarring . to raise the total to 56 % of all scarring , grid cells 1 , 9 , 12 for female right hands , the highest incidence of scarring was found in grid cells 7 and 17 which equated to 28 % of all scarring . figure 3 shows the common positions within the hand for the incidence of scarring , so that the distal region of the hand appears to be more commonly scarred , particularly in relation to the index and middle fingers.fig . 3location of approximately 50 % of scarring on the dorsum of the hand in right and left hands of males and females location of approximately 50 % of scarring on the dorsum of the hand in right and left hands of males and females figure 4 shows the location of what can be described as the medial border ( grid cells 5 , 10 , 15 , 20 and 24 ) , the lateral border ( grid cells 1 , 6 , 11 and 16 ) , the index finger corridor ( grid cells 7 , 12 , 17 and 21 ) and the middle finger corridor ( grid cells 9 , 13 , 18 and 22 ) . an average of 7 % of all scars occur along the medial border , 8.5 % along the lateral border , 31 % in the index finger corridor and 33 % in the middle finger corridor.fig . 4location of the medial and lateral border segments and the index and middle finger corridor regionstable 4percentage incidence of scarring in different regions of the hand for rights and lefts in both sexesmalefemaleaveragerightleftrightleftindex corridor cells 7 , 12 , 17 , 213231382431middle corridor cells 9 , 13 , 18 , 223123453333lateral border cells 1 , 6 , 11 , 16815388.5medial border cells 5 , 10 , 15 , 20 , 24813347 location of the medial and lateral border segments and the index and middle finger corridor regions percentage incidence of scarring in different regions of the hand for rights and lefts in both sexes tables 5 and 6 show the incidence of scars in each of the 24 grid cells separated by scar type and size for males and females , respectively . table 5 shows that , in the male right hand , linear scars are more than twice as common as non - linear scars and mann whitney u tests show this to be highly significantly different ( p < 0.001 ) . small scars are the most common size ( 66 % of all scars in the male right hand ) followed by medium scars ( 26 % of all scars ) , and large scars were least common ( 8 % of all scars ) . in the male left hand , linear scars were more common than non - linear scars by a factor of over 4 , and mann whitney u tests showed this to be highly significantly different ( p < 0.001 ) . linear scars were found most frequently in grid cells 7 , 12 , 17 and 18 whereas non - linear scars were most frequently found in grid cells 1 and 18 . small scars were the most common ( 65 % of all scars ) and were most frequently located in grid cells 12 , 17 and 18 . medium scars were less common ( 24 % of all scars in the male left hand ) and were most commonly found in grid cells 7 and 11 whereas large scars were the least common ( 11 % of all scars ) and occurred most frequently in grid cell 1.table 5number of scars ( linear and non - linear ) in male right and left hands , in each cell , separated according to scar sizecell no.male rightmale leftlinearnon - linearsmallmediumlargelinearnon - linearsmallmediumlarge1400401389572312111036343121373948324223012002052121010100620200404007131455265151248331325461291610178112613501000000403011182451154710212152125023218431312514211218321430120202001553611142853163130172810171611242132226441816714812482642191029218062020703406472121175175014414402256110015311612357111013085024202001141230total181701656620282662268438table 6number of scars ( linear and non - linear ) in female right and left hands , separate according to scar sizecell no.female rightfemale leftlinearlon - linearsmallmediumlargelinearnon - linearsmallmediumlarge1501311010022231021300351141534314000002001150000000000600000000007941021301208313011000196160115600100000001100115050020110125252022220135252030300143030010100153011120110161111010100179493121210185261030300192011000000200000000000212130020200223122050140230000000000240000000000total732265237381333153 number of scars ( linear and non - linear ) in male right and left hands , in each cell , separated according to scar size number of scars ( linear and non - linear ) in female right and left hands , separate according to scar size table 6 shows that , in the female right hand , linear scars are more than three times as common as non - linear scars , and mann whitney u tests showed this to be highly significant ( p < 0.001 ) . linear scars are most commonly found in grid cells 7 and 17 , and although non - linear do not have a high occurrence , they tend to be found in the same grid cells as linear scars . small scars are the most common size ( 68 % of all scars in the female right hand ) followed by medium scars ( 24 % of all scars ) , and then large scars were least common ( 7 % of all scars ) . small scars were most frequently found in grid cells 7 and 17 with medium and large scars not showing a particularly high incidence in any grid cell . in the female left hand , linear scars were more common than non - linear scars by a factor of almost 3 , and again this reached statistical significance ( p < 0.01 ) . linear scars were found most frequently in grid cells 3 and 22 whilst non - linear scars were most frequently found in grid cell 9 . small scars were the most common ( 65 % of all scars in the female left hand ) and were most frequently located in grid cell 9 . medium scars were less common ( 29 % of all scars ) , and large scars were the least common ( 6 % of all scars ) . the incidence of medium and large scars did not permit identification of a most frequent grid cell . only three individuals presented with identifiable surgical scars ( 1.3 % of the total sample ) , and all were found in males and classified as large scars . one was located on the left hand in grid cell 3 , and the other two were located on the right and in grid cells 3 and 4 . the phrase the hand is the window onto the mind has been attributed to the german philosopher immanuel kant from around 1798 [ 34 , 35 ] , and certainly , the analysis of the hand has intrigued science and the public alike through their portrayal in both art and literature . that the intimate anatomy of the hand can be of investigative value was exploited by conan doyle in his novel a study in scarlet where holmes pontificated that by a man 's finger - nails , by his coat - sleeve , by his boots , by his trouser - knees , by the callosities of his forefinger and thumb , by his expression , by his shirt - cuff by each of these things a man 's calling is plainly revealed.. whilst neither finger callosities nor nails form the core of this investigation , it is evident that the persistent scars on the back of the hand paint an interesting picture in terms of marks that can be utilised as present or absent morphological features in the comparison of images for the purposes of verification of identity . previous research has indicated that males are more likely to present a larger number of scars than females , and their location will be influenced by the dominance of the hand [ 12 , 15 , 19 ] . interestingly , although males showed a greater incidence on their left hands , females showed a greater incidence on their right hands . this is unlikely to be explained by hand dominance as it was shown in table 1 that the percentage of hand dominance was similar in the two groups and agreed with the general literature . it is possible that this result reflects differences in the type of injuries sustained by males and females [ 12 , 18 ] , and although this seems like a very stereotypical explanation , it is difficult to find a reasonable alternative other than males are more likely to scar their non - dominant hand as a result of perhaps injury caused by an implement in the dominant hand , whereas female scarring is less implement - based and more accidental in aetiology as described by hill et al . it would appear from this research that , perhaps as expected , surgical scars are rare within the data base sample , and when they do occur , they are most commonly represented in cells 3 and 4 . on the dorsum of the hand , surgical intervention scars occur most frequently over the joint regions to address arthroplasty , but in this research , the most common surgical scars were in the region over the intercarpal joints . the database is not necessarily representative of the general population , and it is important that the database be increased significantly so that comparisons may be made within appropriate populational sectors . for the purposes of verification of identity , it has been shown that non - linear scars are less common as are scars in excess of 6 mm . therefore , whilst in isolation , the larger and non - linear scars may prove to be of greater value ; the pattern of location of two or more small scars has proved to be of considerable importance in the formation of a two - dimensional locational map . contrary to the findings of rosberg and dahlin , the medial and lateral borders of the hand are not the most common location for the persistence of scarring in this sample . however , the highest incidence of scarring occurred along the index and middle corridor regions , where 64 % of all scarring in the hand was located . for the purposes of image comparison , scars are a useful trait to examine for presence or absence , similarity or differences in shape , size and position . they are most commonly accidental in nature and therefore not predictable per se , although the incidence of scarring along the index and middle fingers is likely to be higher than in any other part of the hand . in males , scars are more likely to occur in the index and middle finger corridors of the non - dominant hand , and in the majority of casework undertaken , we have identified that it is the left non - dominant hand of the perpetrator that tends to be captured in images , and in the majority of cases , it is the thumb , index and middle finger regions that are most frequently observed . therefore , it is exceptionally important to be aware of the incidence of scarring in this region in the general population and how that might relate to the profile of the offender hand [ 37 , 38 ] . it is accepted that the occupational origin of the individuals within a database will be important as it is likely that the more manual work that is undertaken then the greater will be the incidence of scarring . the database utilised in this communication is of considerable value , but ongoing additions to the resource from different societal sectors will allow future inferences to be made regarding the incidence of scarring in different groups . it is essential that the anatomical interpretation of features of the hand is fully documented if expert opinions are to be proffered on the likelihood of two individuals showing matching features .

metal - ceramic restorations have been successfully applied in restorative dentistry due to high fracture strength . however , achieving a natural translucency is more difficult with metal - ceramic restorations than all - ceramic restorations because metal substructures prevent light transmission in metal - ceramic restorations . this has led to an increase in use of all - ceramic restorations . among all - ceramic restorations , however , high translucency of a restoration is not always an advantage , for instance in cases with discolored teeth , colored core materials and cast metal post and cores . in these clinical conditions , a restorative material with optimal masking ability of the underlying substructures a method to evaluate the masking ability of restorative materials is to measure color differences in cie lab color system . in this color system , the l * , a * and b * color parameters and e refer to lightness , red - green value , yellow - blue value , and color difference , respectively , which can be measured by a spectrophotometer . the e color difference is calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 which is the most commonly used formula for e . this formula can detect even a small amount of color difference between two objects [ 9 , 10 ] . as spectrophotometers can detect even the slightest amount of color difference not perceptible by the human eye , perceptual and clinically acceptable thresholds have been defined based on e values . it has been reported that the clinically acceptable threshold is more than the perceptual threshold , because of optical conditions of oral environment . if the e ( color difference ) between two objects is more than the perceptual threshold , a color mismatch will be detected by the human eye . the perceptual threshold has been reported from e=1 to e=5.5 in different studies [ 1214 ] . if a restorative material has an ideal masking ability , e will be equal to zero when placing the material on white and black substrates , namely that the material has a similar color on different substrates , and the substrates can not affect the color of the material . from a clinical point of view , if e of a material on different backgrounds is less than the threshold , it can thoroughly mask the background color . several studies have surveyed the optical properties of different ceramic materials [ 8 , 1518 ] . some studies have evaluated various factors affecting the final color of zirconia restorations including dental substrates [ 1922 ] , luting agents [ 21 , 23 ] , ceramic veneers [ 2426 ] , glaze , and firing process . suputtamongkol et al , reported that the background color could affect the overall color of posterior zirconia restorations on a metal post and core or a prefabricated post and a composite core ; however , color changes did not exceed the perceptual threshold . choi and razzoog assessed the masking ability of a zirconia ceramic ( zrc ) with and without the veneering porcelain and revealed that the zirconium oxide coping material alone had some degree of masking ability . oh and kim disclosed that abutment shade , ceramic thickness and coping type affected the resulting color of zirconia restorations . vichi et al , evaluated the effects of different substrates with different colors on the final color of all - ceramic ips - empress glass - ceramic restorations , and advised to take the color of substrate into consideration in cases with ceramic thickness of less than 1 mm . masking ability of a zrc is related to its color coverage over underlying substructures including cement and dental substrates . it may not be feasible to mask a substrate with cements , because different shades do not exist for all cements . thus , it seems that the color masking ability of a zrc on different substrates may be an important factor affecting the esthetic results of zirconia - based restorations . on the other hand , posts and cores , which are widely applied in restorative dentistry to restore severely damaged teeth the effects of these core materials ( as substrates ) on masking ability of zrcs ( as substructures in zirconia - based restorations ) have to be clearly determined . therefore , this in vitro study aimed to evaluate the effect of three different core materials including a nickel - chromium alloy ( nca ) , a non - precious gold alloy ( npga ) , and a zrc on masking ability of a zrc . the null hypothesis was that the zrc would mask the three aforementioned core materials with no significant difference . the discs were placed over four substrates including a white ( w ) substrate ( as the control ) , nca , npga and zrc . the procedure was conducted as follows : specimen preparation : a computer - aided design / computer - aided manufacturing system ( coritec 250i , imes - icore gmbh , eiterfeld , germany ) milled zirconia blanks ( luminesse high strength / low translucency , 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate zirconia discs . the zirconia blanks had 37% translucency and were used to fabricate zirconia frameworks in zirconia - based restorations . the discs had 0.5 mm thickness and 10 mm diameter [ 20 , 23 ] . all the zirconia discs were sintered at 1520c for a 12-hour process in a sintering furnace ( isint ht , imes - icore gmbh , eiterfeld , germany ) . a digital micrometer ( 293 mdc - mx lite ; mitutoyo corporation , tokyo , japan ) with the accuracy of 0.002 mm an adjustment / polishing kit ( bruxzir ; glidewell direct , irvine , ca , usa ) was used to reduce the thicknesses according to the afore - mentioned acceptable range . in case of lack of acceptable thickness the zirconia discs were polished , cleaned in an ultrasonic bath ( elmasonic s-30 ; dentec , north shore , australia ) containing 98% ethanol for 15 minutes , and were finally dried . the zirconia disc specimens were prepared as such , and had natural white color of zirconia ( fig . 1).substrate preparation : in order to fabricate the w substrate with the dimensions of 10 mm in diameter and 10 mm in height , a white teflon material ( ptfe , omnia plastica spa , busto arsizio , italy ) was milled . then , two cylindrical acrylic resin ( duralay , reliance dental mgf co. , worth , il , usa ) patterns were formed according to the afore - mentioned dimensions . the patterns were cast by a nca ( verabond v ; alba dent , fairfield , ca , usa ) and a npga ( alba dent , fairfield , ca , usa ) to fabricate nca and npga substrates , respectively . an adjusting / polishing kit ( np alloy adjustment kit ; shofu inc . , kyoto , japan ) was employed to polish the alloy substrates . in order to fabricate the zrc substrate , a cylindrical pattern was virtually designed by a software program ( solidworks 2015 , solidworks corp . , the same computer - aided design / computer - aided manufacturing system milled a zirconia blank ( luminesse high strength 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate a zirconia substrate based on the above - mentioned virtual design . the zirconia substrate was dipped in a2 shade color liquid ( dd bio zx2 monolithic zero lzdd ; dental direkt gmbh , spenge , germany ) for 10 seconds . the zirconia substrate was sintered at 1520c for a 12-hour process in the same sintering furnace , and was then polished by the same polishing kit . the four substrates of w , nca , npga , and zrc were prepared as such . all the substrates were cleaned in the same ultrasonic bath containing 98% ethanol for 15 minutes ( fig . 2 ) color measurement : a spectrophotometer ( spectroshade micro , mht , verona , italy ) was employed for spectrometric measurements . a putty silicone material ( speedex ; coltene , altstatten , switzerland ) was adapted to the mouth piece of the spectrophotometer to match the conditions of spectrophotometry for all specimens and to prevent external lights . the specimens were located at the center of this putty mold . before each measurement , the spectro - photometer was calibrated by the white and green calibration plates , respectively . the discs were placed over the substrates with a water drop in - between to prevent the refraction of light . each disc was placed on each of the substrates , and the color measurements were made . all the color measurements were made at the center of specimens marked by a pen on the monitor screen of spectrophotometer . the color parameters of l * , a * , and b * were recorded for each specimen . additionally , e values were calculated to determine the e of disc on the substrates . in order to compare the color of specimens on the nca , npga , and zrc substrates with their color on the w substrate , the e values were measured in three situations including : w - nca , w - npga , and w - zrc . the e w - nca e w - npga , and e w - zrc values were calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 specimen preparation : a computer - aided design / computer - aided manufacturing system ( coritec 250i , imes - icore gmbh , eiterfeld , germany ) milled zirconia blanks ( luminesse high strength / low translucency , 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate zirconia discs . the zirconia blanks had 37% translucency and were used to fabricate zirconia frameworks in zirconia - based restorations . the discs had 0.5 mm thickness and 10 mm diameter [ 20 , 23 ] . all the zirconia discs were sintered at 1520c for a 12-hour process in a sintering furnace ( isint ht , imes - icore gmbh , eiterfeld , germany ) . a digital micrometer ( 293 mdc - mx lite ; mitutoyo corporation , tokyo , japan ) with the accuracy of 0.002 mm an adjustment / polishing kit ( bruxzir ; glidewell direct , irvine , ca , usa ) was used to reduce the thicknesses according to the afore - mentioned acceptable range . in case of lack of acceptable thickness the zirconia discs were polished , cleaned in an ultrasonic bath ( elmasonic s-30 ; dentec , north shore , australia ) containing 98% ethanol for 15 minutes , and were finally dried . the zirconia disc specimens were prepared as such , and had natural white color of zirconia ( fig . substrate preparation : in order to fabricate the w substrate with the dimensions of 10 mm in diameter and 10 mm in height , a white teflon material ( ptfe , omnia plastica spa , busto arsizio , italy ) was milled . then , two cylindrical acrylic resin ( duralay , reliance dental mgf co. , worth , il , usa ) patterns were formed according to the afore - mentioned dimensions . the patterns were cast by a nca ( verabond v ; alba dent , fairfield , ca , usa ) and a npga ( alba dent , fairfield , ca , usa ) to fabricate nca and npga substrates , respectively . an adjusting / polishing kit ( np alloy adjustment kit ; shofu inc . , kyoto , japan ) was employed to polish the alloy substrates . in order to fabricate the zrc substrate , a cylindrical pattern was virtually designed by a software program ( solidworks 2015 , solidworks corp . , the same computer - aided design / computer - aided manufacturing system milled a zirconia blank ( luminesse high strength 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate a zirconia substrate based on the above - mentioned virtual design . the zirconia substrate was dipped in a2 shade color liquid ( dd bio zx2 monolithic zero lzdd ; dental direkt gmbh , spenge , germany ) for 10 seconds . the zirconia substrate was sintered at 1520c for a 12-hour process in the same sintering furnace , and was then polished by the same polishing kit . the four substrates of w , nca , npga , and zrc were prepared as such . all the substrates were cleaned in the same ultrasonic bath containing 98% ethanol for 15 minutes ( fig . 2 ) color measurement : a spectrophotometer ( spectroshade micro , mht , verona , italy ) was employed for spectrometric measurements . a putty silicone material ( speedex ; coltene , altstatten , switzerland ) was adapted to the mouth piece of the spectrophotometer to match the conditions of spectrophotometry for all specimens and to prevent external lights . the specimens were located at the center of this putty mold . before each measurement , the spectro - photometer was calibrated by the white and green calibration plates , respectively . the discs were placed over the substrates with a water drop in - between to prevent the refraction of light . each disc was placed on each of the substrates , and the color measurements were made . all the color measurements were made at the center of specimens marked by a pen on the monitor screen of spectrophotometer . the color parameters of l * , a * , and b * were recorded for each specimen . additionally , e values were calculated to determine the e of disc on the substrates . in order to compare the color of specimens on the nca , npga , and zrc substrates with their color on the w substrate , the e values were measured in three situations including : w - nca , w - npga , and w - zrc . the e w - nca e w - npga , and e w - zrc values were calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 zirconia disc specimens tested substrates ( from left to right : zirconia ceramic , non - precious gold alloy , nickel - chromium alloy and white substrate ) the perceptual threshold of e=2.6 was considered in this study [ 1214 ] . normal distribution of the data was accepted in all groups by the kolmogorov - smirnov test ( p>0.05 ) . the values of l * , a*and b * for each disc in npga , nca and zrc groups were subtracted from l*white , a*white and b*white values , which were the corresponding values for w substrate . in order to compare the standard l * , a * , b * ( from which , the values of w substrate were subtracted ) and e values among the groups , randomized block anova was employed . stata software ( statacorp lp , lakeway , tx , usa ) compared the e values with the predetermined perceptual threshold of e=2.6 using one - sample t - test . the results were explained according to the measured variables of l * , a * , b * , and e in four sections . l * parameter ( lightness ) : the mean and standard deviation of the l * values for the w , nca , npga and zrc groups were 88.351.46 , 78.241.31 , 78.981.26 , and 82.161.16 units , respectively ( table 1 , fig . 3 ) . statistical analysis showed a significant difference among the groups in this regard ( p<0.0001 ) . pairwise comparisons of the groups using bonferroni test revealed significant differences between the nca and zrc ( p<0.0001 ) , and npga and zrc ( p<0.0001 ) . the color parameter values in the four groups of white substrate ( w ) , nickel - chromium alloy ( nca ) , non - precious gold alloy ( npga ) and zirconia ceramic ( zrc ) the mean l * values of the groups a * parameter ( red - green value ) : the mean and standard deviation of the a * values for the w , nca , npga and zrc groups were 0.400.42 , 0.040.52 , 0.600.42 , and 1.520.38 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test revealed significant differences between nca and npga ( p<0.0001 ) , nca and zrc ( p<0.0001 ) and npga and zrc ( p<0.0001 ) . the a * values increased in all the groups compared to the control . the increase in the a the mean a * values of the groups b * parameter ( yellow - blue value ) : the mean and standard deviation of the b * values for the w , nca , npga , and zrc groups were 3.380.36 , 1.921.01 , 3.860.49 , and 4.880.57 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test showed significant differences between nca and npga ( p<0.0001 ) , nca and zrc ( p<0.0001 ) , and npga and zrc ( p<0.001 ) . zrc had the highest b * value , and nca had the lowest b * value among the groups . the mean b * values of the groups e ( color difference ) : the mean and standard deviation of the e values for the nca , npga and zrc groups were 10.262.43 , 9.451.74 , and 6.701.91 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test showed significant differences between nca and zrc ( p<0.0001 ) , and npga and zrc ( p=0.001 ) . the difference between nca and npga was not statistically significant ( p=0.65 ) . in order to compare the e values of the groups with the predetermined perceptual threshold of e=2.6 , the null hypothesis of e 2.6 was rejected for all the groups ( p<0.0001 ) . the present study evaluated the color parameters of l * , a * , b * , and e for zirconia disc specimens on four different substrates including w , nca , npga and zrc . statistical analysis indicated significant differences among the groups in the l * , a * , b * and e values . the examined zrc on the tested substrates showed perceptible color change namely that the examined zrc could not thoroughly mask the tested substrates . the l * ( lightness ) values decreased in all the groups compared with the control . the change in l * value shows the impact of the substrates on the zirconia specimens . this can be explained by the optical properties of zrc , which is a semi - translucent material . according to the results , metal core materials such as ncas and npgas can decrease the lightness of zrc more than a zirconia core material the a * ( red - green ) values increased in all the groups compared to the control . a reason for this may be the natural a * value of the zrc as a substrate . the tested zirconia substrate ( a2 shade ) positively shifted the a * value towards red . the b * ( yellow - blue ) values changed in the groups depending on the substrates . the nickel - chromium substrate decreased the b * value while the non - precious gold and zirconia substrates increased the b * value compared to the control . this may be due to the yellow color tendency of a2 shade zirconia substrate and npga , which impact on the color of zrc and shift it towards yellow . on the contrary , nca shifted the color of zrc towards blue , and negatively affected the b * value . the e values of all groups were more than the predetermined perceptual threshold of e=2.6 . assessment of the l * , a * and b * values indicated their changes in the substrate groups . thus , color changes were derived from all three color parameters . the l * , a * , and b * values manifested significant differences between the substrate groups . this demonstrated that the quality of the color change was different depending on the substrate . the e values manifested no significant difference between the metal substrates ; however , significant differences existed between the zirconia substrate and the metal substrates , and the least color change belonged to the zirconia substrate . this demonstrated that the quantity of the color change was different depending on the substrate as well . hence , both quality and quantity of the color of zrc were altered by the substrates . the used metal substrates did not differ in terms of the quantity of color change , and both affected the quantity of the color change more than the zirconia substrate . suputtamongkol et al , reported that the color of an underlying substructure could affect the overall color of posterior zirconia - based restorations on a metal post and core or a prefabricated post and a composite core , ranging from e = 1.2 to 3.1 . additionally , slight color changes of zirconia crowns were detected by measuring the e . although some differences exist between the afore - mentioned study and the current research such as zrc brands , thicknesses of ceramics , veneered versus non - veneered zrcs , hypothesized perceptual thresholds and substrate types , both studies showed that zrc could not thoroughly mask the underlying materials . oh and kim in an in vitro study of color masking ability assessed the effects of abutment shade , ceramic thickness and coping type on the final color of zirconia restorations . the abutments were fabricated of gold alloy , base metal ( nickel - chromium ) alloy , and four different shades of composite resins . in their study , the average e value of lava specimens between the a2 shade composite resin and gold alloy abutments was higher than that between the a2 shade composite resin and other abutments , and was close to e=5.5 . a similar result was obtained in the current study , although the substrates and zrcs used in the two studies were different . according to the study by oh and kim , gold alloy substrate yielded the highest e among the tested substrates ; however , the npga and the nca yielded close e values in the current study . this dissimilarity in the results may be caused by the color difference of the precious gold alloy used by oh and kim and the npga used in the current study . choi and razzoog evaluated the masking ability of zrc with and without a veneering porcelain . in their study , color differences caused by zrc and the veneering porcelain were compared with substrates alone . they concluded that the non - veneered zrc had some degree of masking ability over different tested substrates ( white , black , gray , and a3 shade tooth - colored substrates ) . however , the current study compared substrate - induced color differences with a zrc over w substrate and concluded that the tested zrc could not mask the examined substrates . choi and razzoog measured the e values between the substrate alone ( as control ) and the zrc over the substrate , while the current study measured the e values between the zrc over w substrate ( as control ) and zrc over the tested substrates . based on the results of our study , the three core materials including nca , npga and zrc can change the color of tested zrc . the color of zrc may be closer to the final color of a restoration than the metal cores . therefore , it is esthetically advised to preferably choose a zrc post and core instead of nca or npga post and cores in zirconia - based restorations and to reduce the core material - induced color change by further core reduction , applying sufficient thickness of the veneering porcelain , and use of proper luting agents in zirconia - based restorations . it should be noted that given that the physical and mechanical properties of zirconia post and core are confirmed in further studies , zirconia post and core should be clinically recommended . this needs future investigations . considering the optical properties of zrc and the thickness of zirconia sub - structures , which is approximately 0.5 mm in a normal case , light transmission through the zirconia structure can be expected in zirconia - based restorations . the present study evaluated the effects of three core materials in this respect . on the other hand , the zirconia core overlaying materials such as porcelain veneers and glaze may influence the final color of zirconia - based restorations , which were not assessed in this study . therefore , evaluation of the effect of other factors in this respect is recommended in future studies . the present study had some limitations such as using a specific brand of zrc , an uncolored zrc , and a specific brand of composite resin . within the limitations of this study , it was concluded that the tested zrc could not thoroughly mask the three core materials of nca , npga , and zrc .

objectives : masking ability of a restorative material plays a role in hiding colored substructures ; however , the masking ability of zirconia ceramic ( zrc ) has not yet been clearly understood in zirconia - based restorations . this study evaluated the effect of three different core materials on masking ability of a zrc.materials and methods : ten zirconia disc samples , 0.5 mm in thickness and 10 mm in diameter , were fabricated . a white ( w ) substrate ( control ) and three substrates of nickel - chromium alloy ( nca ) , non - precious gold alloy ( npga ) , and zrc were prepared . the zirconia discs were placed on the four types of substrates for spectrophotometry . the l * , a * , and b * values of the specimens were measured by a spectrophotometer and color change ( e ) values were calculated to determine color differences between the test and control groups and were then compared with the perceptual threshold . randomized block anova and bonferroni test analyzed the data . a significance level of 0.05 was considered.results:the mean and standard deviation values of e for nca , npga , and zrc groups were 10.262.43 , 9.451.74 , and 6.701.91 units , respectively . significant differences were found in the e values between zrc and the other two experimental groups ( nca and npga ; p<0.0001 and p=0.001 , respectively ) . the e values for the groups were more than the predetermined perceptual threshold.conclusions:within the limitations of this study , it was concluded that the tested zrc could not well mask the examined core materials .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSION

among all - ceramic restorations , however , high translucency of a restoration is not always an advantage , for instance in cases with discolored teeth , colored core materials and cast metal post and cores . in these clinical conditions , a restorative material with optimal masking ability of the underlying substructures a method to evaluate the masking ability of restorative materials is to measure color differences in cie lab color system . in this color system , the l * , a * and b * color parameters and e refer to lightness , red - green value , yellow - blue value , and color difference , respectively , which can be measured by a spectrophotometer . it has been reported that the clinically acceptable threshold is more than the perceptual threshold , because of optical conditions of oral environment . if the e ( color difference ) between two objects is more than the perceptual threshold , a color mismatch will be detected by the human eye . if a restorative material has an ideal masking ability , e will be equal to zero when placing the material on white and black substrates , namely that the material has a similar color on different substrates , and the substrates can not affect the color of the material . from a clinical point of view , if e of a material on different backgrounds is less than the threshold , it can thoroughly mask the background color . some studies have evaluated various factors affecting the final color of zirconia restorations including dental substrates [ 1922 ] , luting agents [ 21 , 23 ] , ceramic veneers [ 2426 ] , glaze , and firing process . suputtamongkol et al , reported that the background color could affect the overall color of posterior zirconia restorations on a metal post and core or a prefabricated post and a composite core ; however , color changes did not exceed the perceptual threshold . choi and razzoog assessed the masking ability of a zirconia ceramic ( zrc ) with and without the veneering porcelain and revealed that the zirconium oxide coping material alone had some degree of masking ability . vichi et al , evaluated the effects of different substrates with different colors on the final color of all - ceramic ips - empress glass - ceramic restorations , and advised to take the color of substrate into consideration in cases with ceramic thickness of less than 1 mm . masking ability of a zrc is related to its color coverage over underlying substructures including cement and dental substrates . thus , it seems that the color masking ability of a zrc on different substrates may be an important factor affecting the esthetic results of zirconia - based restorations . on the other hand , posts and cores , which are widely applied in restorative dentistry to restore severely damaged teeth the effects of these core materials ( as substrates ) on masking ability of zrcs ( as substructures in zirconia - based restorations ) have to be clearly determined . therefore , this in vitro study aimed to evaluate the effect of three different core materials including a nickel - chromium alloy ( nca ) , a non - precious gold alloy ( npga ) , and a zrc on masking ability of a zrc . the null hypothesis was that the zrc would mask the three aforementioned core materials with no significant difference . the discs were placed over four substrates including a white ( w ) substrate ( as the control ) , nca , npga and zrc . the zirconia blanks had 37% translucency and were used to fabricate zirconia frameworks in zirconia - based restorations . the discs had 0.5 mm thickness and 10 mm diameter [ 20 , 23 ] . all the zirconia discs were sintered at 1520c for a 12-hour process in a sintering furnace ( isint ht , imes - icore gmbh , eiterfeld , germany ) . in case of lack of acceptable thickness the zirconia discs were polished , cleaned in an ultrasonic bath ( elmasonic s-30 ; dentec , north shore , australia ) containing 98% ethanol for 15 minutes , and were finally dried . the zirconia disc specimens were prepared as such , and had natural white color of zirconia ( fig . 1).substrate preparation : in order to fabricate the w substrate with the dimensions of 10 mm in diameter and 10 mm in height , a white teflon material ( ptfe , omnia plastica spa , busto arsizio , italy ) was milled . the patterns were cast by a nca ( verabond v ; alba dent , fairfield , ca , usa ) and a npga ( alba dent , fairfield , ca , usa ) to fabricate nca and npga substrates , respectively . the zirconia substrate was sintered at 1520c for a 12-hour process in the same sintering furnace , and was then polished by the same polishing kit . the four substrates of w , nca , npga , and zrc were prepared as such . the specimens were located at the center of this putty mold . each disc was placed on each of the substrates , and the color measurements were made . the color parameters of l * , a * , and b * were recorded for each specimen . additionally , e values were calculated to determine the e of disc on the substrates . in order to compare the color of specimens on the nca , npga , and zrc substrates with their color on the w substrate , the e values were measured in three situations including : w - nca , w - npga , and w - zrc . the e w - nca e w - npga , and e w - zrc values were calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 specimen preparation : a computer - aided design / computer - aided manufacturing system ( coritec 250i , imes - icore gmbh , eiterfeld , germany ) milled zirconia blanks ( luminesse high strength / low translucency , 98 mm discs # 5113 ; talladium , valencia , ca , usa ) to fabricate zirconia discs . the zirconia blanks had 37% translucency and were used to fabricate zirconia frameworks in zirconia - based restorations . the discs had 0.5 mm thickness and 10 mm diameter [ 20 , 23 ] . all the zirconia discs were sintered at 1520c for a 12-hour process in a sintering furnace ( isint ht , imes - icore gmbh , eiterfeld , germany ) . a digital micrometer ( 293 mdc - mx lite ; mitutoyo corporation , tokyo , japan ) with the accuracy of 0.002 mm an adjustment / polishing kit ( bruxzir ; glidewell direct , irvine , ca , usa ) was used to reduce the thicknesses according to the afore - mentioned acceptable range . in case of lack of acceptable thickness the zirconia discs were polished , cleaned in an ultrasonic bath ( elmasonic s-30 ; dentec , north shore , australia ) containing 98% ethanol for 15 minutes , and were finally dried . the zirconia disc specimens were prepared as such , and had natural white color of zirconia ( fig . substrate preparation : in order to fabricate the w substrate with the dimensions of 10 mm in diameter and 10 mm in height , a white teflon material ( ptfe , omnia plastica spa , busto arsizio , italy ) was milled . the patterns were cast by a nca ( verabond v ; alba dent , fairfield , ca , usa ) and a npga ( alba dent , fairfield , ca , usa ) to fabricate nca and npga substrates , respectively . the zirconia substrate was sintered at 1520c for a 12-hour process in the same sintering furnace , and was then polished by the same polishing kit . the four substrates of w , nca , npga , and zrc were prepared as such . the specimens were located at the center of this putty mold . each disc was placed on each of the substrates , and the color measurements were made . the color parameters of l * , a * , and b * were recorded for each specimen . additionally , e values were calculated to determine the e of disc on the substrates . in order to compare the color of specimens on the nca , npga , and zrc substrates with their color on the w substrate , the e values were measured in three situations including : w - nca , w - npga , and w - zrc . the e w - nca e w - npga , and e w - zrc values were calculated using this formula : e*ab=[(l*2l*1)2+(a*2a*1)2+(b*2b*1)2]1/2 zirconia disc specimens tested substrates ( from left to right : zirconia ceramic , non - precious gold alloy , nickel - chromium alloy and white substrate ) the perceptual threshold of e=2.6 was considered in this study [ 1214 ] . the values of l * , a*and b * for each disc in npga , nca and zrc groups were subtracted from l*white , a*white and b*white values , which were the corresponding values for w substrate . in order to compare the standard l * , a * , b * ( from which , the values of w substrate were subtracted ) and e values among the groups , randomized block anova was employed . stata software ( statacorp lp , lakeway , tx , usa ) compared the e values with the predetermined perceptual threshold of e=2.6 using one - sample t - test . the results were explained according to the measured variables of l * , a * , b * , and e in four sections . l * parameter ( lightness ) : the mean and standard deviation of the l * values for the w , nca , npga and zrc groups were 88.351.46 , 78.241.31 , 78.981.26 , and 82.161.16 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test revealed significant differences between the nca and zrc ( p<0.0001 ) , and npga and zrc ( p<0.0001 ) . the color parameter values in the four groups of white substrate ( w ) , nickel - chromium alloy ( nca ) , non - precious gold alloy ( npga ) and zirconia ceramic ( zrc ) the mean l * values of the groups a * parameter ( red - green value ) : the mean and standard deviation of the a * values for the w , nca , npga and zrc groups were 0.400.42 , 0.040.52 , 0.600.42 , and 1.520.38 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test revealed significant differences between nca and npga ( p<0.0001 ) , nca and zrc ( p<0.0001 ) and npga and zrc ( p<0.0001 ) . the a * values increased in all the groups compared to the control . the increase in the a the mean a * values of the groups b * parameter ( yellow - blue value ) : the mean and standard deviation of the b * values for the w , nca , npga , and zrc groups were 3.380.36 , 1.921.01 , 3.860.49 , and 4.880.57 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test showed significant differences between nca and npga ( p<0.0001 ) , nca and zrc ( p<0.0001 ) , and npga and zrc ( p<0.001 ) . zrc had the highest b * value , and nca had the lowest b * value among the groups . the mean b * values of the groups e ( color difference ) : the mean and standard deviation of the e values for the nca , npga and zrc groups were 10.262.43 , 9.451.74 , and 6.701.91 units , respectively ( table 1 , fig . pairwise comparisons of the groups using bonferroni test showed significant differences between nca and zrc ( p<0.0001 ) , and npga and zrc ( p=0.001 ) . in order to compare the e values of the groups with the predetermined perceptual threshold of e=2.6 , the null hypothesis of e 2.6 was rejected for all the groups ( p<0.0001 ) . the present study evaluated the color parameters of l * , a * , b * , and e for zirconia disc specimens on four different substrates including w , nca , npga and zrc . statistical analysis indicated significant differences among the groups in the l * , a * , b * and e values . the examined zrc on the tested substrates showed perceptible color change namely that the examined zrc could not thoroughly mask the tested substrates . the l * ( lightness ) values decreased in all the groups compared with the control . the change in l * value shows the impact of the substrates on the zirconia specimens . according to the results , metal core materials such as ncas and npgas can decrease the lightness of zrc more than a zirconia core material the a * ( red - green ) values increased in all the groups compared to the control . the tested zirconia substrate ( a2 shade ) positively shifted the a * value towards red . the b * ( yellow - blue ) values changed in the groups depending on the substrates . the nickel - chromium substrate decreased the b * value while the non - precious gold and zirconia substrates increased the b * value compared to the control . this may be due to the yellow color tendency of a2 shade zirconia substrate and npga , which impact on the color of zrc and shift it towards yellow . on the contrary , nca shifted the color of zrc towards blue , and negatively affected the b * value . the e values of all groups were more than the predetermined perceptual threshold of e=2.6 . assessment of the l * , a * and b * values indicated their changes in the substrate groups . the l * , a * , and b * values manifested significant differences between the substrate groups . this demonstrated that the quality of the color change was different depending on the substrate . the e values manifested no significant difference between the metal substrates ; however , significant differences existed between the zirconia substrate and the metal substrates , and the least color change belonged to the zirconia substrate . this demonstrated that the quantity of the color change was different depending on the substrate as well . the used metal substrates did not differ in terms of the quantity of color change , and both affected the quantity of the color change more than the zirconia substrate . suputtamongkol et al , reported that the color of an underlying substructure could affect the overall color of posterior zirconia - based restorations on a metal post and core or a prefabricated post and a composite core , ranging from e = 1.2 to 3.1 . although some differences exist between the afore - mentioned study and the current research such as zrc brands , thicknesses of ceramics , veneered versus non - veneered zrcs , hypothesized perceptual thresholds and substrate types , both studies showed that zrc could not thoroughly mask the underlying materials . oh and kim in an in vitro study of color masking ability assessed the effects of abutment shade , ceramic thickness and coping type on the final color of zirconia restorations . the abutments were fabricated of gold alloy , base metal ( nickel - chromium ) alloy , and four different shades of composite resins . in their study , the average e value of lava specimens between the a2 shade composite resin and gold alloy abutments was higher than that between the a2 shade composite resin and other abutments , and was close to e=5.5 . according to the study by oh and kim , gold alloy substrate yielded the highest e among the tested substrates ; however , the npga and the nca yielded close e values in the current study . this dissimilarity in the results may be caused by the color difference of the precious gold alloy used by oh and kim and the npga used in the current study . choi and razzoog evaluated the masking ability of zrc with and without a veneering porcelain . in their study , color differences caused by zrc and the veneering porcelain were compared with substrates alone . they concluded that the non - veneered zrc had some degree of masking ability over different tested substrates ( white , black , gray , and a3 shade tooth - colored substrates ) . however , the current study compared substrate - induced color differences with a zrc over w substrate and concluded that the tested zrc could not mask the examined substrates . choi and razzoog measured the e values between the substrate alone ( as control ) and the zrc over the substrate , while the current study measured the e values between the zrc over w substrate ( as control ) and zrc over the tested substrates . based on the results of our study , the three core materials including nca , npga and zrc can change the color of tested zrc . therefore , it is esthetically advised to preferably choose a zrc post and core instead of nca or npga post and cores in zirconia - based restorations and to reduce the core material - induced color change by further core reduction , applying sufficient thickness of the veneering porcelain , and use of proper luting agents in zirconia - based restorations . considering the optical properties of zrc and the thickness of zirconia sub - structures , which is approximately 0.5 mm in a normal case , light transmission through the zirconia structure can be expected in zirconia - based restorations . the present study evaluated the effects of three core materials in this respect . on the other hand , the zirconia core overlaying materials such as porcelain veneers and glaze may influence the final color of zirconia - based restorations , which were not assessed in this study . therefore , evaluation of the effect of other factors in this respect is recommended in future studies . within the limitations of this study , it was concluded that the tested zrc could not thoroughly mask the three core materials of nca , npga , and zrc .

solvent polarity strongly affects the mechanism of photoinduced electron transfer ( et ) reactions . in polar solvents , the process usually involves complete et from the electron donor to the acceptor , thereby forming spin - correlated radical ion pairs ( rips ) . in nonpolar solvents , the formation of excited - state charge transfer complexes , so - called exciplexes , is often observed . expectedly , in moderately polar solvents , both quenching reactions may contribute simultaneously and radical ions can result from direct et as well as dissociation of the exciplex . due to similar spectral footprints of ion pairs and exciplexes , the magnetic field effect ( mfe ) on the exciplex emission provides a versatile tool for the study of this intricate dynamics . the approach has been applied to homogeneous solvents as well as under conditions of preferential solvation , which often occur in binary solvent mixtures of solvents of vastly different polarity . the term preferential solvation subsumes nonspecific and specific interactions of solutes with specific components of binary solvent mixtures . it often induces microheterogeneity in the solvent , which is characterized by the spatially nonuniform distribution of the solvent components in the vicinity of polar or ionic solutes . these microclusters of polar solvent molecules have a pronounced effect on the mutual diffusion and reactivity of radical ion pairs and , thus , the mfes they elicit . so far , microheterogeneous solvation has only been studied by steady - state mfe measurements . this severely limits the insights in the radical pair dynamics in these systems , because only time - resolved mfe measurements allow one to discriminate the initial reaction products . the mfe relevant to this study results from the radical pair mechanism . in short , the overall singlet and triplet states of the radical pair are coherently interconverted by the hyperfine interactions ( hfis ) of the magnetic nuclei of the radicals . an external magnetic field will remove the degeneracy of the three electronic triplet sublevels ( t0 and t ) of the spin - correlated pair ( in the absence of significant exchange or electron electron dipolar coupling ) . when the energy separation between these states exceeds the size of the mixing interactions , t can not mix with the singlet state s. in this way , the external magnetic field reduces the probability of intersystem crossing in the radical pair and , thus , alters the relative concentrations of singlet and triplet reaction products ( see scheme 1a ) . the hyperfine - induced spin mixing between s and t0 and t can only efficiently proceed when the electron exchange interaction , which energetically splits the s from the t - states , is smaller in magnitude than the hyperfine interactions . owing to the fact that the electron exchange interaction depends exponentially on the distance between the radical ions , this implies that only diffusively separated radical ions can undergo s / t - conversion . eventually , the singlet rip concentration can be detected through the emission of the exciplex , which is produced by singlet rip cage recombination ( see scheme 1b ) . in the absence of an external magnetic field ( b0 = 0 ) , the singlet ( s ) and all three triplet ( t0, ) states are interconverted by the radical hyperfine interactions ( hfi ) . in the presence of a sufficiently large magnetic field b0 , the t states can not mix with the s state . the red and orange arrows refer to the radiative processes of the exciplex and the locally excited fluorophore emissions , respectively . as a consequence of this mechanism , any factor influencing the rip molecular dynamics may affect mfe features such as the magnitude of the mfe observed under saturating field conditions and the b1/2 value , which gives the field intensity delivering half the saturation mfe . in this way , the exciplex system can serve as a magneto - fluorescent probe , sensitive to the local dielectric heterogeneities of binary solvents . indeed , using magnetic - field - affected - reaction - yield ( mary ) line broadening measurements , the b1/2 values were found to vary strongly with the solvent composition in microheterogeneous solvents . solvent mixtures composed of toluene ( to ) and dimethyl sulfoxide ( dmso ) have been studied in detail . studies of the magnetic isotope effect revealed no direct transfer of spin density on solvent molecules . instead , the observed effects were entirely due to the radical pair dynamics . in general , the rp lifetime is reduced in the microheterogeneous to / dmso environment due to an enhanced cage - effect facilitating rp reencounters . as the dmso concentration and thus the relative macroscopic permittivity , s , is increased , the lifetime of the rip increases due to a smaller depth of the dielectric traps . this is a consequence of the rips evading swift recombination by their diffusive escape from the trap . accordingly , the extrapolated b1/2 values in the limit of zero donor concentration , b1/2(0 ) , are larger than expected for long - lived rps but decrease with increasing macroscopic dielectric constant , in contrast to what is seen in homogeneous solvents . nath and co - workers have recently suggested that two populations of exciplexes exist for the pyrene / n , n - dimethylaniline donor acceptor system in comparably polar thf / dmf and benzene / acetonitrile solvent mixtures . the authors argue that the effect is attributed to ( incomplete ) relaxation of the solvent shell surrounding the contact ion pair . the aforementioned steady - state approaches usually do not take the exciplex kinetics into account . the mechanism of fluorescence quenching by electron transfer is , however , ambiguous ; i.e. , the initial quenching products are a rip via distant et ( pathway 2b of scheme 1b ) or the exciplex ( pathway 2a ) . several studies have illustrated the use of time - resolved mfes of the exciplex to identify the initial quenching products in homogeneous solvents and solvent mixtures . in this work , we describe the solvent polarity dependence of the time - resolved mfe in homogeneous and micro - heterogeneous binary solvent mixtures as measured by time - correlated single photon counting ( tcspc ) . the well - studied donor n , n - dimethylaniline ( quencher)/9,10-dimethylanthracene ( fluorophore ) serves as a magnetosensitive probe ( see scheme 1c ) . our study gives detailed insights into the peculiar rp dynamics in microheterogeneous solvents . throughout this work , we use the terms preferential solvation and microheterogeneous medium interchangeably , although , strictly speaking , the former refers to specific and nonspecific solute solvent interactions , while the latter signifies the nonuniform solvent structure , which the former induce . in the present context n , n - dimethylaniline ( dma , aldrich 99.5% ) was distilled under reduced pressure and subsequently handled under an argon atmosphere . the concentration of the fluorophore was constant at 1.5 10 m , while that of the quencher was 0.06 m. solutions were prepared in septa - sealed fused silica cuvettes . all solutions were purged with solvent - saturated nitrogen gas for 15 min to remove dissolved oxygen before adding the quencher through the septum using hamilton syringes . a series of solvent mixtures of propyl acetate ( pa , aldrich 99.5% , distilled under reduced pressure , s = 6 ; this and all subsequently reported values refer to a temperature of 295 k ) and butyronitrile ( bn , fluka 99% , distilled under reduced pressure , s = 24.7 ) and of toluene ( to , fluka 99% , distilled under reduced pressure , s = 2.38 ) and dimethyl sulfoxide ( dmso , aldrich 99.9% , used as received , s = 46.4 ) with widely varying dielectric constants , s , were prepared . the dielectric constants of these mixtures are given by eq 1 and eq 2 for the pa / bn and to / dmso systems , respectively ( at 295 k):12here , wpa is the mass fraction of pa and xdmso is the mole fraction of dmso . the homogeneous mixtures of pa / bn allow systematic variation of the solvent dielectric constants within a range of 624.7 ( at 295 k ) while keeping the viscosity ( ) and the refractive index ( n ) almost constant . the pekar factor , = ( 1/ 1/s ) ( 1/n 1/s ) , which governs the outer - sphere electron transfer reorganization energy , increases only moderately with increasing permittivity . for the microheterogeneous mixtures of to / dmso , the solvent dielectric constant was varied from 4.3 to 15.5 . the viscosity and the bulk pekar factor of to / dmso mixtures slightly increase with increasing dmso concentration . we are not aware of a microheterogeneous solvent mixture for which this could be avoided . using the experimental setup described in ref ( 32 ) , the exciplex emission was recorded at 550 nm under continuous excitation of the sample at 374 nm . for each sample , the fluorescence intensities were acquired three times under conditions of zero and saturating magnetic field exposition for 60 s each . field - on and field - off measurements were recorded in an alternating fashion . the excitation slit width was 2 nm , the emission slit width 6 nm , and the spectrometer time constant 1 s. measurements were conducted at 295 k. the three repetitions were analyzed independently , and the experimental errors were obtained according to the method described in ref ( 32 ) . the absolute mfe , ss , was evaluated from3here , i(em , bsat ) and i(em , b0 = 0 ) are the mean intensities at em in a saturated magnetic field ( bsat = 62 mt ) and in the absence of an additional magnetic field , respectively . if(em , b0 ) is the residual emission of the locally excited fluorophore at em in the absence of quencher . ic and i0 are the intensities of prompt emission of the fluorophore in the presence and absence of the quencher , respectively , which have been obtained from the decomposition of the fluorescence spectra . time - resolved data of the mfes on the exciplex were obtained by the tcspc technique . in order to investigate the effect of an external magnetic field on the exciplex emission ( figure 1 ) , a saturating magnetic field ( the fluorophore is excited at 374 nm by a laser diode ( picoquant , ldh - p - c-405 ) , and a 550 nm long - pass filter in front of the detector ensured that only the exciplex luminescence was detected . a detailed description of the experimental setup is given in refs ( 1 ) and ( 2 ) . time - resolved exciplex emission ( upper panel ) of the 9,10-dimethylanthracene ( 1.5 10 m)/n , n - dimethylaniline ( 0.06 m ) system in a to / dmso mixture at s = 7.3 in the absence ( gray ) and presence ( blue ) of an external magnetic field ( 62 mt ) monitored with a long - pass filter ( lp550 ) after pulsed excitation at 374 nm . in the presence of an external magnetic field , the delayed emission of the exciplex is enhanced . the time evolution of the magnetic field effect i(t ) ( lower panel ) was obtained by taking the difference of the exciplex emission time traces in the absence and presence of an external magnetic field . the red solid line is calculated from the model , eq 6 . in the lower panel , t = 0 ns coincides with the pulse excitation ; in the upper panel , the emission traces are shifted by approximately 15 ns to longer times . the raw data of a typical time - resolved mfe measurement are shown in figure 1 . the time traces rise with a time constant of approximately 2 ns , almost independent of the magnetic field intensity . the decay kinetics of the exciplex emission includes the dissociation into free ions and recombination giving rise to delayed exciplex emission . the time - resolved mfe ( tr - mfe ) is given as the difference in the exciplex emission intensity , i(t ) , in the absence and presence of the external , biasing magnetic field:4 in order to determine the mfe , the amplitudes within the first nanosecond after the excitation pulse were matched . we integrated the time traces so obtained to determine the steady - state mfe of the exciplex , tr , from the time - resolved mfe measurements:5scheme 1b shows the pertinent reaction pathways for fluorescence quenching by electron transfer . the abscissa can be expressed as the projection of a two - dimensional reaction coordinate comprising the distance between the fluorophore and the quencher and the outer - sphere electron transfer reaction coordinate . an increase in the intensity of the exciplex emission ( 6 ) is due to the enhancement of the population of the singlet rip , which can reform the exciplex via ( 4 ) . the singlet rip can be indirectly generated via exciplex dissociation ( 3 ) , e.g. , following a quenching reaction yielding the exciplex ( 2a ) , or directly , via a remote electron transfer reaction ( 2b ) . note that the et reactions preserve the overall spin of the reactant pair ; i.e. , both the exciplex and the rip are formed as singlet entities . the external magnetic field gives rise to an increase in the singlet rip probability and hence the exciplex via pathway 4 . as a consequence of the exciplex dissociation here being a slow process , the mfes generated by the direct et route ( 2b ) will be observed on a faster time scale than those formed by the exciplex route ( 2a ) . in this way , time - resolved mfes of the exciplex emission can distinguish the reaction channels populating the magneto - sensitive rp state ( 2b versus 2a ) . to simulate the experimental data , we used the model introduced in ref ( 2 ) . in this model , the probability that the radical pair system exists as an exciplex , e(t , b0 ) , is given by6where i is the probability that the rip is initially generated ( pathway 2b in scheme 1b ) , kd denotes the rate constant of exciplex dissociation , ri is the distance of rip formation via distant et , and r(t , b0|ri ) is the probability that a rip formed at distance ri at t = 0 has recombined by time t. re refers to the contact distance of the fluorophore and quencher , at which the transition of the rip to the exciplex ( or contact ion pair ) occurs . e = 1 i is the initial exciplex probability ( pathway 2a in scheme 1b ) , and e is the intrinsic exciplex lifetime ( without dissociation ) . the contributions to the exciplex probability in eq 6 can be described as follows : the first term is the probability that the exciplex is formed initially , while the second term accounts for the probability that the initially generated rip reforms an exciplex at t. the third term describes the probability that the exciplex dissociated at time is reformed at t , and the last term models the depopulation of the exciplex by dissociation ( kd ) and radiative / nonradiative decay with the rate constant . r(t , b0|ri ) is field dependent and has been calculated in the low - viscosity limit as described in detail in refs ( 1 ) and ( 2 ) . the radical pair was assumed to diffuse in a potential of mean force approximately accounting for the microheterogeneous environment . this potential was calculated following an approach based on the d e0 theorem as outlined in refs ( 18 ) and ( 20 ) . note that more elaborate schemes of treating the diffusion - influenced reversible exciplex kinetics have been derived on the basis of the integral encounter theory . while also comprehensively applicable to highly viscous mixtures , this approach is difficult to employ for radical pairs with a multitude of magnetic nuclei ( as is the case here ) and beyond the scope of the current presentation . figure 2 illustrates the microheterogeneous medium structure to clarify typical length scales and the extent of the local enrichment of the polar component as predicted on the basis of the continuum model from ref ( 20 ) . illustration of the structure of the microheterogeneous solvent surrounding a radical ion pair ( contact distance : 6.8 ) as predicted by the continuum solvation model described in ref ( 18 ) . ( a ) local mole fraction of dmso in dmso / toluene mixtures of bulk concentration xdmso = 0.2 ( left ; s = 7.3 ) and 0.4 ( right ; s = 13 ) . ( b ) deviation of the local dielectric constant from the macroscopic dielectric constant for the two solvent mixtures from part a. the plots show contours through a plane containing the two radical centers . figure 3 illustrates the dependence of the mfe of the dmant / dma system on the solvent dielectric constant , s , of the microheterogeneous to / dmso mixture as determined from steady - state ( ss ) and time - resolved ( tr ) measurements ( upper panel ) . the lower panel gives a comparison of the s dependence for the binary solvent mixtures of pa / bn ( homogeneous ) and to / dmso ( microheterogeneous ) . this observation suggests that in both types of experiments the same photochemical reactions are probed . in fact , in view of the low light intensities and low concentrations employed in these experiments , bulk processes such as f - pair reactions and processes involving fluorophore triplets ( triplet triplet and triplet doublet pair reactions ) are not expected to contribute to the observed mfes here . instead , the mfes on the exciplex result exclusively from the effect of an external magnetic field on the s / t mixing in the geminate radical pair . the onset of the mfe is found at s = 6 ( with = 0.1% ) and s = 4.3 ( with = 1.6% ) in the pa / bn and to / dmso mixture , respectively . in the homogeneous pa / bn mixtures , the mfe is small for s < 10 but rises sharply for s 13 to attain its maximum value around s 18 ( 11% ) . for larger solvent polarities , it decreases slightly as a larger population of the radical pairs dissociates indefinitely without reencountering ( within the coherence time of the radical pair ) . in the microheterogeneous to / dmso mixtures , the onset of the mfe occurs at a lower bulk permittivity and the maximal value ( 14.5% ) is already observed at s = 8.3 . in essence , the mfe appears to report a more polar environment than would be expected on the basis of the bulk permittivity . these peculiarities in the mfes can be explained on the basis of a dielectric enrichment of the polar component ( dmso ) in the vicinity of the magneto - sensitive rip . as a consequence , the effective dielectric constant , eff , of the enriched solvation shell this specific solvation increases the reencounter probability of the geminate rp without impeding the spin - conversion by too tight binding , which in homogeneous solvents of low polarity is seen to hamper the mfe by confining the radical pairs to configurations with large exchange coupling . as a consequence , larger mfes are possible in the microheterogeneous environments as compared to homogeneous solutions . solvent permittivity dependence of the magnetic field effect of the 9,10-dimethylanthracene / n , n - dimethylaniline exciplex determined from steady - state ( blue filled triangles ) and time - resolved mfe ( red filled stars with barely visible error bars reflecting the statistical error of the registration process ) measurements in solvent mixtures of to / dmso ( upper panel ) . the lower panel compares the mfe in the homogeneous and microheterogeneous solvent mixtures . for various dielectric constants , the time - resolved mfes are shown in figure 4 together with least - squares fits applying the model given by eq 6 . the maximum of the tr - mfes occurs in the range from 10 to 75 ns after photoexcitation , with larger values occurring at lower dielectric constants . for all samples , the mfes reached the noise level of the experiment within 400 ns after excitation . experimental time - dependent magnetic field effects ( i ) at different dielectric constants , s , in pa / bn ( top panel ) and to / dmso ( middle panel ) solvent mixtures for the system 9,10-dimethylanthracene / n , n - dimethylaniline . the bottom panel compares the tr - mfes of the exciplex in pa / bn and to / dmso mixtures of the same dielectric constant ( s = 13 ) . the exciplex lifetime , e , is one of the central parameters characterizing the time evolution of the mfe . we have determined e from the initial decay of the tcspc traces of the exciplex , for which the rp recombination is negligible . figure 5 shows that the apparent exciplex lifetime decreases with increasing bulk dielectric constant , s , in both binary solvent mixtures . furthermore , as expected in view of the above discussion , the exciplex lifetimes are considerably smaller in the to / dmso mixtures as compared to the iso - bulk dielectric pa / bn mixtures . note also that the exciplex is an excited charge - transfer complex with correspondingly large dipole moment . as a consequence , its formation is expected to enhance the diffusive enrichment of polar solvent molecules in its surrounding . in this way , the exciplex can act as a catalyst to its own dissociation ( pathway 3 in scheme 1b ) . as the exciplex lifetime is considerably larger than the characteristic time of solvation , these dynamic aspects can however not be resolved here . permittivity dependence of the exciplex lifetimes of the 9,10-dimethylanthracene / n , n - dimethylaniline exciplex in pa / bn ( red circles ) and to / dmso mixtures ( blue squares ) . the exciplex kinetics are also characterized by the association constant ka = ka / kd ( top panel in figure 6 ) and the exciplex dissociation quantum yield d = kde ( middle panel in figure 6 ) . d was estimated from the dependence of e on dielectric constants assuming that the intrinsic , radiative and nonradiative decay rates are independent of solvent composition . ka has a strong effect on the shape , i.e. , the temporal evolution , of the time - resolved mfes and can thus be extracted from the experimental data by least - squares fitting . following this approach as detailed in ref ( 2 ) , ka and d were obtained as a function of s . in general , d increases with s , likely because polar environments reduce the exciplex stabilization free energy by increasing the reorganization energy . this effect is indeed found and much more pronounced in the microheterogeneous environment as compared to the homogeneous one . for to / dmso , the exciplex dissociation quantum yield approaches d = 1 for a bulk dielectric constant as low as s = 8 , while for pa / bn mixtures this is only the case at s 22 . dependence of the association constant , ka , the dissociation quantum yield of the exciplex , d , and the initial rip formation , i , on the dielectric constants , s , of homogeneous ( pa / bn ) and microheterogeneous ( to / dmso ) solvent mixtures . the filled circles and squares with error bars have been determined from the experimental data by modeling the tr - mfe . likewise , the association constant ka is expected to decrease with polarity , as is clearly evidenced in figure 6 for the homogeneous pa / bn solutions . while ka in general also decreases with increasing polarity for to / dmso , it is nearly constant in the range from s = 8 to 12 . this surprising behavior suggests that ka increases in this region in parallel to kd such that ka is approximately constant . this can likely be attributed to an increased association rate brought about by the microheterogeneous solvation impeding the separation of the radical ions with respect to the homogeneous environment . just as ka , the probability of the initial rip formation , i , governs the time evolution of mfes of the exciplex and was extracted from the experimental data by least - squares fitting . the extracted i values are also shown in figure 6 as a function of the dielectric constant . the data reveal that , for the dmant / dma system with moderate free energy of charge separation ( get = 0.28 ev ) , the direct exciplex formation ( pathway 2a in scheme 1b ) contributes at all dielectric constants in homogeneous and microheterogeneous environments . furthermore , the probability of distant et quenching increases with increasing solvent polarity . note that the requirement to detect the exciplex luminescence limits the accessible s range to the indicated range ; beyond the respective limits , the exciplex emission is insufficient for the accurate determination of i . in both solvent mixtures , for to / dmso , this is the case for s 9 , and for pa / bn , for s 20 , in agreement with the general bearing . distant electron transfer quenching is more favored in polar solutions , where it leads to a better solvation of the reaction products ( rips ) . this holds true despite a minor increase in the reorganization energy resulting from the increasing polarity . in the low - viscous solutions studied here , the diffusive approach of the reactants is fast enough compared to the intrinsic electron transfer rate to always form a contact complex ( exciplex ) at the contact distance . as a consequence , i is smaller than unity , even for the most polar and hardly emissive solutions . in this work , we have employed time - resolved mfe measurements to study the exciplex and rip dynamics of the dmant / dma system in homogeneous and microheterogeneous solvent mixtures of various bulk permittivities . we use a model accounting for the reversible exciplex dissociation to identify the peculiarities resulting from microheterogeneous solvent environments in solvent mixtures of to and dmso . the exciplex dynamics and the initial quenching products depend strongly on the solvent properties . in microheterogeneous solvents , specific solvation of the exciplex and rip gives rise to an environment that is significantly more polar than that observed in homogeneous solvents of comparable bulk permittivity . for low permittivities , this brings about a larger dissociation quantum yield of the exciplex , a lower exciplex lifetime , and a larger probability that the system undergoes distant electron transfer quenching . specific solvation furthermore impedes the radical separation such that , in the permittivity region from 7 to 15 , the association constant decays only slowly with the solvent polarity in the to / dmso mixtures . in both environments , the probability of the initial formation of rips is always less than unity ; i.e. , the exciplex formation contributes at all s . in microheterogeneous solution , the local concentration of dmso in the solvation shell around the exciplexes and rips plays an important role in the exciplex kinetics and the fluorescence quenching mechanism . together with earlier works , this study demonstrates that time - resolved mfe studies have the potential to provide detailed insights into the reaction dynamics of rips and exciplexes . since the reaction dynamics of the geminate radical pair are very sensitive to small changes in the local dielectric environment , the technique is here recognized as a versatile tool in revealing and characterizing the phenomenon of specific solvation in solvent mixtures .

external magnetic fields can impact recombination yields of photoinduced electron transfer reactions by affecting the spin dynamics in transient , spin - correlated radical pair intermediates . for exciplex - forming donor acceptor systems , this magnetic field effect ( mfe ) can be investigated sensitively by studying the delayed recombination fluorescence . here , we investigate the effect of preferential solvation in microheterogeneous solvent mixtures on the radical pair dynamics of the system 9,10-dimethylanthracene ( fluorophore)/n , n - dimethylaniline ( quencher ) by means of time - resolved magnetic field effect ( tr - mfe ) measurements , wherein the exciplex emission is recorded in the absence and the presence of an external magnetic field using time - correlated single photon counting ( tcspc ) . in microheterogeneous environments , the mfe of the exciplex emission occurs on a faster time scale than in iso - dielectric homogeneous solvents . in addition , the local polarity reported by the exciplex is enhanced compared to homogeneous solvent mixtures of the same macroscopic permittivity . detailed analyses of the tr - mfe reveal that the quenching reaction directly yielding the radical ion pair is favored in microheterogeneous environments . this is in stark contrast to homogeneous media , for which the mfe predominantly involves direct formation of the exciplex , its subsequent dissociation to the magneto - sensitive radical pair , and re - encounters . these observations provide evidence for polar microdomains and enhanced caging , which are shown to have a significant impact on the reaction dynamics in microheterogeneous binary solvents .

Introduction Experimental Section Results and Discussion Conclusions

in polar solvents , the process usually involves complete et from the electron donor to the acceptor , thereby forming spin - correlated radical ion pairs ( rips ) . due to similar spectral footprints of ion pairs and exciplexes , the magnetic field effect ( mfe ) on the exciplex emission provides a versatile tool for the study of this intricate dynamics . the approach has been applied to homogeneous solvents as well as under conditions of preferential solvation , which often occur in binary solvent mixtures of solvents of vastly different polarity . it often induces microheterogeneity in the solvent , which is characterized by the spatially nonuniform distribution of the solvent components in the vicinity of polar or ionic solutes . these microclusters of polar solvent molecules have a pronounced effect on the mutual diffusion and reactivity of radical ion pairs and , thus , the mfes they elicit . this severely limits the insights in the radical pair dynamics in these systems , because only time - resolved mfe measurements allow one to discriminate the initial reaction products . in short , the overall singlet and triplet states of the radical pair are coherently interconverted by the hyperfine interactions ( hfis ) of the magnetic nuclei of the radicals . an external magnetic field will remove the degeneracy of the three electronic triplet sublevels ( t0 and t ) of the spin - correlated pair ( in the absence of significant exchange or electron electron dipolar coupling ) . when the energy separation between these states exceeds the size of the mixing interactions , t can not mix with the singlet state s. in this way , the external magnetic field reduces the probability of intersystem crossing in the radical pair and , thus , alters the relative concentrations of singlet and triplet reaction products ( see scheme 1a ) . owing to the fact that the electron exchange interaction depends exponentially on the distance between the radical ions , this implies that only diffusively separated radical ions can undergo s / t - conversion . eventually , the singlet rip concentration can be detected through the emission of the exciplex , which is produced by singlet rip cage recombination ( see scheme 1b ) . in the absence of an external magnetic field ( b0 = 0 ) , the singlet ( s ) and all three triplet ( t0, ) states are interconverted by the radical hyperfine interactions ( hfi ) . in the presence of a sufficiently large magnetic field b0 , the t states can not mix with the s state . the red and orange arrows refer to the radiative processes of the exciplex and the locally excited fluorophore emissions , respectively . as a consequence of this mechanism , any factor influencing the rip molecular dynamics may affect mfe features such as the magnitude of the mfe observed under saturating field conditions and the b1/2 value , which gives the field intensity delivering half the saturation mfe . in this way , the exciplex system can serve as a magneto - fluorescent probe , sensitive to the local dielectric heterogeneities of binary solvents . instead , the observed effects were entirely due to the radical pair dynamics . accordingly , the extrapolated b1/2 values in the limit of zero donor concentration , b1/2(0 ) , are larger than expected for long - lived rps but decrease with increasing macroscopic dielectric constant , in contrast to what is seen in homogeneous solvents . nath and co - workers have recently suggested that two populations of exciplexes exist for the pyrene / n , n - dimethylaniline donor acceptor system in comparably polar thf / dmf and benzene / acetonitrile solvent mixtures . the authors argue that the effect is attributed to ( incomplete ) relaxation of the solvent shell surrounding the contact ion pair . several studies have illustrated the use of time - resolved mfes of the exciplex to identify the initial quenching products in homogeneous solvents and solvent mixtures . in this work , we describe the solvent polarity dependence of the time - resolved mfe in homogeneous and micro - heterogeneous binary solvent mixtures as measured by time - correlated single photon counting ( tcspc ) . the well - studied donor n , n - dimethylaniline ( quencher)/9,10-dimethylanthracene ( fluorophore ) serves as a magnetosensitive probe ( see scheme 1c ) . throughout this work , we use the terms preferential solvation and microheterogeneous medium interchangeably , although , strictly speaking , the former refers to specific and nonspecific solute solvent interactions , while the latter signifies the nonuniform solvent structure , which the former induce . in the present context n , n - dimethylaniline ( dma , aldrich 99.5% ) was distilled under reduced pressure and subsequently handled under an argon atmosphere . using the experimental setup described in ref ( 32 ) , the exciplex emission was recorded at 550 nm under continuous excitation of the sample at 374 nm . the excitation slit width was 2 nm , the emission slit width 6 nm , and the spectrometer time constant 1 s. measurements were conducted at 295 k. the three repetitions were analyzed independently , and the experimental errors were obtained according to the method described in ref ( 32 ) . the absolute mfe , ss , was evaluated from3here , i(em , bsat ) and i(em , b0 = 0 ) are the mean intensities at em in a saturated magnetic field ( bsat = 62 mt ) and in the absence of an additional magnetic field , respectively . ic and i0 are the intensities of prompt emission of the fluorophore in the presence and absence of the quencher , respectively , which have been obtained from the decomposition of the fluorescence spectra . time - resolved data of the mfes on the exciplex were obtained by the tcspc technique . in order to investigate the effect of an external magnetic field on the exciplex emission ( figure 1 ) , a saturating magnetic field ( the fluorophore is excited at 374 nm by a laser diode ( picoquant , ldh - p - c-405 ) , and a 550 nm long - pass filter in front of the detector ensured that only the exciplex luminescence was detected . time - resolved exciplex emission ( upper panel ) of the 9,10-dimethylanthracene ( 1.5 10 m)/n , n - dimethylaniline ( 0.06 m ) system in a to / dmso mixture at s = 7.3 in the absence ( gray ) and presence ( blue ) of an external magnetic field ( 62 mt ) monitored with a long - pass filter ( lp550 ) after pulsed excitation at 374 nm . in the presence of an external magnetic field , the delayed emission of the exciplex is enhanced . the time evolution of the magnetic field effect i(t ) ( lower panel ) was obtained by taking the difference of the exciplex emission time traces in the absence and presence of an external magnetic field . the time - resolved mfe ( tr - mfe ) is given as the difference in the exciplex emission intensity , i(t ) , in the absence and presence of the external , biasing magnetic field:4 in order to determine the mfe , the amplitudes within the first nanosecond after the excitation pulse were matched . we integrated the time traces so obtained to determine the steady - state mfe of the exciplex , tr , from the time - resolved mfe measurements:5scheme 1b shows the pertinent reaction pathways for fluorescence quenching by electron transfer . an increase in the intensity of the exciplex emission ( 6 ) is due to the enhancement of the population of the singlet rip , which can reform the exciplex via ( 4 ) . , following a quenching reaction yielding the exciplex ( 2a ) , or directly , via a remote electron transfer reaction ( 2b ) . the external magnetic field gives rise to an increase in the singlet rip probability and hence the exciplex via pathway 4 . as a consequence of the exciplex dissociation here being a slow process , the mfes generated by the direct et route ( 2b ) will be observed on a faster time scale than those formed by the exciplex route ( 2a ) . in this way , time - resolved mfes of the exciplex emission can distinguish the reaction channels populating the magneto - sensitive rp state ( 2b versus 2a ) . in this model , the probability that the radical pair system exists as an exciplex , e(t , b0 ) , is given by6where i is the probability that the rip is initially generated ( pathway 2b in scheme 1b ) , kd denotes the rate constant of exciplex dissociation , ri is the distance of rip formation via distant et , and r(t , b0|ri ) is the probability that a rip formed at distance ri at t = 0 has recombined by time t. re refers to the contact distance of the fluorophore and quencher , at which the transition of the rip to the exciplex ( or contact ion pair ) occurs . the contributions to the exciplex probability in eq 6 can be described as follows : the first term is the probability that the exciplex is formed initially , while the second term accounts for the probability that the initially generated rip reforms an exciplex at t. the third term describes the probability that the exciplex dissociated at time is reformed at t , and the last term models the depopulation of the exciplex by dissociation ( kd ) and radiative / nonradiative decay with the rate constant . figure 2 illustrates the microheterogeneous medium structure to clarify typical length scales and the extent of the local enrichment of the polar component as predicted on the basis of the continuum model from ref ( 20 ) . illustration of the structure of the microheterogeneous solvent surrounding a radical ion pair ( contact distance : 6.8 ) as predicted by the continuum solvation model described in ref ( 18 ) . figure 3 illustrates the dependence of the mfe of the dmant / dma system on the solvent dielectric constant , s , of the microheterogeneous to / dmso mixture as determined from steady - state ( ss ) and time - resolved ( tr ) measurements ( upper panel ) . instead , the mfes on the exciplex result exclusively from the effect of an external magnetic field on the s / t mixing in the geminate radical pair . the onset of the mfe is found at s = 6 ( with = 0.1% ) and s = 4.3 ( with = 1.6% ) in the pa / bn and to / dmso mixture , respectively . in the microheterogeneous to / dmso mixtures , the onset of the mfe occurs at a lower bulk permittivity and the maximal value ( 14.5% ) is already observed at s = 8.3 . in essence , the mfe appears to report a more polar environment than would be expected on the basis of the bulk permittivity . these peculiarities in the mfes can be explained on the basis of a dielectric enrichment of the polar component ( dmso ) in the vicinity of the magneto - sensitive rip . as a consequence , the effective dielectric constant , eff , of the enriched solvation shell this specific solvation increases the reencounter probability of the geminate rp without impeding the spin - conversion by too tight binding , which in homogeneous solvents of low polarity is seen to hamper the mfe by confining the radical pairs to configurations with large exchange coupling . as a consequence , larger mfes are possible in the microheterogeneous environments as compared to homogeneous solutions . solvent permittivity dependence of the magnetic field effect of the 9,10-dimethylanthracene / n , n - dimethylaniline exciplex determined from steady - state ( blue filled triangles ) and time - resolved mfe ( red filled stars with barely visible error bars reflecting the statistical error of the registration process ) measurements in solvent mixtures of to / dmso ( upper panel ) . the lower panel compares the mfe in the homogeneous and microheterogeneous solvent mixtures . for various dielectric constants , the time - resolved mfes are shown in figure 4 together with least - squares fits applying the model given by eq 6 . the maximum of the tr - mfes occurs in the range from 10 to 75 ns after photoexcitation , with larger values occurring at lower dielectric constants . experimental time - dependent magnetic field effects ( i ) at different dielectric constants , s , in pa / bn ( top panel ) and to / dmso ( middle panel ) solvent mixtures for the system 9,10-dimethylanthracene / n , n - dimethylaniline . the bottom panel compares the tr - mfes of the exciplex in pa / bn and to / dmso mixtures of the same dielectric constant ( s = 13 ) . we have determined e from the initial decay of the tcspc traces of the exciplex , for which the rp recombination is negligible . furthermore , as expected in view of the above discussion , the exciplex lifetimes are considerably smaller in the to / dmso mixtures as compared to the iso - bulk dielectric pa / bn mixtures . permittivity dependence of the exciplex lifetimes of the 9,10-dimethylanthracene / n , n - dimethylaniline exciplex in pa / bn ( red circles ) and to / dmso mixtures ( blue squares ) . , the temporal evolution , of the time - resolved mfes and can thus be extracted from the experimental data by least - squares fitting . this effect is indeed found and much more pronounced in the microheterogeneous environment as compared to the homogeneous one . for to / dmso , the exciplex dissociation quantum yield approaches d = 1 for a bulk dielectric constant as low as s = 8 , while for pa / bn mixtures this is only the case at s 22 . dependence of the association constant , ka , the dissociation quantum yield of the exciplex , d , and the initial rip formation , i , on the dielectric constants , s , of homogeneous ( pa / bn ) and microheterogeneous ( to / dmso ) solvent mixtures . the filled circles and squares with error bars have been determined from the experimental data by modeling the tr - mfe . this can likely be attributed to an increased association rate brought about by the microheterogeneous solvation impeding the separation of the radical ions with respect to the homogeneous environment . the data reveal that , for the dmant / dma system with moderate free energy of charge separation ( get = 0.28 ev ) , the direct exciplex formation ( pathway 2a in scheme 1b ) contributes at all dielectric constants in homogeneous and microheterogeneous environments . note that the requirement to detect the exciplex luminescence limits the accessible s range to the indicated range ; beyond the respective limits , the exciplex emission is insufficient for the accurate determination of i . in both solvent mixtures , for to / dmso , this is the case for s 9 , and for pa / bn , for s 20 , in agreement with the general bearing . distant electron transfer quenching is more favored in polar solutions , where it leads to a better solvation of the reaction products ( rips ) . in the low - viscous solutions studied here , the diffusive approach of the reactants is fast enough compared to the intrinsic electron transfer rate to always form a contact complex ( exciplex ) at the contact distance . in this work , we have employed time - resolved mfe measurements to study the exciplex and rip dynamics of the dmant / dma system in homogeneous and microheterogeneous solvent mixtures of various bulk permittivities . we use a model accounting for the reversible exciplex dissociation to identify the peculiarities resulting from microheterogeneous solvent environments in solvent mixtures of to and dmso . the exciplex dynamics and the initial quenching products depend strongly on the solvent properties . in microheterogeneous solvents , specific solvation of the exciplex and rip gives rise to an environment that is significantly more polar than that observed in homogeneous solvents of comparable bulk permittivity . for low permittivities , this brings about a larger dissociation quantum yield of the exciplex , a lower exciplex lifetime , and a larger probability that the system undergoes distant electron transfer quenching . in both environments , the probability of the initial formation of rips is always less than unity ; i.e. in microheterogeneous solution , the local concentration of dmso in the solvation shell around the exciplexes and rips plays an important role in the exciplex kinetics and the fluorescence quenching mechanism . together with earlier works , this study demonstrates that time - resolved mfe studies have the potential to provide detailed insights into the reaction dynamics of rips and exciplexes . since the reaction dynamics of the geminate radical pair are very sensitive to small changes in the local dielectric environment , the technique is here recognized as a versatile tool in revealing and characterizing the phenomenon of specific solvation in solvent mixtures .

estrogenic contaminants , including steroidal estrogens as well as a variety of anthropogenic chemicals , are commonly detected in aquatic environments due to inputs from wastewater and have gained notoriety as endocrine disrupting compounds ( edcs ) . exposure of male fish to estrogenic pollutants is linked to numerous adverse reproductive effects , including the development of testicular oocytes , reduced sperm counts and sperm motility , testicular fibrosis , and reduced fecundity . in addition to these apical end points , widely studied proximal biomarkers of estrogenic edc exposure in male fish include hepatic induction of the egg precursor proteins vitellogenin ( vtg ) and choriogenin ( chg ) , which are normally produced only by females in response to circulating serum estrogen . effects of estrogenic compounds are mediated in large part through nuclear estrogen receptors ( ers ) , which regulate genomic responses via action as ligand - activated transcription factors . much interest has been garnered in determining the roles of multiple er subtypes in modulating estrogenic responses in fish . while mammals have been found to have two nuclear er subtypes ( er and er ) , teleost fish have at least three ( er , er1 , and er2 ) , with the second er subtype having arisen as a result of a genome duplication event in the teleost lineage . these three er subtypes have been shown in many cases to have distinctive tissue distribution patterns , dissimilar ligand affinities , and different patterns of gene regulation following ligand exposure . such differences offer evidence that these receptors have nonredundant physiological functions . a fourth er subtype , er2 , has additionally been identified in rainbow trout ( oncorhynchus mykiss ) as well as some cyprinid species , likely attributable to a single and more recent gene duplication event in these species . there remains significant debate surrounding the respective roles of piscine er subtypes in regulating vtg and chg induction in response to estrogenic ligands . studies in a variety of fish species have shown that vtg induction is accompanied by a sharp increase in hepatic er expression and little change of hepatic er subtype expression , implying that er is the principle receptor mediating regulation of the vtg gene . furthermore , er1 and er2 of largemouth bass ( micropterus salmoides ) have been found to have an inhibitory effect on recombinant er-mediated transcription in vitro . conversely , recent studies using gene knockdown in goldfish ( carassius auratus ) primary hepatocytes and zebrafish ( danio rerio ) embryos demonstrate that er1 and/or er2 subtypes are required for estrogen - mediated upregulation of hepatic er as well as vtg induction . this emerging model suggests that er subtypes play a critical role in vitellogenesis in the normal reproductive cycle of females , as well as in the estrogenic response of male fish exposed to edcs . the subfunctionalization of ers presents an interesting challenge to the use of in vitro estrogen screening assays as ecological risk assessment tools for aquatic environments . classical estrogen screening assays , e.g. , the yeast estrogen screen ( yes ) , the t47d - kbluc assay , as well as the er transcriptional assays used by the u.s . epa endocrine disruptor screening program ( edsp ) , report activation of the human ( h ) er and/or er and thus are inherently anthropocentric in terms of their molecular targets . nevertheless , results from these assays are commonly extrapolated to other species , including fish . these assays are also often used to assess the estrogenic potency of aquatic environmental samples , which is reported in terms of 17-estradiol ( e2 ) equivalents ( eeq ) . such standardized assays offer a rapid , sensitive , and cost - effective means of screening for the presence of estrogenic compounds and thus have great utility for hazard characterization . however , given the complex involvement of multiple er subtypes in modulating estrogenic response in fish , the relationship between assay - derived eeqs and in vivo effects is unlikely to be straightforward . in this study , the ability of assay - derived eeqs to recapitulate estrogenic effects in fish was examined , using japanese medaka ( oryzias latipes ) as a model . stock solutions of estrogenic compounds were prepared at concentrations determined to be of equal potency in the yes , which reports activation of the her. test compounds included e2 , estrone ( e1 ) , 17-estradiol ( e2 ) , and estriol ( e3 ) , all steroidal estrogen species that are commonly detected in wastewater effluents . also tested was an extract from the anaerobic waste lagoon of a commercial swine operation , a potently estrogenic environmental matrix that contains a mixture of estrogen species , with e1 being the predominant estrogenic compound in the waste . effects of these yes - calibrated solutions in medaka following waterborne exposure were determined using quantitative real - time pcr ( qpcr ) , and affinity of these solutions for medaka ( m ) er , mer1 , and mer2 was determined using competitive ligand binding assays . additionally , in order to compare the yes to another classical screening assay that reports both her and her , the same yes - calibrated stock solutions were also tested in the t47d - kbluc assay . the same stock solutions were used across all experiments in this study , with stocks stored at 20 c in order to preserve chemical integrity . e2 , e1 , e2 , and e3 standards were purchased from steraloids inc . the e2 standard was dissolved in ethanol and diluted to a concentration of 8.8 m . all other estrogen standards were dissolved in ethanol and diluted to levels found to have an eeq of 8.8 m 5% in the yes assay , as described below . swine lagoon extract was prepared from the anaerobic lagoon slurry of a commercial swine sow operation , which receives waste from approximately 2500 gestating sows . details on the field site and extraction procedure are provided in supporting information ; see also yost et al . estrogen concentrations in all stock solutions , determined using liquid chromatography / tandem mass spectrometry , are provided in supplementary table si-1 . the yes utilizes a recombinant yeast line that expresses her , as well as a -galactosidase reporter driven by estrogen responsive elements ( ere ) . for the assay , yeast cells were dosed with a serial dilution of e2 stock solution alongside a serial dilution of e1 , e2 , e3 , or lagoon extract stock solution ; details are in supporting information . response curves were fit using graphpad prism version 6.0 for mac os x ( graphpad software , la jolla , california , usa , www.graphpad.com ) . eeqs were calculated as the ratio of the concentration of e2 that evoked a half - maximal response ( ec50e2 ) to the dilution factor of e1 , e2 , e3 , or lagoon extract stock solution that evoked a half - maximal response ( df50 ) . all stock solutions were confirmed to have the same eeq 5% in the yes assay , based on average values from 3 or 4 runs of the assay . were subsequently run in the t47d - kbluc estrogen screening assay , which utilizes a t47d human breast cancer cell line that maintains endogenous levels of her and her and stably expresses a luciferase reporter driven by a triplet ere . for the assay , cells were dosed with a serial dilution of e2 stock solution alongside a serial dilution of e1 , e2 , e3 , or lagoon extract stock solution in rpmi 1640 media ( sigma aldrich , st . louis , mo ; 5% dextran - coated charcoal - treated fetal bovine serum vol / vol ) ; details are in supporting information . sigmoid concentration response curves were fit using graphpad prism software , and eeq for each stock solution was calculated as a ratio of ec50e2 to df50 . final eeqs were calculated on the basis of average values from 2 or 3 runs of the assay . adult male medaka , between six and eight months of age , were obtained from the breeding colony at ncsu environmental and molecular toxicology ( description in supporting information ) . fish were exposed for 7 days to e2 at 0.64 nm ( 174 ng / l ; actual concentration ) ; to e1 , e2 , e3 , or swine lagoon extract , each at a yes - derived eeq of 0.64 nm 5% ; or to a negative ( ethanol ) control . exposures were conducted in 2-l glass beakers containing 1 l of exposure media . for each treatment , 12 fish were randomly distributed between four replicate beakers , 3 fish per beaker . exposure media were prepared daily by spiking 4 l of rearing media ( 5.1 mm nacl , 0.12 mm kcl , 0.198 mm mgso47h20 , and 0.081 mm cacl22h20 in picopure water ) with estrogen stock solution or with ethanol , for a final ethanol concentration of > 0.01% in all media . freshly prepared batches of media were then aliquoted equally between quadruplicate beakers in each treatment . treatments were maintained by static renewal , with 100% renewal of media every 24 h. survival rate through the experiment was 7591% , with no significant relationship between survival and treatment . at 7 days , all fish were euthanized with tricaine methanesulfonate in accordance with the iacuc - approved protocol . livers were excised , transferred to cryovials , immediately frozen in liquid nitrogen , and then moved to storage at 80 c . total rna was isolated from individual medaka livers using rna - bee reagent ( isotex diagnostics , friendswood , tx ) according to the manufacturer s protocol . rna integrity was assessed using the agilent 2100 bioanalyzer and rna 6000 nano kit ( agilent technologies , santa clara , ca ) . all samples were found to have rna integrity numbers of 9 or greater , indicating high - quality rna . cdna was then synthesized using 2 g rna with the high capacity cdna reverse transcription kit ( applied biosystems , grand island , ny ) . vtg-1 ( ab064320 ) and vtg-2 ( ab074891 ) are two distinct polypeptides that are precursors for the vtg phospholipoproteins in egg yolk . chg - h ( d89609 ) , chg - h minor ( chg - hm ) ( ab025967 ) , and chg - l ( af500194 ) are the three glycoproteins that comprise the zona pellucida ( zp ) , which is the thick inner layer that makes up the bulk of the egg envelope ; chg - h and chg - hm are identified as being in the zpb protein family , while chg - l is in the zpc protein family . mer ( ab033491.1 ) , mer1 ( nm_001104702.1 ) , and mer2 ( nm_001128512.1 ) are the three medaka nuclear estrogen receptor subtypes . vtg and chg primers were designed according to zhang et al , and 18s primers were designed according zhang et al . see supporting information for primer sequences , validation procedures , and qpcr reaction conditions . to quantify relative gene expression , the threshold cycle ( ct ) for 18s amplification 18s expression did not vary significantly with treatment . to determine fold change in each target gene following estrogen exposure , mean ct of the negative control group was subtracted from ct of each sample to yield ct . fold change in target gene expression was then calculated using 2 . as an additional comparison , the relative levels of the three mers were compared within the negative control treatment and within the e2 treatment , using mer expression as a calibrator . mean mer ct within a treatment was subtracted from the ct of each mer in the same treatment , and fold expression relative to mer was then calculated using 2 . using graphpad prism , target gene expression in each estrogen treatment was compared to gene expression in the negative control and e2 treatments using unpaired t test ( = 0.05 ) . correlation ( r ) between expression of each mer subtype and other target genes was then determined using linear regression . to compare relative expression of mers within the control and e2 treatments , the expression of each mer subtype was compared to the expression of mer within the same treatment using unpaired t test ( = 0.05 ) . bacterial lysates containing full - length mer , mer1 , or mer2 proteins were produced using bacterial expression systems , and assays were carried out as described by hawkins et al . , saturation binding analysis was first performed by incubating lysate with a range of [ 3h]e2 concentrations between 0.519 nm , and kd values for specific binding to each receptor were determined using graphpad prism . competition analysis was then performed on all mer subtypes by incubating lysate with a saturating concentration of [ 3h]e2 ( 23 nm , determined from saturation analysis ) and a range of competing analyte concentrations . sigmoidal competition curves were fit to specific binding data , and the concentration of each steroidal estrogen competitor that inhibited 50% of [ 3h]e2 binding ( ic50 ) was determined for each mer using graphpad prism , using the steps outlined in supporting information . relative binding affinity ( rba ) was calculated as the ratio of the ic50 of e2 to the ic50 of other steroidal estrogen competitors . for lagoon extract , the concentration factor of sample extract that inhibited 50% of [ 3h]e2 binding ( cf50 ) was determined for each mer . each assay was performed at least twice in order to calculate final kd , ic50 , and cf50 values . estrogen and lagoon extract stock solutions used in this study were all found to have yes - derived eeqs of 8.8 m 5% ( figure 1 ; values in supplementary table si-1 ) . estrogenic potency ( eeq ) of the steroidal estrogen and lagoon extract stock solutions in the yes and t47d - kbluc estrogen screening assays . mean standard error of the mean ( sem ) is shown ( n = 3 or 4 for yes assay ; n = 2 or 3 for t47d - kbluc assay ) . stock solutions that had been calibrated to be equipotent in the yes were subsequently tested in the t47d - kbluc assay . results indicate that the yes - calibrated stock solutions were not equipotent in the t47d - kbluc ( figure 1 ; values in supplementary table si-1 ) . e1 and lagoon slurry extract were both slightly more potent in the t47d - kbluc versus the yes , and e3 was approximately 10-fold more potent in the t47d - kbluc versus the yes . conversely , the potency of e2 was 3.3-fold less in the t47d - kbluc versus the yes . as with the yes , the same e2 stock solution at a concentration of 8.8 m was used as the calibration standard in this assay . fold change in hepatic gene expression in male japanese as expected , the expression of vtg and chg was upregulated with estrogen exposure ( figure 2a ) ; however , magnitude of gene induction was strikingly and often significantly different between exposures . gene induction by e2 exposure averaged 27,004-fold ( vtg-1 ) , 73,735-fold ( vtg-2 ) , 12,730-fold ( chg - h ) , 3,796-fold ( chg - hm ) , and 324-fold comparatively , induction of these genes in e1 exposures was 3682% of that induced by e2 and in lagoon extract exposures was 1659% of that induced by e2. as shown in figure 2a , the difference between these exposures and e2 was often statistically significant ; this was especially true for lagoon extract exposure . meanwhile , gene induction by e2 was only 215% of that evoked by e2 , making e2 the least potent of these estrogen treatments in the medaka . expression of vtg / chg was always significantly lower in e2 exposures relative to e2 and was not significantly different from the negative control for vtg-1 and vtg-2 induction . conversely , e3 exposure almost always elicited the greatest magnitude of response of all the estrogens , often significantly greater than e2 , with upregulation in vtg and chg that was 91241% of that observed in e2-exposed fish . fold change ( log scale ) in hepatic expression of ( a ) vtg and chg genes and ( b ) er genes in medaka exposed to e2 , e1 , e2 , e3 , or swine lagoon extract at a yes - derived eeq of 0.64 nm . significant difference between each exposure and the negative control is given by the letters a ( not significantly different from control ) or b ( significantly different from control ) ( p < 0.05 ) . additionally , significant differences between each exposure and the e2 exposure group are indicated by asterisks . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 , and * * * * p < 0.0001 ) . relative to the negative control , expression of mer was significantly upregulated by all estrogen exposures ( figure 2b ) . magnitude of mer upregulation was not significantly different between e2 ( 72-fold induction ) , e1 ( 56-fold induction ) , lagoon extract ( 59-fold induction ) , and e3 ( 67-fold induction ) exposures ; however , induction by e2 exposures ( 24-fold induction ) was significantly lower than that induced by e2. meanwhile , all estrogen exposures resulted in significant downregulation ( 2- to 5-fold ) of hepatic mer1 and mer2 expression relative to negative control . a significant correlation was observed between mer expression and expression of vtg / chg genes ( p < 0.0001 for all ) , with linear regression analysis indicating r of 0.5405 for correlation of mer and vtg-1 , and r ranging from 0.8065 to 0.8551 for correlation of mer and vtg-2/chg genes . no linear correlation was observed between expression of mer and mer1 ( r = 1.83 10 ; p = 0.9913 ) or mer2 ( r = 8.86 10 ; p = 0.8109 ) . similarly , linear correlation was poor between mer1 and vtg-1 ( r = 0.002872 , p = 0.6667 ) , vtg-2 ( r = 0.05925 ; p = 0.0472 ) , chg - l ( r = 0.04323 ; p = 0.0914 ) , chg - h ( r = 0.05959 ; p = 0.0465 ) , and chg - hm ( r = 0.06816 ; p = 0.0328 ) ; and mer2 and vtg-1 ( r = 0.002861 ; p = 0.6673 ) , vtg-2 ( r = 0.04610 ; p = 0.08100 ) , chg - l ( r = 0.03588 ; p = 0.3588 ) , chg - h ( r = 0.04187 ; p = 0.0967 ) , and chg - hm ( r = 0.04876 ; p = 0.0725 ) . expression of the two mer subtypes , however , was highly correlated ( r = 0.7713 ; p < 0.0001 ) . using mer expression as a calibrator , it was estimated that expression of mer2 in control fish was significantly greater ( av 30-fold ) than mer ( figure 3a ) , making mer2 the most highly expressed er subtype in control male medaka liver . average mer1 expression in control fish was also significantly greater than that of er ( av 8-fold ) . following estrogen exposure , the receptor population shifted dramatically . mer became the most highly expressed hepatic er subtype in e2-exposed fish , with expression significantly elevated an average of 23-fold above mer1 and 5-fold above mer2 ( figure 3b ) . relative hepatic expression of the three mer subtypes , normalized to mer expression , in ( a ) male medaka from the negative control group and ( b ) male medaka exposed to e2. mean sem ( n = 9 ) is shown . asterisks indicate significant difference relative to mer expression ( p > 0.05 ) . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 , and * * * * p < 0.0001 . saturation binding curves for the three mers are shown in supplementary figure si-1 , and competitive binding curves are shown in supplementary figure si-2 . saturation binding analysis with [ 3h]e2 demonstrated that kd values were similar for mer1 ( 1.017 nm ) and mer2 ( 1.107 nm ) , while the kd for mer was higher ( 1.654 nm ) . this indicates that the [ 3h]e2 ligand has a greater affinity for mer1 and mer2 than for mer and that the affinity of this ligand is similar between the two mer subtypes . in competitive ligand binding assays , ic50 s for e2 with each receptor followed the rank order of mer1 mer2 > mer ( table 1 ) . overall , both saturation binding analysis and competitive binding results indicate that e2 has approximately 1.5- to 1.7-fold greater affinity for mer subtypes versus mer. for the swine lagoon extract competitor , cf50 is indicated rather than ic50 . in competitive ligand binding assays , all test compounds including lagoon extract were able to displace [ 3h]e2 binding to all three mers ( supplementary figure si-2 ) . rbas for the five estrogen treatments indicate that e2 is the strongest competitor and e2 or e3 are the weakest competitors for all three mer subtypes ( table 1 ) . however , when ic50 s of each compound are compared across the three receptors , it is evident that each ligand has a unique pattern of binding preferences . in contrast to e2 , ic50 s for e1 followed the rank order of mer > me21 mer2 and indicate that e1 has an approximately 1.7-fold greater affinity for mer than for either of the mer subtypes . similar to e1 , e2 had ic50 s following a rank order of mer > mer1 > mer2 . e3 displayed ic50 s following the rank order of mer1 > mer2 > mer , indicating that , like e2 , this ligand also has greater binding affinity for the mer subtypes compared to mer ; however , while e2 had a similar affinity for the two mer subtypes , e3 had a 1.9-fold higher affinity for mer1 versus mer2 . finally , cf50 s of swine lagoon extract followed the rank order of mer > mer1 mer2 and indicate that the mixture of compounds in this extract has approximately 1.7- to 1.9-fold greater binding affinity for the mer versus the mer subtypes ( table 1 ) . notably , this is the same rank order of binding affinity observed for e1 , which is the predominant species of steroidal estrogen found in the lagoon extract ( supplementary table si-1 ) . piscine responses to estrogenic edc exposure are driven by the complex interplay of ligand interactions with multiple er subtypes , which may be difficult to predict using classical her - based transactivation assays . in this study , qpcr results clearly indicate a discrepancy between the activity of various steroidal estrogens in the yes assay and effects on exposed male medaka . this discordance is not unexpected , given the inherent challenges of extrapolating not only from an in vitro system to a living organism but also between human and medaka molecular targets . while er ligand binding domains are well conserved evolutionarily , several key amino acid changes have been identified in er ligand binding pockets of teleost fish relative to humans , which may suggest functional differences . nevertheless , rbas of steroidal estrogens for her have been reported to follow the rank order of e2 ( 100% ) > e1 ( 60% ) > e2 ( 58% ) > e3 ( 14% ) , which is similar to those reported here for mer. likewise , ec50 's for the in vitro transactivation of recombinant mer have been reported to follow the rank order of e2 > e1 > e3 . our laboratory has determined that the relative potencies of steroidal estrogens in the yes follow the same rank order of e2 ( 100% ) > e1 ( 47% ) > e2 ( 2.9% ) > e3 ( 0.76% ) . these similarities suggest that sequence differences between her and mer are not likely a predominant factor contributing to discordance between yes - derived eeqs and vtg / chg induction in medaka for the suite of compounds examined in this study . indeed , other studies have indicated that ligand specificity of er for common xenoextrogens is often well conserved across species , but it should be noted that this might not be the case for all compounds . for instance , a recent study demonstrated that several subtype - specific ligands of mammalian ers did not maintain the same selectivity in mozambique tilapia ( oreochromis mossambicus ) , indicating that agonistic characteristics can not always be extrapolated between species . the yes assay is also notably unable to detect the estrogenic activity of chlorinated chemicals , which has led to its exclusion from the u.s . however , this limitation is not applicable to the suite of estrogens used in this study . although numerous other variables could potentially come into play , such as metabolic differences or variations in nuclear receptor coactivators between yeast cells and medaka hepatocytes , ligand binding data suggest that lack of recapitulation of eeqs in vivo may be due in part to ligand interactions with er subtypes , which are not accounted for by the yes . of note , ligand interactions with plasma membrane - bound ers ( e.g. , gpr30 ) in vivo may also contribute to our observed differences , but this mechanism is not considered here . the potential involvement of such receptors in this response should not be conclusively discounted , although it has been demonstrated in rainbow trout that the synthetic estrogen 17-ethynylestradiol ( ee2 ) does not stimulate vtg induction via membrane - bound ers . in medaka , the magnitude of vtg and chg mrna induction elicited by estrogen exposure followed the consistent rank order of e3 > e2 > e1 lagoon extract > e2. expression of vtg and chg genes was highly correlated with mer expression and poorly correlated with mer subtype expression , which is consistent with observations in other studies . however , when data from competitive ligand binding assays are compared with medaka gene expression data , it is evident that the compounds that elicited the most robust biomarker induction in medaka , i.e. , e2 and e3 , both exhibited preferential binding affinity for the two mer subtypes over mer. in contrast , e2 , e1 , and swine lagoon extract , all of which elicited comparatively weak responses in the exposed medaka , exhibited greater affinity for mer than for mer subtypes . these binding preferences are similar to those reported in a closely related teleost species , atlantic croaker ( micropogonias undulates ) . the correlation between er affinity and vtg / chg induction is particularly interesting in light of recent findings indicating a primary role for er subtypes in the initiation of vitellogenesis . nelson and habibi used selective gene knockdown on goldfish ( gf ) primary hepatocytes to examine the functional roles of er subtypes on vtg and gfer mrna expression . gfer1 was found necessary for maintaining baseline expression of gfer , and both gfer subtypes contributed to upregulation of gfer and vtg following estrogen exposure . the authors speculated that gfer-mediated upregulation of gfer primes hepatocytes for further stimulation by estrogen , switching the liver into the mode for vtg production . a more recent study by griffin et al . took a similar approach using gene knockdown in zebrafish ( zf ) embryos and determined that both zfer and zferb ( formerly known as zfer1 ) were needed to induce zfer and vtg , while the role of zfera ( formerly zfer2 ) was unclear . these authors envisioned a scenario in which zfer and zferb act cooperatively to upregulate zfer and vtg upon estrogen stimulation . furthermore , knockdown of zferb also blocked induction of brain aromatase , an enzyme critically involved in teleost sexual differentiation . nelson and habibi reported er1 to be the most highly expressed hepatic er subtype in male and early recrudescent female goldfish and er to be the most highly expressed of these receptors in females approaching sexual maturity . in this study , we found mer2 to be the most highly expressed er subtype in adult male medaka liver , which is consistent with previous reports in medaka . following 7-day exposure to estrogens , mer was significantly upregulated and mer subtypes were significantly downregulated relative to negative controls , shifting the receptor population so that mer was the most highly expressed hepatic er subtype . interestingly , these expression patterns are similar to that observed in female fish in response to natural fluctuations in circulating estradiol levels . in seasonal spawning species such as largemouth bass and rainbow trout , females have been demonstrated to have elevated hepatic expression of er2 during the early vitellogenic stages of the reproductive cycle , while hepatic er reaches peak expression during the later stages of vtg production and oocyte maturation . hepatic expression of er1 is reported to be relatively static throughout the reproductive cycle of females in these species ; however , slight but significant changes in er1 expression are positively correlated with er expression , supporting the hypothesis that er1 regulates baseline expression of er. while one study reported largemouth bass er subtypes to be less sensitive than er to e2-mediated transactivation , studies in a variety of other fish species find that er1 and/or er2 have greater binding affinity for e2 and greater sensitivity to e2-mediated transactivation relative to er , which is consistent with binding results in our study . this heightened responsiveness to the endogenous er ligand perhaps also supports a role for er subtypes in the generation of the estrogenic response . the hypothesis regarding er as an inducer of hepatic er contrasts with reports that er can oppose er-mediated transcription . using human and largemouth bass er transactivation assays , it has been shown that the addition or coexpression of er attenuates the transcriptional activity of er. this has been attributed in part to the formation of er/ heterodimers that possess limited transactivational capacity and indicates that the ratio of these subtypes within cells is a critical determinant of transcriptional activity . er and er have been found to have opposing actions in a number of scenarios , including regulation of the cyclin d promoter , and transcriptional activation at activating protein 1 ( ap1 ) and stimulating protein 1 ( sp1 ) response elements . these trends may represent tissue- and/or species - specific differences in functional relationships between nuclear er subtypes . notwithstanding , it is noted that e3 exposure in our study generated both the greatest magnitude of vtg / chg upregulation and greatest magnitude of er1/er2 downregulation . the inverse relationship between these genes potentially indicates oppression of er-mediated vtg / chg expression by er subtypes . however , such a relationship was observed only for e3 and not for the other estrogen exposures . although no firm conclusions can be drawn , it is possible that either or both of these hypotheses regarding the functional relationship of er/er played into the results observed in our study . the contrasting nature of these hypotheses begs the question of whether the functional roles of multiple er subtypes differ throughout the chronology of the piscine estrogenic response , perhaps with er subtypes playing a supporting role of er following initial exposure to estrogen , but a different role in primed hepatocytes . since the swine lagoon extract employed in our study is representative of a suite of compounds that could reasonably be encountered in surface waters adjacent to livestock operations , it was of particular interest that the medaka response to this exposure often differed significantly from the response to e2. e1 is the most abundant steroidal estrogen in the lagoon extract , and comparison of gene expression results demonstrates that e1 and the lagoon extract elicited similar magnitudes of vtg and chg mrna induction . likewise , e1 and swine lagoon extract exhibited nearly identical binding behavior , with both treatments having slightly less than 2-fold greater affinity for mer versus the mer subtypes . given this correlation , e1 seems to be the principle compound driving vtg and chg induction in medaka exposed to this estrogenic mixture . the hormone composition in this swine lagoon extract is typical of many livestock waste facilities , with e1 being by far the predominant estrogen species present in the waste . e1 is also often found to be the most abundant steroidal estrogen in municipal wastewater effluents and impacted surface waters and thus is arguably one of the most widespread estrogenic edcs in aquatic environments . our results suggest that caution should be taken when representing the estrogenic potency of these effluents and surface waters using e2 as a calibration reference ( i.e. , assay - derived eeqs ) . while a direct comparison was not made between the t47d - kbluc assay and medaka gene expression , eeqs derived using the two screening assays suggest that the t47d - kbluc may be more predictive than the yes of vtg / chg induction by this suite of compounds . e2 was less potent and e3 was more potent in the t47d - kbluc versus in the yes , mirroring the vtg and chg gene expression responses observed in medaka . cells have been shown using western blot to express slightly higher endogenous levels of er relative to er. as male medaka also possess higher baseline hepatic levels of er relative to er , the relative levels of these receptors in t47d cells may enhance the translational capacity of this assay to vtg / chg induction in these fish . other cellular factors might also be expected to play a role , such as different suites of endogenous nuclear receptor coregulators in human cells versus yeast cells . conversely , e1 and lagoon slurry extract were slightly more potent in the t47d - kbluc versus in the yes , which does not reflect the effects of these compounds in medaka . this may be due to the cellular expression of e2 dehydrogenase , an enzyme highly expressed in t47d cells that may increase estrogenic potency via the formation of e2 from e1 . notably , a recent study found that municipal wastewater effluent elicited far greater effects in fathead minnows ( pimephales promelas ) than predicted on the basis of the t47d - kbluc assay ; the authors speculated that this was likely due to enterohepatic recirculation of ee2 in fish . such effects are less likely for swine effluent , as synthetic hormones are not used in u.s . , results herein indicate that significant discrepancies exist between the yes assay and the induction of estrogenic biomarkers in a well - characterized model species , japanese medaka . vtg and chg are widely used biomarkers of endocrine disruption , with the vtg biomarker demonstrated via meta analysis to have a significant quantitative relationship with fecundity in both female and male fish . results of our study should not discount the advantages of the yes assay : the yes is arguably easier and less expensive than vertebrate cell - based assays such as the t47d - kbluc and fits into the framework of the adverse outcome pathway by identifying er activation as an anchoring mode of action . however , results suggest direct extrapolation between yes - derived eeqs and effects in fish may be problematic , particularly if results observed for vtg / chg are potentially emblematic of apical level effects . the t47d - kbluc may offer better predictive capacity for effects in fish , although direct comparison between t47d - kbluc and in vivo effects would be necessary in order to substantiate this observation . perhaps future studies could examine variables including the er:er ratio on the translational capacities of in vitro screening assays to effects in living organisms . another observation in our study is the utility of recombinant protein binding assays to highlight the interactions between ligand and specific receptor subtypes , potentially helping to bridge the gap between screening assays and effects in whole organisms . recombinant proteins could provide a favorable alternative to the cytosolic preparations that are often used for er binding assays , for instance , the rat uterine cytosol that is used to assess er binding in the u.s . another example is the use of trout liver cytosol to assess er binding , which is performed in conjunction with trout liver slice vtg induction to prioritize estrogenic compounds in another tiered approach by the u.s . the use of recombinant proteins could potentially enhance these assays by providing specificity and allowing ligand interactions with individual receptor subtypes to be observed ; these ligand interactions could then be linked to apical effects via further testing in higher tiered assays . given the apparent complex involvement of er and er subtypes in generating the estrogenic response , the inclusion of multiple er subtypes in screening batteries could provide insight into the mechanisms of estrogenic activity , as well as enhance the translational capacities of in vitro assays for risk assessment . this includes the assessment of complex and environmentally relevant mixtures , such as livestock waste effluents .

this study seeks to delineate the ligand interactions that drive biomarker induction in fish exposed to estrogenic pollutants and provide a case study on the capacity of human ( h ) estrogen receptor ( er)-based in vitro screening assays to predict estrogenic effects in aquatic species . adult male japanese medaka ( oryzias latipes ) were exposed to solutions of singular steroidal estrogens or to the estrogenic extract of an anaerobic swine waste lagoon . all exposure concentrations were calibrated to be equipotent based on the yeast estrogen screen ( yes ) , which reports activation of her. these exposures elicited significantly different magnitudes of hepatic vitellogenin and choriogenin gene induction in the male medaka . effects of the same yes - calibrated solutions in the t47d - kbluc assay , which reports activation of her and her , generally recapitulated observations in medaka . using competitive ligand binding assays , it was found that the magnitude of vitellogenin / choriogenin induction by different estrogenic ligands correlated positively with preferential binding affinity for medaka er subtypes , which are highly expressed in male medaka liver prior to estrogen exposure . results support emerging evidence that er subtypes are critically involved in the teleost estrogenic response , with the er:er ratio being of particular importance . accordingly , incorporation of multiple er subtypes into estrogen screening protocols may increase predictive value for the risk assessment of aquatic systems , including complex estrogenic mixtures .

Introduction Materials and Methods Results Discussion

estrogenic contaminants , including steroidal estrogens as well as a variety of anthropogenic chemicals , are commonly detected in aquatic environments due to inputs from wastewater and have gained notoriety as endocrine disrupting compounds ( edcs ) . exposure of male fish to estrogenic pollutants is linked to numerous adverse reproductive effects , including the development of testicular oocytes , reduced sperm counts and sperm motility , testicular fibrosis , and reduced fecundity . in addition to these apical end points , widely studied proximal biomarkers of estrogenic edc exposure in male fish include hepatic induction of the egg precursor proteins vitellogenin ( vtg ) and choriogenin ( chg ) , which are normally produced only by females in response to circulating serum estrogen . effects of estrogenic compounds are mediated in large part through nuclear estrogen receptors ( ers ) , which regulate genomic responses via action as ligand - activated transcription factors . much interest has been garnered in determining the roles of multiple er subtypes in modulating estrogenic responses in fish . while mammals have been found to have two nuclear er subtypes ( er and er ) , teleost fish have at least three ( er , er1 , and er2 ) , with the second er subtype having arisen as a result of a genome duplication event in the teleost lineage . there remains significant debate surrounding the respective roles of piscine er subtypes in regulating vtg and chg induction in response to estrogenic ligands . studies in a variety of fish species have shown that vtg induction is accompanied by a sharp increase in hepatic er expression and little change of hepatic er subtype expression , implying that er is the principle receptor mediating regulation of the vtg gene . this emerging model suggests that er subtypes play a critical role in vitellogenesis in the normal reproductive cycle of females , as well as in the estrogenic response of male fish exposed to edcs . the subfunctionalization of ers presents an interesting challenge to the use of in vitro estrogen screening assays as ecological risk assessment tools for aquatic environments . classical estrogen screening assays , e.g. , the yeast estrogen screen ( yes ) , the t47d - kbluc assay , as well as the er transcriptional assays used by the u.s . epa endocrine disruptor screening program ( edsp ) , report activation of the human ( h ) er and/or er and thus are inherently anthropocentric in terms of their molecular targets . these assays are also often used to assess the estrogenic potency of aquatic environmental samples , which is reported in terms of 17-estradiol ( e2 ) equivalents ( eeq ) . however , given the complex involvement of multiple er subtypes in modulating estrogenic response in fish , the relationship between assay - derived eeqs and in vivo effects is unlikely to be straightforward . in this study , the ability of assay - derived eeqs to recapitulate estrogenic effects in fish was examined , using japanese medaka ( oryzias latipes ) as a model . stock solutions of estrogenic compounds were prepared at concentrations determined to be of equal potency in the yes , which reports activation of the her. also tested was an extract from the anaerobic waste lagoon of a commercial swine operation , a potently estrogenic environmental matrix that contains a mixture of estrogen species , with e1 being the predominant estrogenic compound in the waste . effects of these yes - calibrated solutions in medaka following waterborne exposure were determined using quantitative real - time pcr ( qpcr ) , and affinity of these solutions for medaka ( m ) er , mer1 , and mer2 was determined using competitive ligand binding assays . additionally , in order to compare the yes to another classical screening assay that reports both her and her , the same yes - calibrated stock solutions were also tested in the t47d - kbluc assay . the same stock solutions were used across all experiments in this study , with stocks stored at 20 c in order to preserve chemical integrity . all stock solutions were confirmed to have the same eeq 5% in the yes assay , based on average values from 3 or 4 runs of the assay . were subsequently run in the t47d - kbluc estrogen screening assay , which utilizes a t47d human breast cancer cell line that maintains endogenous levels of her and her and stably expresses a luciferase reporter driven by a triplet ere . fish were exposed for 7 days to e2 at 0.64 nm ( 174 ng / l ; actual concentration ) ; to e1 , e2 , e3 , or swine lagoon extract , each at a yes - derived eeq of 0.64 nm 5% ; or to a negative ( ethanol ) control . chg - h ( d89609 ) , chg - h minor ( chg - hm ) ( ab025967 ) , and chg - l ( af500194 ) are the three glycoproteins that comprise the zona pellucida ( zp ) , which is the thick inner layer that makes up the bulk of the egg envelope ; chg - h and chg - hm are identified as being in the zpb protein family , while chg - l is in the zpc protein family . relative binding affinity ( rba ) was calculated as the ratio of the ic50 of e2 to the ic50 of other steroidal estrogen competitors . estrogen and lagoon extract stock solutions used in this study were all found to have yes - derived eeqs of 8.8 m 5% ( figure 1 ; values in supplementary table si-1 ) . estrogenic potency ( eeq ) of the steroidal estrogen and lagoon extract stock solutions in the yes and t47d - kbluc estrogen screening assays . mean standard error of the mean ( sem ) is shown ( n = 3 or 4 for yes assay ; n = 2 or 3 for t47d - kbluc assay ) . stock solutions that had been calibrated to be equipotent in the yes were subsequently tested in the t47d - kbluc assay . results indicate that the yes - calibrated stock solutions were not equipotent in the t47d - kbluc ( figure 1 ; values in supplementary table si-1 ) . e1 and lagoon slurry extract were both slightly more potent in the t47d - kbluc versus the yes , and e3 was approximately 10-fold more potent in the t47d - kbluc versus the yes . conversely , the potency of e2 was 3.3-fold less in the t47d - kbluc versus the yes . fold change in hepatic gene expression in male japanese as expected , the expression of vtg and chg was upregulated with estrogen exposure ( figure 2a ) ; however , magnitude of gene induction was strikingly and often significantly different between exposures . gene induction by e2 exposure averaged 27,004-fold ( vtg-1 ) , 73,735-fold ( vtg-2 ) , 12,730-fold ( chg - h ) , 3,796-fold ( chg - hm ) , and 324-fold comparatively , induction of these genes in e1 exposures was 3682% of that induced by e2 and in lagoon extract exposures was 1659% of that induced by e2. meanwhile , gene induction by e2 was only 215% of that evoked by e2 , making e2 the least potent of these estrogen treatments in the medaka . conversely , e3 exposure almost always elicited the greatest magnitude of response of all the estrogens , often significantly greater than e2 , with upregulation in vtg and chg that was 91241% of that observed in e2-exposed fish . fold change ( log scale ) in hepatic expression of ( a ) vtg and chg genes and ( b ) er genes in medaka exposed to e2 , e1 , e2 , e3 , or swine lagoon extract at a yes - derived eeq of 0.64 nm . magnitude of mer upregulation was not significantly different between e2 ( 72-fold induction ) , e1 ( 56-fold induction ) , lagoon extract ( 59-fold induction ) , and e3 ( 67-fold induction ) exposures ; however , induction by e2 exposures ( 24-fold induction ) was significantly lower than that induced by e2. using mer expression as a calibrator , it was estimated that expression of mer2 in control fish was significantly greater ( av 30-fold ) than mer ( figure 3a ) , making mer2 the most highly expressed er subtype in control male medaka liver . mer became the most highly expressed hepatic er subtype in e2-exposed fish , with expression significantly elevated an average of 23-fold above mer1 and 5-fold above mer2 ( figure 3b ) . relative hepatic expression of the three mer subtypes , normalized to mer expression , in ( a ) male medaka from the negative control group and ( b ) male medaka exposed to e2. in competitive ligand binding assays , ic50 s for e2 with each receptor followed the rank order of mer1 mer2 > mer ( table 1 ) . in competitive ligand binding assays , all test compounds including lagoon extract were able to displace [ 3h]e2 binding to all three mers ( supplementary figure si-2 ) . e3 displayed ic50 s following the rank order of mer1 > mer2 > mer , indicating that , like e2 , this ligand also has greater binding affinity for the mer subtypes compared to mer ; however , while e2 had a similar affinity for the two mer subtypes , e3 had a 1.9-fold higher affinity for mer1 versus mer2 . finally , cf50 s of swine lagoon extract followed the rank order of mer > mer1 mer2 and indicate that the mixture of compounds in this extract has approximately 1.7- to 1.9-fold greater binding affinity for the mer versus the mer subtypes ( table 1 ) . notably , this is the same rank order of binding affinity observed for e1 , which is the predominant species of steroidal estrogen found in the lagoon extract ( supplementary table si-1 ) . piscine responses to estrogenic edc exposure are driven by the complex interplay of ligand interactions with multiple er subtypes , which may be difficult to predict using classical her - based transactivation assays . in this study , qpcr results clearly indicate a discrepancy between the activity of various steroidal estrogens in the yes assay and effects on exposed male medaka . nevertheless , rbas of steroidal estrogens for her have been reported to follow the rank order of e2 ( 100% ) > e1 ( 60% ) > e2 ( 58% ) > e3 ( 14% ) , which is similar to those reported here for mer. likewise , ec50 's for the in vitro transactivation of recombinant mer have been reported to follow the rank order of e2 > e1 > e3 . our laboratory has determined that the relative potencies of steroidal estrogens in the yes follow the same rank order of e2 ( 100% ) > e1 ( 47% ) > e2 ( 2.9% ) > e3 ( 0.76% ) . these similarities suggest that sequence differences between her and mer are not likely a predominant factor contributing to discordance between yes - derived eeqs and vtg / chg induction in medaka for the suite of compounds examined in this study . although numerous other variables could potentially come into play , such as metabolic differences or variations in nuclear receptor coactivators between yeast cells and medaka hepatocytes , ligand binding data suggest that lack of recapitulation of eeqs in vivo may be due in part to ligand interactions with er subtypes , which are not accounted for by the yes . in medaka , the magnitude of vtg and chg mrna induction elicited by estrogen exposure followed the consistent rank order of e3 > e2 > e1 lagoon extract > e2. however , when data from competitive ligand binding assays are compared with medaka gene expression data , it is evident that the compounds that elicited the most robust biomarker induction in medaka , i.e. , e2 and e3 , both exhibited preferential binding affinity for the two mer subtypes over mer. the correlation between er affinity and vtg / chg induction is particularly interesting in light of recent findings indicating a primary role for er subtypes in the initiation of vitellogenesis . nelson and habibi reported er1 to be the most highly expressed hepatic er subtype in male and early recrudescent female goldfish and er to be the most highly expressed of these receptors in females approaching sexual maturity . in this study , we found mer2 to be the most highly expressed er subtype in adult male medaka liver , which is consistent with previous reports in medaka . while one study reported largemouth bass er subtypes to be less sensitive than er to e2-mediated transactivation , studies in a variety of other fish species find that er1 and/or er2 have greater binding affinity for e2 and greater sensitivity to e2-mediated transactivation relative to er , which is consistent with binding results in our study . this heightened responsiveness to the endogenous er ligand perhaps also supports a role for er subtypes in the generation of the estrogenic response . using human and largemouth bass er transactivation assays , it has been shown that the addition or coexpression of er attenuates the transcriptional activity of er. the contrasting nature of these hypotheses begs the question of whether the functional roles of multiple er subtypes differ throughout the chronology of the piscine estrogenic response , perhaps with er subtypes playing a supporting role of er following initial exposure to estrogen , but a different role in primed hepatocytes . since the swine lagoon extract employed in our study is representative of a suite of compounds that could reasonably be encountered in surface waters adjacent to livestock operations , it was of particular interest that the medaka response to this exposure often differed significantly from the response to e2. given this correlation , e1 seems to be the principle compound driving vtg and chg induction in medaka exposed to this estrogenic mixture . e1 is also often found to be the most abundant steroidal estrogen in municipal wastewater effluents and impacted surface waters and thus is arguably one of the most widespread estrogenic edcs in aquatic environments . while a direct comparison was not made between the t47d - kbluc assay and medaka gene expression , eeqs derived using the two screening assays suggest that the t47d - kbluc may be more predictive than the yes of vtg / chg induction by this suite of compounds . e2 was less potent and e3 was more potent in the t47d - kbluc versus in the yes , mirroring the vtg and chg gene expression responses observed in medaka . as male medaka also possess higher baseline hepatic levels of er relative to er , the relative levels of these receptors in t47d cells may enhance the translational capacity of this assay to vtg / chg induction in these fish . conversely , e1 and lagoon slurry extract were slightly more potent in the t47d - kbluc versus in the yes , which does not reflect the effects of these compounds in medaka . this may be due to the cellular expression of e2 dehydrogenase , an enzyme highly expressed in t47d cells that may increase estrogenic potency via the formation of e2 from e1 . notably , a recent study found that municipal wastewater effluent elicited far greater effects in fathead minnows ( pimephales promelas ) than predicted on the basis of the t47d - kbluc assay ; the authors speculated that this was likely due to enterohepatic recirculation of ee2 in fish . results of our study should not discount the advantages of the yes assay : the yes is arguably easier and less expensive than vertebrate cell - based assays such as the t47d - kbluc and fits into the framework of the adverse outcome pathway by identifying er activation as an anchoring mode of action . however , results suggest direct extrapolation between yes - derived eeqs and effects in fish may be problematic , particularly if results observed for vtg / chg are potentially emblematic of apical level effects . the t47d - kbluc may offer better predictive capacity for effects in fish , although direct comparison between t47d - kbluc and in vivo effects would be necessary in order to substantiate this observation . perhaps future studies could examine variables including the er:er ratio on the translational capacities of in vitro screening assays to effects in living organisms . another observation in our study is the utility of recombinant protein binding assays to highlight the interactions between ligand and specific receptor subtypes , potentially helping to bridge the gap between screening assays and effects in whole organisms . recombinant proteins could provide a favorable alternative to the cytosolic preparations that are often used for er binding assays , for instance , the rat uterine cytosol that is used to assess er binding in the u.s . given the apparent complex involvement of er and er subtypes in generating the estrogenic response , the inclusion of multiple er subtypes in screening batteries could provide insight into the mechanisms of estrogenic activity , as well as enhance the translational capacities of in vitro assays for risk assessment .

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

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

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

the modern backbone communication infrastructures , networks , and technologies like gsm , umts , wcdma , wimax , internet , wireless , and bluetooth protocols provide extraordinary high data rates , thus allowing cost - efficient and time - efficient remote delivering of medical data that have been collected from the portable , embedded devices that reside onto the end - users ( monitored patients ) toward remote medical servers , for further processing . furthermore , the pervasiveness and ubiquitous character of modern user - centric nomadic environments , in which the user is the central point surrounded by different types of embedded and small computing devices , like , for example , electrocardiograph ( ecg ) and pulse , oxygen saturation , and blood pressure sensors , add extra requirements concerning security aspects such as the protection of sensitive medical data and measurements , data integrity and confidentiality , and protection of patients privacy . in such environments , it is imperative to design and deploy efficient and effective network architectures as well as a generic , if possible , communication interface targeted to connect the external networks with the smart individual - person ( equipped with different types of smart embedded medical devices ( emds ) ) satisfying the basic security requirements for real - time , secure data communication , and protection of sensitive medical data and measurements , data integrity and confidentiality , and protection of the monitored patient 's privacy . it is an xml - based standard that is designed to communicate machine - readable license information in an ubiquitous , unambiguous , and secure manner between peer entities . although mpeg-21 is a standard that is used for sharing multimedia digital content , under well - defined terms , we argue that it could be used for protection of medical data and user 's privacy in a pervasive healthcare environment , assuming that the digital content is the medical data generated by the emds . in our work , we propose a generic security framework based on the mpeg-21 standard adopted for wearable emds thus filling an existing gap which made these devices and the data acquired by them vulnerable to any kind of attacks . although the mpeg-21 standard has been adopted for delivery of multimedia content , we argue that it can be used in our architecture in order to enhance users ' privacy and to enhance security requirements that are applied in such environments . moreover , since the medical measurements that taken from patients ' emds can include different data types , besides raw data , like , for example , a snapshot of electrocardiograph data in a jpeg file , or a video file of electrocardiograph data , the mpeg-21 standard can help us in content delivery , with security enhancement toward different types of end - users . our endeavor demonstrates that no general - purpose home server is required for processing medical data before sent toward end - users , that is , doctors , medical staff , thus making the proposed architecture a good candidate for portable devices in pervasive healthcare environments exposing limited resources . we strongly believe that the outcome of this work contributes toward the establishment of a standard context - aware security environment in which parts of sensitive personal information can be viewed only by valid users even though many users can have access to them . more specifically , section 2 gives a presentation of the relevant work , while section 3 analyzes the emerging of emds security requirements in combination with mpeg-21 standard . consistent with the major challenge to provide good quality and reliable health care services to an increasing number of people utilizing limited financial and human resources , they propose a person - based health care system which consists of wearable commercial of - the - shelf nodes which are already used in the hospital environment , and they are capable of sensing and processing blood - oxygen , blood - pressure , ecgs , and other vital signs and can be seamlessly integrated into wireless personal area networks ( wpans ) for ubiquitous real - time patient monitoring . their architecture lacks safety , security , and privacy considerations , which may lead to serious breaches to architecture 's and emds functionalities or to users ' privacy . venkatasubramanian and gupta made a survey on security solutions for pervasive healthcare environments focusing on securing of data collected by emds , securing the communications between emds and investigation of mechanisms for controlling the access to medical data . they propose the use of cryptographic primitives , where measurements of physiological values are used for cryptographic keys , eliminating thus the necessity for key distribution , for securing data , and for the establishment of secure communications between two entities . concerning the access control to medical data , they survey methods that are based on role - based access control ( rbac ) , extending it for usage in pervasive healthcare environments . in this work , the author does not provide a clear architecture that implements the usage of mpeg-21 and also he does not use the mpeg-21 intellectual property management and protection ( ipmp ) components for the protection of medical records , but he mentions its use as a future and open research issue . the primary function of a pervasive healthcare architecture is to collect , to process , and to store medical data from different types of emds , which are located on the patient , locally into general - purpose computing devices for further handling or to remote - located servers . in order to identify the security mechanisms that should be deployed into the proposed architecture for pervasive healthcare with the utilization of emds , we must identify and classify ( i ) possible attackers and malicious users of the aforementioned environment ; ( ii ) safety , security , and privacy requirements . provide a short classification of possible malicious users and adversaries in a pervasive healthcare environment , identifying active adversaries which are malicious users that have explicit interference and physical access to an emd ( i.e. such attackers include healthcare personnel , nurses , doctors , it professionals , and even the patients themselves . security is a general requirement in modern computing environments , but in pervasive healthcare systems security is an imperative requirement because those systems handle very sensitive data like medical data and electronic personal records ( eprs ) . in order to identify the security aspects and requirements in such environments , we have to identify the threat model ; some of the most significant threats for pervasive healthcare environments are ( a ) nonauthorized access to patient 's medical data , that is , a nurse and a doctor must have different authorities and access control to medical data ; ( b ) intentional alteration of medical data , thus leading to incorrect diagnosis and patient 's treatment ; ( c ) disclosure of medical data to third parties ( e.g. such goals are ( a ) accessibility to medical data , that is , ensuring that only appropriate entities must have access to emds and their medical data ; ( b ) accuracy of measurements and data , that is , all data and measurements taken from the emd must be accurate ; ( c ) traceability and identification of emds , that is , an emd must have mechanisms that allow it to make its presence clear to authorized entities , whenever it is necessary ; ( d ) maintenance and reconfigurability , that is , authorized personnel may alter emds configuration , locally or remotely ; finally , ( e ) resources efficiency , that is , extend emds battery life , through minimization of power consumption . those goals are not different from traditional security requirements of computing systems that basically rely on the attainment of authentication and authorization , data confidentiality , data integrity , availability and protection of users ' privacy , and have to be extended and possibly reviewed in the context of pervasive healthcare environments . two basic subcategories of authorization can be distinguished ( a ) personal authorization , that is , specific people or groups of people may access patients ' data and perform specific action over it ; ( b ) role - based authorization , that is , a person may have access to medical data based on a specific role that he has , for example , a doctor , a nurse , a caregiver . mechanisms ensure that when there is an interchange of data between two peer entities , the received data and the original ones are the same , and that no intermediate alteration has occurred , for example , through interference of an eavesdropper . data confidentiality , it assures that stored or transmitted data are well protected from possible disclosure . privacy , it can be defined as an entity 's ability to control how , when , and to what extent personal information about the entity will be communicated to third parties . extending privacy in the context of pervasive healthcare environments , we refer to ( i ) emd - existence and emd - type privacy , that is , an emd should not make its presence and its type clear to an unauthorized party , since the user ( patient ) might not want to know nonauthorized users what type of emd they carry with them and what type of functions it serves . moreover , data confidentiality is achieved through cryptographic mechanisms , that is , sensitive data are encrypted and data integrity is achieved transparently by the doctor 's devices that have access to the data . finally , the utilization of a drm mechanism through the mpeg-21 framework helps in the protection of user 's privacy , that is , different groups of users have access to different types of medical data . mpeg-21 is a new standard which has been proposed for primary use in the context of multimedia world , allowing the seamless , transparent , and universal delivery of multimedia content to the end - user , thus solving any interoperability issues . a component by itself is not an item , but the components are the basic building blocks of items , and ( iii ) descriptors which are metadata that accompany a resource , containing information that concerns all or part of the specific resource . the security problems may arise from the fact that the digital item 's description , that is , its structure , contents , attributes , and metadata , is a clear xml document and it is easily visible to anyone and vulnerable to nonauthorized usage . due to that fact , the mpeg-21 includes a part named intellectual property management and protection ( ipmp ) which provide mechanisms for protection of digital item . those tools are not predescribed by the standard , but each user , vendor , and so forth , may define and implement his own set of tools which perform basic security functions like encryption / decryption algorithms , authentication and data integrity mechanisms , watermarking , and fingerprinting . with the use of mpeg-21 ipmp components , we may protect the whole di or a part of it through the encapsulation of the original didl elements that we want to protect , with additional information ( ipmp info ) that refer to mechanisms and tools for the protection of the original elements . the ipmpdidl : info element contains information about protection and usage rules of the digital content , which may be categorized to ( i ) information about protection of the whole digital item , which is included in the child element ipmpdidl : ipmpgeneralinfodescriptor and ( ii ) information about protection of a certain part of the digital item 's hierarchy , which is included in the child element ipmpdidl : ipmpinfodescriptor , see figure 2 . moreover , the license can be used to convey some other sensitive data , like , for example , decryption keys for the encrypted ( protected ) contents of the digital item . then , the generated file is sent to a data server which is located at the patient 's hospital and it is stored to a database . if it is necessary , the patient 's doctor may extract data from that file remotely through his pda device , acting and informing accordingly the patient . after the network is configured , the personal server application is initiated in order to manage the network , that is , channel and time synchronization , data acquisition , data processing , data fusion , and so forth . furthermore , upon the availability of a communication channel , the personal server establishes a secure link toward tier 3 and transmits reports or files which are being further processed and integrated in the patient 's health record . as soon as the medical data measurements are collected by the emds , they are sent toward the pda ( tier 2 ) which is responsible for the creation of the mpeg-21-protected container that contains the medical measurements with appropriate usage licenses , as it can be seen in figure 5 . in steps 14 , medical data measurements from different user - located emds ( ecg , blood pressure and oxygen saturation , and pulse rate ) are taken and sent toward the pda , which in turn , aggregates them and creates an initial packaged data file that contains the previous data measurements . afterwards , the packaged file is amended to further processing in order to be encoded according to mpeg-21 terms , with appropriate metadata , protection information , and patient 's instructions ( relevant usage licenses ) ( step 5 ) . then , the new mpeg-21-based packaged file is sent to the hospital 's servers for further handling , that is , storage in data servers , and informing physicians and doctors about patient 's situation ( step 6 ) . in the following figures , we depict an abstract view of an mpeg-21 digital item that contains emds measurements , figure 6 , and the schematic encapsulation of them into an ipmp container , see figure 7 . in section 4 , we have emphasized that in such pervasive healthcare environments the protection of patient 's privacy and medical data confidentiality are key issues . we identify three groups of people that should have different access rights to the protected contents ( i ) the it supervisors which are responsible for storing the received packaged files to databases , ( b ) the specialized medical personnel like , for example , the nurses , and ( c ) the doctors , who are responsible for the attendance of the patients . for example , the it stuff may just see the patient 's identification number , ( patid ) , which is unique for each patient , in order to store the packaged file in data server and update patient 's electronic record . on the other hand , a nurse or trained medical stuff must be able to see some details about the patient , for example , a part of patients ' personal data like surname , name , and maybe an initial diagnosis , which may have automatically taken part at patient 's home after the occurrences of data measurements , in order to inform the patient 's doctor for further handling of the patient 's situation . finally , the patient 's doctor may have full access to contents of mpeg-21-packaged files , that is , patients personal data , detailed medical data measurements taken by emds , time of measurements , and initial automated diagnosis . so the generated mpeg-21 container contains three different items ; the first item is unprotected and contains a text data file with the patient 's identification number , which can be used for storage of the file to a database for further handling , and some other data , like the time that the file was created at the user 's pda . the license also contains the key which has been used from the patient 's pda for the encryption of the medical data file . we assume that each patient and each of the doctors and trained medical staff have a prearranged set of public - private keys ; those key pairs are assigned by the main hospital 's server and can be revoked at any time since there is continuous communication between the hospital 's server and the patient 's ( home ) server . so the license for data usage can be encrypted using the public key of the entity with which it is related , for example , the doctor with whom the patient is related . in order to access the protected items ' contents , the end - user must decrypt the license with his private key , retrieve the decryption key from the license , and decrypt the encrypted contents . following , in figure 8 , we depict a schematic view of the encapsulated mpeg-21-based data file which is transmitted from patient 's home server toward hospital data servers . finally , in figure 9 , we present an xml - view of the mpeg-21 ipmp - protected container that contains the three prementioned items . even if one malicious user has access to the remote location of the tool , he needs some cryptographic sensitive information like , for example , a decryption key for decoding the protected contents ; the key for the decryption is included into the encrypted license that accompanies each item , which furthermore defines the usage rules of the contents . we strongly believe that the outcome of this work contributes toward the establishment of a standard security environment in which parts of sensitive personal information can be viewed only by valid users even though many users can have access to them . moreover , our endeavor demonstrates that no general - purpose home server is required for processing of medical data before sent toward end - users , that is , doctors , medical staff , thus making the proposed architecture a good candidate for portable devices in pervasive healthcare environments exposing limited resources . in our proposal , we use the mpeg-21 standards ' ipmp components in order to enhance users ' privacy and achieve security requirements that are applied in such environments . as a matter of fact furthermore , with respect to future work , we are planning to exploit some other elements of the mpeg-21 standard which , for example , would allow us to include ( in line ) into the ipmp - protected digital item the protection tools that are used to govern the protected item rather than remote referenced them , and also we could use some other elements of mpeg-21 ipmp that allow digitally authentication of the protection tools , that is , each tool carries a digital signature which validated each time the tool is used , before appliance of them to the protected contents . moreover , as the mpeg-21 is a quite new open standard that can be used for protection of sensitive data with the use of drm , as a future work , we are investigating the identification of possible leaks and vulnerabilities of the standard , that may lead to attacks and a compromised system . at this time , we assume that the hospital 's server is a trusted one , and that the devices on which those data are viewed are also safe and trusted , and that there is no possibility for data storage or alteration .

medical - grade skin adhesives , eyeglasses , and tissue undercuts have been traditionally used in the rehabilitation of patients with extra - oral defects . however , with the development and advancement of craniofacial implants and greater predictable aesthetics , improved prosthesis retention and stability is now a reality.1,2 a retentive matrix to hold clips and magnets is mandatory when using implant retained extra - oral prosthesis . this retentive matrix is made using acrylic resin to which the elastomer of extra - oral prosthesis is processed . this demands acrylic resin matrix to be securely bonded to flexible soft material of prosthesis . that means that denture resins may be used as a rigid base into which retention components are embedded , while the facial surface supports the silicone component of facial prosthesis . the rigid base or framework is usually fabricated with auto - polymerizing , heat - polymerizing , or visible light - curing denture resin . so it is apparent that for a serviceable and functional prosthesis , adequate bond strength is vital.1,3 the bond of silicone elastomer to the acrylic resin component must be sufficiently tenacious to withstand the substantial forces acting upon the bond interface , not only during placement and removal of the prosthesis , but also during mould opening and deflasking procedures as this is the weakest link in the restoration.2 maxillofacial silicone elastomers are dimethyl siloxane polymers and have different chemical structure from that of pmma denture base resin . it is likely that adhesive primers have an organic solvent and an adhesive agent that reacts with both silicone and resin materials.4 they activate the surfaces via etching or promoting hydrogen bonding and covalent coupling , increasing the wettability of the substrate and by impregnating the surface layer with the polymeric ingredients.5 singer et al.6 found that bond strength was enhanced using a combination of mdx 4 - 4210 silicone and either s-2260 or a-4040 primers . primer 1205 showed better bond strength than 1205 irrespective of the polymerization method or primer reaction time.7 polyzois8 reported that the curing method ( microwave irradiation and dry heat ) did not significantly affect the bond strength of silicone elastomers to acrylic denture resin unlike , the type of silicone elastomers . frangou et al.9 stated that the compatibility and affinity of primer composition with the selected silicone elastomer is important for efficient bonding . polyzois and frangou2 found that higher bond strength was achieved when the resin base was finished with 80-grit sic paper than when it was finished with 240 , 260 , 600 or 1000-grit sic paper . in another study , frangou et al.9 also claimed that when the bonding surface of the resin specimens were finished with a polishing machine using 80-grit sic paper under a constant flow of water , the bond strength increased . on the contrary , amin et al.10 reported that sandblasting the acrylic resin base weakened the bond between the resin and the silicone . similar results were reported by miami et al.4 who proved that roughening of the denture surface with air- particle abrasion was not effective for enhancing failure load and maintaining longevity of the silicone and acrylic resin . further studies should be carried out to find the proper preparation of resin surfaces to increase the bond strength between silicone elastomer and resin bases . there is a lack of evidence that compares the effect of primers on silicone - acrylic bond strength using various surface characteristics . the aim of this study was to evaluate the effect of 3 different commercially available bonding agents and surface alterations on the bond strength of silicone to acrylic resin with the null hypothesis that no difference would exist in the bond strength between them . the bond strength between heat polymerizing acrylic resin ( trevalon h ; dentsply , mumbai , india ) and silicone elastomer ( cosmesil m511 ; principality , uk ) was evaluated using 3 different commercially available bonding agents . g611 platinum primer ( principality medical ; newport , uk ) , a-330 gold platinum primer ( factor 2 ; phoenix , az , usa ) , and cyanoacrylates resin ( fevi kwik ; pidilite industries , mumbai , india . ) were used as bonding agents . the bond strength between silicone elastomer and heat polymerized acrylic resin substrate was tested using a acrylic resin blank as a test specimen with the dimension of 75 mm 10 mm 3 mm . silicone elastomer was bonded to a part of specified area on the acrylic resin blank . a total of 96 test specimens were fabricated , which were then divided into 4 main groups ( a , b , c and ct ) with 24 specimens each . primer g611 constituted the group a , primer a-330 constituted the group b , cyanoacrylate resin group c and the ct group represented the control group without any primer . these groups were further divided into subgroups 1 , 2 , and 3 with 8 specimens each . specimens of subgroup 1 had no surface alteration ( plain ) on the acrylic resin blank , subgroup 2 specimens had retentive beads on the area of acrylic resin blank where the silicone elastomer was bonded and specimens of the subgroup 3 had retentive holes . prefabricated acrylic sheet of 3 mm thickness was cut into required dimension of 75 mm ( length ) 10 mm ( width ) . the mold cavity , thus obtained was used to fabricate the heat polymerizing acrylic resin blank . the heat polymerizing acrylic resin was packed into the mold and the plain resin blanks without any surface alteration were fabricated for the subgroup 1 ( fig . each blank was measured and marking was done at a length of 25 mm where silicone had to be bonded . in the area of 25 mm 10 mm of the blank , adhesive was applied for the retentive beads which were provided by the manufacturer and the measured amount ( 0.01 mg ) of retentive beads ( retentionen , renfert , chicago , il , usa ) were spread evenly throughout the marked area and allowed to dry . the modified sheets with beads were then flasked , packed , polymerized and polished ( fig . after the acrylic blanks were finished and polished , they were measured and marked for a length of 25 mm 10 mm , where silicone had to be bonded . horizontal lines were marked in this area at a distance of 3 mm and vertical lines were marked at a distance of 2.5 mm . at the intersection of these lines , 24 holes were made of diameter 1.5 mm and depth of 0.5 mm using a round tungsten carbide bur of diameter 1.5 mm ( fig . 3 ) . on the acrylic resin blanks fabricated ( with and without surface characterization ) above , another acrylic blank of the same dimension ( 75 mm 10 mm 3 mm in length , width and thickness respectively ) was overlapped and the borders were sealed neatly with wax to seal the gap between the two blanks . the fused acrylic blanks were then flasked with the first pour covering till the junction of the two blanks . the plaster was allowed to set and later petroleum jelly was applied all over followed by which the second pour was done using dental stone and the flask was clamped and stone was allowed to set . later the flask was opened , dewaxed and the overlapped acrylic blank from the upper member of the flask was removed from the mold . the lower member of the flask contained the acrylic blank with the required surface characteristic and the upper member of the flask contained mold of overlaped resin blank in which silicone was packed . next , an adhesive tape was applied to define the area over which the silicone elastomer was to be bonded to the acrylic substrate . the tape covered an area of 50 mm 10 mm leaving an uncovered area of 25 mm10 mm ( surface characterization was done ) where the silicone had to be bonded to the acrylic substrate . the uncovered area of acrylic blank was cleaned with acetone and then left to air dry . different primers were applied to their corresponding group specimens according to the manufacturer 's instruction and cosmesil m511 silicone elastomer was packed and cured according to manufacturer 's instructions . only for the group c the silicone was packed without the application of primer . after finishing of the cured silicone strip , a uniform layer of cyanoacrylate resin was applied on the area of acrylic resin blank to be bonded to silicone and the cured silicone strip was immediately placed in its correct position on the acrylic resin blank and a weight of 1 kg was placed on the specimens for 15 minutes , then the silicone had bonded to the resin blank . all the test groups were subjected to an 180 peel strength test on hounsfeild universal testing machine ( ht-400 ) . the test was carried out according to the astm d-903 specifications . in each specimens , the silicone strip was bonded to acrylic denture base at one end ( 25 mm 10 mm 3 mm ) and left free at the other ( 50 mm 10 mm 3 mm ) . the free end of the strip was turned back at 180 so that the hard acrylic base was clamped in the lower clamp and the soft free silicone strip was gripped in the upper clamp ( fig . peel strength ( n / mm ) was determined using the formula peel strength = f / w ( 1 + / 2 + 1 ) where f = maximum force recorded ( n ) ; w = width of specimens ( mm ) ; = extension ratio of silicone elastomer ( the ratio of stretched to primary length ) the results obtained were then subjected to statistical analysis using a 4 3 general factorial analysis of variance ( 2-way anova ) . pair wise comparison of 4 groups and 3 altered surfaces with respect to bond strength was analyzed by using scheff multiple comparison test procedures . the statistical analysis was performed using stata 10.0 ( stata corp lp , san diego , tx , usa ) statistical software . for each specimen , 0.01 mg of retentive beads of 0.6 mm diameter were used . prior to flasking the prefabricated acrylic sheet were modified . each blank was measured and marking was done at a length of 25 mm where silicone had to be bonded . in the area of 25 mm 10 mm of the blank , adhesive was applied for the retentive beads which were provided by the manufacturer and the measured amount ( 0.01 mg ) of retentive beads ( retentionen , renfert , chicago , il , usa ) were spread evenly throughout the marked area and allowed to dry . the modified sheets with beads were then flasked , packed , polymerized and polished ( fig . after the acrylic blanks were finished and polished , they were measured and marked for a length of 25 mm 10 mm , where silicone had to be bonded . horizontal lines were marked in this area at a distance of 3 mm and vertical lines were marked at a distance of 2.5 mm . at the intersection of these lines , 24 holes were made of diameter 1.5 mm and depth of 0.5 mm using a round tungsten carbide bur of diameter 1.5 mm ( fig . on the acrylic resin blanks fabricated ( with and without surface characterization ) above , another acrylic blank of the same dimension ( 75 mm 10 mm 3 mm in length , width and thickness respectively ) was overlapped and the borders were sealed neatly with wax to seal the gap between the two blanks . the fused acrylic blanks were then flasked with the first pour covering till the junction of the two blanks . the plaster was allowed to set and later petroleum jelly was applied all over followed by which the second pour was done using dental stone and the flask was clamped and stone was allowed to set . later the flask was opened , dewaxed and the overlapped acrylic blank from the upper member of the flask was removed from the mold . the lower member of the flask contained the acrylic blank with the required surface characteristic and the upper member of the flask contained mold of overlaped resin blank in which silicone was packed . next , an adhesive tape was applied to define the area over which the silicone elastomer was to be bonded to the acrylic substrate . the tape covered an area of 50 mm 10 mm leaving an uncovered area of 25 mm10 mm ( surface characterization was done ) where the silicone had to be bonded to the acrylic substrate . the uncovered area of acrylic blank was cleaned with acetone and then left to air dry . the procedure mentioned above different primers were applied to their corresponding group specimens according to the manufacturer 's instruction and cosmesil m511 silicone elastomer was packed and cured according to manufacturer 's instructions . only for the group c the silicone was packed without the application of primer . after finishing of the cured silicone strip , a uniform layer of cyanoacrylate resin was applied on the area of acrylic resin blank to be bonded to silicone and the cured silicone strip was immediately placed in its correct position on the acrylic resin blank and a weight of 1 kg was placed on the specimens for 15 minutes , then the silicone had bonded to the resin blank . all the test groups were subjected to an 180 peel strength test on hounsfeild universal testing machine ( ht-400 ) . the test was carried out according to the astm d-903 specifications . in each specimens , the silicone strip was bonded to acrylic denture base at one end ( 25 mm 10 mm 3 mm ) and left free at the other ( 50 mm 10 mm 3 mm ) . the free end of the strip was turned back at 180 so that the hard acrylic base was clamped in the lower clamp and the soft free silicone strip was gripped in the upper clamp ( fig . peel strength ( n / mm ) was determined using the formula peel strength = f / w ( 1 + / 2 + 1 ) where f = maximum force recorded ( n ) ; w = width of specimens ( mm ) ; = extension ratio of silicone elastomer ( the ratio of stretched to primary length ) the results obtained were then subjected to statistical analysis using a 4 3 general factorial analysis of variance ( 2-way anova ) . pair wise comparison of 4 groups and 3 altered surfaces with respect to bond strength was analyzed by using scheff multiple comparison test procedures . the statistical analysis was performed using stata 10.0 ( stata corp lp , san diego , tx , usa ) statistical software . the groups of primers g611 and a-330 g failed either adhesively or cohesively while the control group and those of cyanoacrylate showed adhesive bond failure . the mean and standard deviations of the adhesion in peel force measurements are presented in table 1 . the primer a-330 g with retentive holes showed the maximum mean peel strength among all the groups ( 6.50 0.52 mpa ) . the control group without surface characterization / plain showed the least mean peel strength ( 0.15 0.23 mpa ) . table 2 represents the two way interactions between the primers and surface characterizations using 2-way anova . there was statistically significant difference between the peel strengths of different primers used ( p=.0000 * ) at 5% level of significance and also between the different surface characterizations used ( p=.0320 * ) at 5% level of significance . this analysis also revealed that two way interaction , primers by surface characterizations was highly significant ( p=.0001 * ) at 5% level of significance . further pair wise scheff multiple post hoc analysis was done for all the 4 primers . for all the surfaces , significant difference was seen between all the primers and control group ( p=.0000 * ) at 5% level of significance . significant difference was also seen between primers g611 and a-330 g when compared to cyanoacrylate ( p=.0000 * ) at 5% level of significance as displayed in table 3 . as presented in table 4 the pair wise comparison also revealed , significant difference between peel strength of retentive holes and that of plain surface of all the primers ( p=.0343 * ) at 5% level of significance . the bond of elastomer to the acrylic resin component must be sufficiently tenacious to withstand the substantial forces acting upon the bond interface , not only during placement and removal of the prosthesis , but also during mold opening and deflasking procedures.2 however , the chemical structure of maxillofacial silicone elastomers ( dimethyl siloxane polymers ) and pmma denture base resin is different , exhibiting poor bond characteristics.2,11 hence , primers are provided to increase the bond strength between silicone elastomer and acrylic resin thereby preventing delamination of silicone and enhancing the longevity of the prosthesis . they increase the bond strength by activating the surfaces via etching or promoting hydrogen bonding and covalent coupling , increasing the wettability of the substrate and by impregnating the surface layer with the polymeric ingredients.5 bond strength can be measured and evaluated by tensile test , shear test and peel test.12 a horizontal component of detaching forces are generated when the patient removes the craniofacial implant retained prosthesis . this type of force is well simulated in the peel test.3,4 for this reason , 180 peel test was used in this study to evaluate the bond strength . in the present study , test showed that specimens of primer g-611 and primer a-330 g were separated either cohesively or adhesively , whereas all test specimens of cyanoacrylate and control group showed adhesive type of bond failure . for cohesive failures , the peel bond strength between the silicone and the denture base was stronger than the strength of the silicone material . although the peel test has the advantage of being the only method in which failure proceeds at a controlled rate and the peel force is a direct measure of the work of detachment , cohesive peel bond test failures should be interpreted with caution . it is likely that the cohesive failures were initiated by small imperfections or voids in the silicone mixture . specimens with both the primer g-611 and a-330 g showed good bond strength with mean peel strength of 5.11 n / mm and 5.21 n / mm , respectively . although previous study conducted by hatamleh and watts1 , showed lower bond strength of 1.30 n / mm and 2.36 n / mm , they stated a-330 g produced the maximum peel strength among the primers , which was in accordance with our study . the increased bond strength of a-330 primer could be due to increased chemical affinity in comparison to g-611 as they vary in composition . specimens with cyanoacrylate produced bond strength of 2.65 n / mm which was significantly less than the bond strength produced by g-611 and a-330 g primers . yet it produced satisfactory bond strength in which the result being 0.15 n / mm without the use of primers . in surface characteristics where retentive holes were used , the maximum peel bond strength was produced with primer g-611 and primer a-330 g in comparison to plain acrylic resin substrate . this is in accordance with the study conducted by craig and gibbons.13 they advocated a roughened surface to improve the adhesive bond . they reported that adhesive values obtained by roughening were approximately double those of smooth surfaces because of a slightly irregular surface provided mechanical locking for the soft material . on the contrary jagger et al.14 claimed that roughening the resin surface with an acrylic bur weakened the bond because of the stress concentration caused by discontinuities of the surface and entrapped air or gas at the interface , which could further weaken the bond by the created voids . however , if the surface roughening is done in a definite pattern , with adequate intervals of plain surface and roughness , then the possibility of stress concentration and weakening of the acrylic resin substrate may be reduced . also , if the packing of silicone into the mold is done with appropriate equalization of pressure ensuring the proper flow of silicone elastomer into the depressions created on the acrylic substrate , air or gas entrapment will be prevented . increase in bond strength with primer g-611 and primer a-330 g with retentive holes could be due to the mechanical interlocking of peg like extension of silicone material into the holes made on acrylic substrate , also the provision of holes provided increased surface area for the silicone elastomer to bond with acrylic resin substrate . similarly , surface characteristics of retentive beads showed increased bond strength although it was not significant in comparison to plain surface with specimens of g-611 and a-330 g , which is in accordance with a study conducted by taft et al.,3 who found that the mean force required in adhesion in peel test for 1205 primer without beads was 35.7 n and for 1205 primer with beads was 40.2 n. the results showed increase in bond strength with beads but showed no statistical significance . decrease in the bond strength with surface characteristics in cyanoacrylate specimens could be due to the method of sample fabrication , where silicone is peeled from the acrylic substrate from the bonding area , bonding agent is applied and the silicone elastomer had to be placed back in the same original position so that the irregularities , elevations and depressions of the two materials will correspond to each other , which is highly difficult . this produced an area of voids and spaces between the acrylic and silicone elastomer , producing a weak bond between the two . this study gives scope for further research to evaluate , the biocompatibility of using cyanoacrylate as a bonding agent in maxillofacial prosthesis . since the prosthesis is worn extraorally the effect of outdoor weathering on the bond strength between silicone and acrylic resin with respect to the bonding agent need to be evaluated . depth of 0.5 mm holes was found to improve the bond strength , but further research is needed to evaluate the variation of bond strength as the depth of the retentive holes varies . further research is also required to evaluate the long term effect of cyanoacrylate on both acrylic resin and silicone elastomer along with its mode of bonding . coloring pigments added to silicone can change surface properties of silicone , which in turn , can affect its bond strength and therefore need to be evaluated as well . primers g-611 and a-330 produced much better bond strength between silicone elastomer and acrylic resin than cyanoacrylate.amongst all the primers tested , a-330 g produced the highest bond strength and was the most compatible with cosmesil m511 silicone.among the primers , the use of retentive holes significantly increased the bond strength in comparison to specimens with no surface characteristics.surface characterization used in case of cyanoacrylate was found to decrease the bond strength unlike in case of primers g-611 and a-330g.as satisfactory bond strength with cyanoacrylate was achieved though it was less than the primers g-611 and a-330 g . primers g-611 and a-330 produced much better bond strength between silicone elastomer and acrylic resin than cyanoacrylate . amongst all the primers tested , a-330 g produced the highest bond strength and was the most compatible with cosmesil m511 silicone . among the primers , the use of retentive holes significantly increased the bond strength in comparison to specimens with no surface characteristics . surface characterization used in case of cyanoacrylate was found to decrease the bond strength unlike in case of primers g-611 and a-330 g . as satisfactory bond strength with cyanoacrylate

purposethe aim of the study was to evaluate the effect of 3 silicone primers and 3 surface characterization of acrylic resin surface on bond strength between silicone elastomer and acrylic resin.materials and methods96 cosmesil silicones bonded to heat - curing acrylic resin were fabricated with the dimension of 75 10 3 mm . the 3 primers used in this study were g611 platinum primer , a-330 gold platinum primer , and cyanoacrylates resin . specimens without primer were used as control . the 3 types of surface characterization done were retentive holes with 1.5 mm in diameter and 0.5 mm deep , retentive beads of 0.6 mm diameter and the third type which was plain without any characterization . the specimens were then checked for bond strength by subjecting them to 180 peel test on a universal testing machine . the obtained results were then subjected to statistical analysis using 2-way anova and scheff multiple post hoc procedures . the statistical significance was set at 5% level of significance.resultsthe maximum bond strength was seen for samples in which a-330 g primer was used followed by g611 primer . the control group showed the minimum bond strength . surface characterization of retentive holes increased the bond strength considerably as compared to retentive beads and samples without any surface characterization.conclusionwithin the limitations of the study , a-330 g primer was more compatible with cosmesil m511 silicone and has better bonding of cosmesil to acrylic resin . retentive holes made on acrylic surface increased the bond strength considerably than those without any surface characterization .

INTRODUCTION MATERIALS AND METHODS Fabrication of acrylic resin blank for the subgroup 2 Fabrication of acrylic resin blank for the subgroup 3 Preparation of mold for silicone elastomer packing Preparation done prior to packing silicone RESULTS DISCUSSION CONCLUSION

medical - grade skin adhesives , eyeglasses , and tissue undercuts have been traditionally used in the rehabilitation of patients with extra - oral defects . the rigid base or framework is usually fabricated with auto - polymerizing , heat - polymerizing , or visible light - curing denture resin . so it is apparent that for a serviceable and functional prosthesis , adequate bond strength is vital.1,3 the bond of silicone elastomer to the acrylic resin component must be sufficiently tenacious to withstand the substantial forces acting upon the bond interface , not only during placement and removal of the prosthesis , but also during mould opening and deflasking procedures as this is the weakest link in the restoration.2 maxillofacial silicone elastomers are dimethyl siloxane polymers and have different chemical structure from that of pmma denture base resin . it is likely that adhesive primers have an organic solvent and an adhesive agent that reacts with both silicone and resin materials.4 they activate the surfaces via etching or promoting hydrogen bonding and covalent coupling , increasing the wettability of the substrate and by impregnating the surface layer with the polymeric ingredients.5 singer et al.6 found that bond strength was enhanced using a combination of mdx 4 - 4210 silicone and either s-2260 or a-4040 primers . primer 1205 showed better bond strength than 1205 irrespective of the polymerization method or primer reaction time.7 polyzois8 reported that the curing method ( microwave irradiation and dry heat ) did not significantly affect the bond strength of silicone elastomers to acrylic denture resin unlike , the type of silicone elastomers . frangou et al.9 stated that the compatibility and affinity of primer composition with the selected silicone elastomer is important for efficient bonding . polyzois and frangou2 found that higher bond strength was achieved when the resin base was finished with 80-grit sic paper than when it was finished with 240 , 260 , 600 or 1000-grit sic paper . in another study , frangou et al.9 also claimed that when the bonding surface of the resin specimens were finished with a polishing machine using 80-grit sic paper under a constant flow of water , the bond strength increased . on the contrary , amin et al.10 reported that sandblasting the acrylic resin base weakened the bond between the resin and the silicone . similar results were reported by miami et al.4 who proved that roughening of the denture surface with air- particle abrasion was not effective for enhancing failure load and maintaining longevity of the silicone and acrylic resin . further studies should be carried out to find the proper preparation of resin surfaces to increase the bond strength between silicone elastomer and resin bases . there is a lack of evidence that compares the effect of primers on silicone - acrylic bond strength using various surface characteristics . the aim of this study was to evaluate the effect of 3 different commercially available bonding agents and surface alterations on the bond strength of silicone to acrylic resin with the null hypothesis that no difference would exist in the bond strength between them . the bond strength between heat polymerizing acrylic resin ( trevalon h ; dentsply , mumbai , india ) and silicone elastomer ( cosmesil m511 ; principality , uk ) was evaluated using 3 different commercially available bonding agents . g611 platinum primer ( principality medical ; newport , uk ) , a-330 gold platinum primer ( factor 2 ; phoenix , az , usa ) , and cyanoacrylates resin ( fevi kwik ; pidilite industries , mumbai , india . ) the bond strength between silicone elastomer and heat polymerized acrylic resin substrate was tested using a acrylic resin blank as a test specimen with the dimension of 75 mm 10 mm 3 mm . silicone elastomer was bonded to a part of specified area on the acrylic resin blank . a total of 96 test specimens were fabricated , which were then divided into 4 main groups ( a , b , c and ct ) with 24 specimens each . primer g611 constituted the group a , primer a-330 constituted the group b , cyanoacrylate resin group c and the ct group represented the control group without any primer . these groups were further divided into subgroups 1 , 2 , and 3 with 8 specimens each . specimens of subgroup 1 had no surface alteration ( plain ) on the acrylic resin blank , subgroup 2 specimens had retentive beads on the area of acrylic resin blank where the silicone elastomer was bonded and specimens of the subgroup 3 had retentive holes . prefabricated acrylic sheet of 3 mm thickness was cut into required dimension of 75 mm ( length ) 10 mm ( width ) . the mold cavity , thus obtained was used to fabricate the heat polymerizing acrylic resin blank . the heat polymerizing acrylic resin was packed into the mold and the plain resin blanks without any surface alteration were fabricated for the subgroup 1 ( fig . in the area of 25 mm 10 mm of the blank , adhesive was applied for the retentive beads which were provided by the manufacturer and the measured amount ( 0.01 mg ) of retentive beads ( retentionen , renfert , chicago , il , usa ) were spread evenly throughout the marked area and allowed to dry . horizontal lines were marked in this area at a distance of 3 mm and vertical lines were marked at a distance of 2.5 mm . at the intersection of these lines , 24 holes were made of diameter 1.5 mm and depth of 0.5 mm using a round tungsten carbide bur of diameter 1.5 mm ( fig . on the acrylic resin blanks fabricated ( with and without surface characterization ) above , another acrylic blank of the same dimension ( 75 mm 10 mm 3 mm in length , width and thickness respectively ) was overlapped and the borders were sealed neatly with wax to seal the gap between the two blanks . the fused acrylic blanks were then flasked with the first pour covering till the junction of the two blanks . the plaster was allowed to set and later petroleum jelly was applied all over followed by which the second pour was done using dental stone and the flask was clamped and stone was allowed to set . the lower member of the flask contained the acrylic blank with the required surface characteristic and the upper member of the flask contained mold of overlaped resin blank in which silicone was packed . next , an adhesive tape was applied to define the area over which the silicone elastomer was to be bonded to the acrylic substrate . the tape covered an area of 50 mm 10 mm leaving an uncovered area of 25 mm10 mm ( surface characterization was done ) where the silicone had to be bonded to the acrylic substrate . different primers were applied to their corresponding group specimens according to the manufacturer 's instruction and cosmesil m511 silicone elastomer was packed and cured according to manufacturer 's instructions . after finishing of the cured silicone strip , a uniform layer of cyanoacrylate resin was applied on the area of acrylic resin blank to be bonded to silicone and the cured silicone strip was immediately placed in its correct position on the acrylic resin blank and a weight of 1 kg was placed on the specimens for 15 minutes , then the silicone had bonded to the resin blank . all the test groups were subjected to an 180 peel strength test on hounsfeild universal testing machine ( ht-400 ) . in each specimens , the silicone strip was bonded to acrylic denture base at one end ( 25 mm 10 mm 3 mm ) and left free at the other ( 50 mm 10 mm 3 mm ) . the free end of the strip was turned back at 180 so that the hard acrylic base was clamped in the lower clamp and the soft free silicone strip was gripped in the upper clamp ( fig . peel strength ( n / mm ) was determined using the formula peel strength = f / w ( 1 + / 2 + 1 ) where f = maximum force recorded ( n ) ; w = width of specimens ( mm ) ; = extension ratio of silicone elastomer ( the ratio of stretched to primary length ) the results obtained were then subjected to statistical analysis using a 4 3 general factorial analysis of variance ( 2-way anova ) . pair wise comparison of 4 groups and 3 altered surfaces with respect to bond strength was analyzed by using scheff multiple comparison test procedures . for each specimen , 0.01 mg of retentive beads of 0.6 mm diameter were used . in the area of 25 mm 10 mm of the blank , adhesive was applied for the retentive beads which were provided by the manufacturer and the measured amount ( 0.01 mg ) of retentive beads ( retentionen , renfert , chicago , il , usa ) were spread evenly throughout the marked area and allowed to dry . horizontal lines were marked in this area at a distance of 3 mm and vertical lines were marked at a distance of 2.5 mm . at the intersection of these lines , 24 holes were made of diameter 1.5 mm and depth of 0.5 mm using a round tungsten carbide bur of diameter 1.5 mm ( fig . on the acrylic resin blanks fabricated ( with and without surface characterization ) above , another acrylic blank of the same dimension ( 75 mm 10 mm 3 mm in length , width and thickness respectively ) was overlapped and the borders were sealed neatly with wax to seal the gap between the two blanks . the fused acrylic blanks were then flasked with the first pour covering till the junction of the two blanks . the lower member of the flask contained the acrylic blank with the required surface characteristic and the upper member of the flask contained mold of overlaped resin blank in which silicone was packed . next , an adhesive tape was applied to define the area over which the silicone elastomer was to be bonded to the acrylic substrate . the tape covered an area of 50 mm 10 mm leaving an uncovered area of 25 mm10 mm ( surface characterization was done ) where the silicone had to be bonded to the acrylic substrate . the procedure mentioned above different primers were applied to their corresponding group specimens according to the manufacturer 's instruction and cosmesil m511 silicone elastomer was packed and cured according to manufacturer 's instructions . after finishing of the cured silicone strip , a uniform layer of cyanoacrylate resin was applied on the area of acrylic resin blank to be bonded to silicone and the cured silicone strip was immediately placed in its correct position on the acrylic resin blank and a weight of 1 kg was placed on the specimens for 15 minutes , then the silicone had bonded to the resin blank . all the test groups were subjected to an 180 peel strength test on hounsfeild universal testing machine ( ht-400 ) . in each specimens , the silicone strip was bonded to acrylic denture base at one end ( 25 mm 10 mm 3 mm ) and left free at the other ( 50 mm 10 mm 3 mm ) . peel strength ( n / mm ) was determined using the formula peel strength = f / w ( 1 + / 2 + 1 ) where f = maximum force recorded ( n ) ; w = width of specimens ( mm ) ; = extension ratio of silicone elastomer ( the ratio of stretched to primary length ) the results obtained were then subjected to statistical analysis using a 4 3 general factorial analysis of variance ( 2-way anova ) . pair wise comparison of 4 groups and 3 altered surfaces with respect to bond strength was analyzed by using scheff multiple comparison test procedures . the statistical analysis was performed using stata 10.0 ( stata corp lp , san diego , tx , usa ) statistical software . the groups of primers g611 and a-330 g failed either adhesively or cohesively while the control group and those of cyanoacrylate showed adhesive bond failure . the primer a-330 g with retentive holes showed the maximum mean peel strength among all the groups ( 6.50 0.52 mpa ) . the control group without surface characterization / plain showed the least mean peel strength ( 0.15 0.23 mpa ) . table 2 represents the two way interactions between the primers and surface characterizations using 2-way anova . there was statistically significant difference between the peel strengths of different primers used ( p=.0000 * ) at 5% level of significance and also between the different surface characterizations used ( p=.0320 * ) at 5% level of significance . this analysis also revealed that two way interaction , primers by surface characterizations was highly significant ( p=.0001 * ) at 5% level of significance . further pair wise scheff multiple post hoc analysis was done for all the 4 primers . for all the surfaces , significant difference was seen between all the primers and control group ( p=.0000 * ) at 5% level of significance . significant difference was also seen between primers g611 and a-330 g when compared to cyanoacrylate ( p=.0000 * ) at 5% level of significance as displayed in table 3 . as presented in table 4 the pair wise comparison also revealed , significant difference between peel strength of retentive holes and that of plain surface of all the primers ( p=.0343 * ) at 5% level of significance . the bond of elastomer to the acrylic resin component must be sufficiently tenacious to withstand the substantial forces acting upon the bond interface , not only during placement and removal of the prosthesis , but also during mold opening and deflasking procedures.2 however , the chemical structure of maxillofacial silicone elastomers ( dimethyl siloxane polymers ) and pmma denture base resin is different , exhibiting poor bond characteristics.2,11 hence , primers are provided to increase the bond strength between silicone elastomer and acrylic resin thereby preventing delamination of silicone and enhancing the longevity of the prosthesis . they increase the bond strength by activating the surfaces via etching or promoting hydrogen bonding and covalent coupling , increasing the wettability of the substrate and by impregnating the surface layer with the polymeric ingredients.5 bond strength can be measured and evaluated by tensile test , shear test and peel test.12 a horizontal component of detaching forces are generated when the patient removes the craniofacial implant retained prosthesis . this type of force is well simulated in the peel test.3,4 for this reason , 180 peel test was used in this study to evaluate the bond strength . in the present study , test showed that specimens of primer g-611 and primer a-330 g were separated either cohesively or adhesively , whereas all test specimens of cyanoacrylate and control group showed adhesive type of bond failure . for cohesive failures , the peel bond strength between the silicone and the denture base was stronger than the strength of the silicone material . although the peel test has the advantage of being the only method in which failure proceeds at a controlled rate and the peel force is a direct measure of the work of detachment , cohesive peel bond test failures should be interpreted with caution . specimens with both the primer g-611 and a-330 g showed good bond strength with mean peel strength of 5.11 n / mm and 5.21 n / mm , respectively . although previous study conducted by hatamleh and watts1 , showed lower bond strength of 1.30 n / mm and 2.36 n / mm , they stated a-330 g produced the maximum peel strength among the primers , which was in accordance with our study . specimens with cyanoacrylate produced bond strength of 2.65 n / mm which was significantly less than the bond strength produced by g-611 and a-330 g primers . yet it produced satisfactory bond strength in which the result being 0.15 n / mm without the use of primers . in surface characteristics where retentive holes were used , the maximum peel bond strength was produced with primer g-611 and primer a-330 g in comparison to plain acrylic resin substrate . this is in accordance with the study conducted by craig and gibbons.13 they advocated a roughened surface to improve the adhesive bond . on the contrary jagger et al.14 claimed that roughening the resin surface with an acrylic bur weakened the bond because of the stress concentration caused by discontinuities of the surface and entrapped air or gas at the interface , which could further weaken the bond by the created voids . however , if the surface roughening is done in a definite pattern , with adequate intervals of plain surface and roughness , then the possibility of stress concentration and weakening of the acrylic resin substrate may be reduced . increase in bond strength with primer g-611 and primer a-330 g with retentive holes could be due to the mechanical interlocking of peg like extension of silicone material into the holes made on acrylic substrate , also the provision of holes provided increased surface area for the silicone elastomer to bond with acrylic resin substrate . similarly , surface characteristics of retentive beads showed increased bond strength although it was not significant in comparison to plain surface with specimens of g-611 and a-330 g , which is in accordance with a study conducted by taft et al.,3 who found that the mean force required in adhesion in peel test for 1205 primer without beads was 35.7 n and for 1205 primer with beads was 40.2 n. the results showed increase in bond strength with beads but showed no statistical significance . decrease in the bond strength with surface characteristics in cyanoacrylate specimens could be due to the method of sample fabrication , where silicone is peeled from the acrylic substrate from the bonding area , bonding agent is applied and the silicone elastomer had to be placed back in the same original position so that the irregularities , elevations and depressions of the two materials will correspond to each other , which is highly difficult . since the prosthesis is worn extraorally the effect of outdoor weathering on the bond strength between silicone and acrylic resin with respect to the bonding agent need to be evaluated . depth of 0.5 mm holes was found to improve the bond strength , but further research is needed to evaluate the variation of bond strength as the depth of the retentive holes varies . further research is also required to evaluate the long term effect of cyanoacrylate on both acrylic resin and silicone elastomer along with its mode of bonding . primers g-611 and a-330 produced much better bond strength between silicone elastomer and acrylic resin than cyanoacrylate.amongst all the primers tested , a-330 g produced the highest bond strength and was the most compatible with cosmesil m511 silicone.among the primers , the use of retentive holes significantly increased the bond strength in comparison to specimens with no surface characteristics.surface characterization used in case of cyanoacrylate was found to decrease the bond strength unlike in case of primers g-611 and a-330g.as satisfactory bond strength with cyanoacrylate was achieved though it was less than the primers g-611 and a-330 g . primers g-611 and a-330 produced much better bond strength between silicone elastomer and acrylic resin than cyanoacrylate . amongst all the primers tested , a-330 g produced the highest bond strength and was the most compatible with cosmesil m511 silicone . among the primers , the use of retentive holes significantly increased the bond strength in comparison to specimens with no surface characteristics . surface characterization used in case of cyanoacrylate was found to decrease the bond strength unlike in case of primers g-611 and a-330 g .

the cremona study is a population survey carried out in 19901991 in the health district of cremona ( lombardia , italy ) to determine the prevalence of diabetes according to the oral glucose tolerance test ( ogtt ) and world health organization criteria ( 8,9 ) . past medical history , anthropometric measures , and clinical data of subjects were collected by trained interviewers using standardized procedures . a venous blood sample was collected after a 12-h overnight fast , and thereafter a 75-g oral glucose monohydrate was given . heart rate and blood pressure were recorded twice , at the beginning and at the end of the visit , in the sitting position , and after at least 10 min rest using a full automatic noninvasive sphyngomanometer . vital status and time of death were acquired from the regional health registry ( updated to 31 december 2005 ) , and causes of death were classified using the icd-9 ( death codes for cvd are from 401 to 448 and cancer from 140.0 to 208.9 ) . median follow - up was 180 months , and median follow - up of those still alive at 182 months ( 98% of those who were still alive had a minimum follow - up period of 174 months ) . study subjects were divided in four categories based on bmi ( nonobese : < 30 kg / m ; obese : 30 kg / m ) and estimated insulin resistance ( insulin sensitive : homeostasis model assessment of insulin resistance [ homa - ir ] < 2.5 ; insulin resistant 2.5 ) . the cutoff of 2.5 for homa - ir was chosen to compare our data with those recently published by kuk et al . therefore , the four categories were 1 ) the nonobese subjects with normal insulin sensitivity , 2 ) the obese but insulin - sensitive subjects , 3 ) the nonobese but insulin - resistant subjects , and 4 ) the obese and insulin - resistant subjects . baseline anthropometric , clinical , and laboratory features of study groups data are means sd or n ( % ) , unless otherwise indicated . * p < 0.05 vs. obese insulin - resistant subjects ; p < 0.05 vs. nonobese insulin - resistant subjects ; p < 0.05 vs. nonobese insulin - sensitive subjects ; for categorical variables p < 0.05 vs. nonobese and obese insulin - sensitive subjects . diabetes was defined according to the use of oral hypoglycemic agents or insulin and according to the world health organization diagnostic criteria for the ogtt ( basal plasma glucose > 7.8 mmol / l or > 11.1 mmol / l after a 2-h oral glucose load ) . impaired glucose tolerance was defined as basal plasma glucose < 7.8 mmol / l and plasma glucose > 7.8 but < 11 mmol / l after a 2-h oral glucose load . metabolic syndrome was defined accordingly to the definition of the national cholesterol education program adult treatment program iii . blood , serum , and plasma measurements were done as previously described ( 8,9 ) . bmi was calculated as weight in kilograms divided by the square of height in meters and alcohol consumption as grams of alcohol ( glass of wine = 20 g , glass of aperitif = 30 g , and glass of liquor = 80 g ) . homa - ir was calculated as previously described ( 11 ) , and ldl cholesterol was calculated using the friedwald formula . serum insulin , triglycerides , fibrinogen , and glucose had a skewed distribution ; therefore , log - transformed values were used in the analysis . the associations of each investigated risk factor with all - cause , cvd , and cancer mortality were estimated by the cox proportional hazard model , with adjustments for age and sex . multivariate cox regression analysis was performed to adjust the comparisons of mortality among the different subgroups for possible confounding factors . kaplan and meier curves for all - cause mortality were plotted for the four groups , as previously described . the cremona study is a population survey carried out in 19901991 in the health district of cremona ( lombardia , italy ) to determine the prevalence of diabetes according to the oral glucose tolerance test ( ogtt ) and world health organization criteria ( 8,9 ) . past medical history , anthropometric measures , and clinical data of subjects were collected by trained interviewers using standardized procedures . a venous blood sample was collected after a 12-h overnight fast , and thereafter a 75-g oral glucose monohydrate was given . heart rate and blood pressure were recorded twice , at the beginning and at the end of the visit , in the sitting position , and after at least 10 min rest using a full automatic noninvasive sphyngomanometer . vital status and time of death were acquired from the regional health registry ( updated to 31 december 2005 ) , and causes of death were classified using the icd-9 ( death codes for cvd are from 401 to 448 and cancer from 140.0 to 208.9 ) . median follow - up was 180 months , and median follow - up of those still alive at 182 months ( 98% of those who were still alive had a minimum follow - up period of 174 months ) . study subjects were divided in four categories based on bmi ( nonobese : < 30 kg / m ; obese : 30 kg / m ) and estimated insulin resistance ( insulin sensitive : homeostasis model assessment of insulin resistance [ homa - ir ] < 2.5 ; insulin resistant 2.5 ) . the cutoff of 2.5 for homa - ir was chosen to compare our data with those recently published by kuk et al . therefore , the four categories were 1 ) the nonobese subjects with normal insulin sensitivity , 2 ) the obese but insulin - sensitive subjects , 3 ) the nonobese but insulin - resistant subjects , and 4 ) the obese and insulin - resistant subjects . baseline anthropometric , clinical , and laboratory features of study groups data are means sd or n ( % ) , unless otherwise indicated . * p < 0.05 vs. obese insulin - resistant subjects ; p < 0.05 vs. nonobese insulin - resistant subjects ; . for categorical variables p < 0.05 vs. nonobese and obese insulin - sensitive subjects . diabetes was defined according to the use of oral hypoglycemic agents or insulin and according to the world health organization diagnostic criteria for the ogtt ( basal plasma glucose > 7.8 mmol / l or > 11.1 mmol / l after a 2-h oral glucose load ) . impaired glucose tolerance was defined as basal plasma glucose < 7.8 mmol / l and plasma glucose > 7.8 but < 11 mmol / l after a 2-h oral glucose load . metabolic syndrome was defined accordingly to the definition of the national cholesterol education program adult treatment program iii . blood , serum , and plasma measurements were done as previously described ( 8,9 ) . bmi was calculated as weight in kilograms divided by the square of height in meters and alcohol consumption as grams of alcohol ( glass of wine = 20 g , glass of aperitif = 30 g , and glass of liquor = 80 g ) . homa - ir was calculated as previously described ( 11 ) , and ldl cholesterol was calculated using the friedwald formula . serum insulin , triglycerides , fibrinogen , and glucose had a skewed distribution ; therefore , log - transformed values were used in the analysis . the associations of each investigated risk factor with all - cause , cvd , and cancer mortality were estimated by the cox proportional hazard model , with adjustments for age and sex . multivariate cox regression analysis was performed to adjust the comparisons of mortality among the different subgroups for possible confounding factors . kaplan and meier curves for all - cause mortality were plotted for the four groups , as previously described . of 2,011 subjects , 708 were nonobese and insulin sensitive , 923 nonobese and insulin resistant , and 337 obese and insulin resistant . there were a total of 43 obese insulin - sensitive individuals , representing 11.0% ( 95% ci 8.114.5 ) of the obese population and 2.1% ( 1.62.9 ) of the entire population . sex distribution did not differ among all groups , whereas cigarette smoking was more frequent in nonobese insulin - sensitive subjects than all other groups . systolic and diastolic blood pressure , heart rate , plasma glucose , insulin , total cholesterol , hdl cholesterol , triglycerides , transaminases , -glutamyltransferase ( gt ) , alkaline phosphatase ( alp ) , and fibrinogen did not differ between the two insulin - sensitive groups . individuals in the insulin - sensitive groups were younger , had lower heart rates , had higher plasma hdl cholesterol , and had lower fibrinogen and triglycerides , as well as had a lower prevalence of diabetes and metabolic syndrome than insulin - resistant groups . waist circumference was higher in obese insulin - sensitive than nonobese insulin - resistant subjects but were lower than in obese insulin - resistant subjects . systolic and diastolic blood pressure , plasma transaminases , gt , and alp were higher in the two groups of insulin - resistant subjects than in nonobese insulin - sensitive subjects . during the 15-year observation period , age and sex were associated with higher all - cause mortality ( age : hr 1.11 [ 95% ci 1.101.12 ] , p < 0.0001 ; female sex : 0.42 [ 0.350.50 ] , p < 0.0001 ) , mortality for cvd ( age : 1.15 [ 1.131.17 ] , p < 0.0001 ; female sex : 0.40 [ 0.310.53 ] , p < 0.0001 ) , and mortality for cancer ( age : 1.07 [ 1.061.09 ] , p < 0.0001 ; female sex : 0.39 [ 0.290.52 ] , p < 0.0001 ) . all - cause mortality was higher in the obese insulin - resistant subjects ( 31% ) in comparison with the reference group of nonobese insulin - sensitive subjects ( 20% ) ( age- and sex - adjusted hr 1.4 , p = 0.01 ) ( table 2 ) ( fig . 1 ) but not in the obese insulin - sensitive subjects ( 12% ) ( 0.99 , p = 0.97 ) ( table 2 ) ( fig . 1 ) and in the nonobese insulin - resistant subjects ( 26% ) ( 1.11 , p = 0.35 ) ( table 2 ) ( fig . cox proportional hazard model adjusting for age and sex survival by kaplan - meier estimates of all - cause mortality . subjects were divided according to bmi ( nonobese : < 30 kg / m ; obese : 30 kg / m ) and estimated insulin resistance ( insulin sensitive : homa - ir < 2.5 ; insulin resistant : 2.5 ) . at the bottom are the detailed figures of the number at risk for each subgroup of individuals . nob - ir , nonobese insulin - resistant subjects ; nob - is , nonobese insulin - sensitive subjects ( the reference ) ; ob - ir , obese insulin - resistant subjects ; ob - is , obese insulin - sensitive subjects . also , mortality for cvd ( 15% , p = 0.015 ) and cancer ( 12% , p = 0.04 ) ( table 2 ) was higher in the obese insulin - resistant subjects but not in the obese insulin - sensitive subjects ( cvd related : 5% , p = 0.66 , and cancer related : 7% , p = 0.95 ) and nonobese insulin - resistant subjects ( cvd related : 12% , p = 0.29 , and cancer related : 9% , p = 0.64 ) when compared with nonobese insulin - sensitive subjects ( cvd related : 8% and cancer related : 7% ) . because the prevalence of cigarette smoking and baseline plasma ldl cholesterol were different among the groups all - cause mortality remained higher in the obese insulin - resistant subjects ( hr 1.66 [ 95% ci 1.122.46 ] , p = 0.011 ) but not in obese insulin - sensitive subjects ( 0.79 [ 0.193.28 ] , p = 0.75 ) and in nonobese insulin - resistant subjects ( 1.22 [ 0.881.70 ] , p = 0.23 ) when compared with nonobese and insulin - sensitive subjects . the analysis was also repeated after the exclusion of diabetic patients . when compared with nonobese insulin - sensitive ( reference group ) , all - cause mortality tended to be higher in obese insulin - resistant subjects ( hr 1.29 [ 95% ci 0.961.73 ] , p = 0.087 ) but was again not different in obese insulin - sensitive subjects ( 1.01 [ 0.472.17 ] , p = 0.97 ) and nonobese insulin - resistant subjects ( 1.00 [ 0.801.25 ] , p = 0.98 ) . similarly , mortality for cvd and cancer tended to be higher in obese insulin - resistant subjects ( cvd : hr 1.40 [ 95% ci 0.942.11 ] , p = 0.071 ; cancer : 1.46 [ 0.942.27 ] , p = 0.097 ) but was not different in obese insulin - sensitive subjects ( cvd : 0.76 [ 0.1973.11 ] , p = 0.71 ; cancer : 1.05 [ 0.333.56 ] , p = 0.94 ) and nonobese insulin - resistant subjects ( cvd : [ 1.09 [ 0.771.54 ] , p = 0.65 ; cancer : 1.01 [ 0.932.27 ] , p = 0.95 ) than nonobese insulin - sensitive subjects . finally , instead of the preselected homa - ir of 2.5 , we repeated the analysis using cutoff values ( top tertile and top quartile ) for homa - ir obtained from the present study . even in this case , the results did not change ( see the online appendix , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0665/dc1 ) . of 2,011 subjects , 708 were nonobese and insulin sensitive , 923 nonobese and insulin resistant , and 337 obese and insulin resistant . there were a total of 43 obese insulin - sensitive individuals , representing 11.0% ( 95% ci 8.114.5 ) of the obese population and 2.1% ( 1.62.9 ) of the entire population . sex distribution did not differ among all groups , whereas cigarette smoking was more frequent in nonobese insulin - sensitive subjects than all other groups . systolic and diastolic blood pressure , heart rate , plasma glucose , insulin , total cholesterol , hdl cholesterol , triglycerides , transaminases , -glutamyltransferase ( gt ) , alkaline phosphatase ( alp ) , and fibrinogen did not differ between the two insulin - sensitive groups . individuals in the insulin - sensitive groups were younger , had lower heart rates , had higher plasma hdl cholesterol , and had lower fibrinogen and triglycerides , as well as had a lower prevalence of diabetes and metabolic syndrome than insulin - resistant groups . waist circumference was higher in obese insulin - sensitive than nonobese insulin - resistant subjects but were lower than in obese insulin - resistant subjects . systolic and diastolic blood pressure , plasma transaminases , gt , and alp were higher in the two groups of insulin - resistant subjects than in nonobese insulin - sensitive subjects . age and sex were associated with higher all - cause mortality ( age : hr 1.11 [ 95% ci 1.101.12 ] , p < 0.0001 ; female sex : 0.42 [ 0.350.50 ] , p < 0.0001 ) , mortality for cvd ( age : 1.15 [ 1.131.17 ] , p < 0.0001 ; female sex : 0.40 [ 0.310.53 ] , p < 0.0001 ) , and mortality for cancer ( age : 1.07 [ 1.061.09 ] , p < 0.0001 ; female sex : 0.39 [ 0.290.52 ] , p < 0.0001 ) . all - cause mortality was higher in the obese insulin - resistant subjects ( 31% ) in comparison with the reference group of nonobese insulin - sensitive subjects ( 20% ) ( age- and sex - adjusted hr 1.4 , p = 0.01 ) ( table 2 ) ( fig . 1 ) but not in the obese insulin - sensitive subjects ( 12% ) ( 0.99 , p = 0.97 ) ( table 2 ) ( fig . 1 ) and in the nonobese insulin - resistant subjects ( 26% ) ( 1.11 , p = 0.35 ) ( table 2 ) ( fig . 1 ) . cox proportional hazard model adjusting for age and sex survival by kaplan - meier estimates of all - cause mortality . subjects were divided according to bmi ( nonobese : < 30 kg / m ; obese : 30 kg / m ) and estimated insulin resistance ( insulin sensitive : homa - ir < 2.5 ; insulin resistant : 2.5 ) . at the bottom are the detailed figures of the number at risk for each subgroup of individuals . nob - ir , nonobese insulin - resistant subjects ; nob - is , nonobese insulin - sensitive subjects ( the reference ) ; ob - ir , obese insulin - resistant subjects ; ob - is , obese insulin - sensitive subjects . also , mortality for cvd ( 15% , p = 0.015 ) and cancer ( 12% , p = 0.04 ) ( table 2 ) was higher in the obese insulin - resistant subjects but not in the obese insulin - sensitive subjects ( cvd related : 5% , p = 0.66 , and cancer related : 7% , p = 0.95 ) and nonobese insulin - resistant subjects ( cvd related : 12% , p = 0.29 , and cancer related : 9% , p = 0.64 ) when compared with nonobese insulin - sensitive subjects ( cvd related : 8% and cancer related : 7% ) . because the prevalence of cigarette smoking and baseline plasma ldl cholesterol were different among the groups , we performed the analysis adjusting also for these two factors . all - cause mortality remained higher in the obese insulin - resistant subjects ( hr 1.66 [ 95% ci 1.122.46 ] , p = 0.011 ) but not in obese insulin - sensitive subjects ( 0.79 [ 0.193.28 ] , p = 0.75 ) and in nonobese insulin - resistant subjects ( 1.22 [ 0.881.70 ] , p = 0.23 ) when compared with nonobese and insulin - sensitive subjects . the analysis was also repeated after the exclusion of diabetic patients . when compared with nonobese insulin - sensitive ( reference group ) , all - cause mortality tended to be higher in obese insulin - resistant subjects ( hr 1.29 [ 95% ci 0.961.73 ] , p = 0.087 ) but was again not different in obese insulin - sensitive subjects ( 1.01 [ 0.472.17 ] , p = 0.97 ) and nonobese insulin - resistant subjects ( 1.00 [ 0.801.25 ] , p = 0.98 ) . similarly , mortality for cvd and cancer tended to be higher in obese insulin - resistant subjects ( cvd : hr 1.40 [ 95% ci 0.942.11 ] , p = 0.071 ; cancer : 1.46 [ 0.942.27 ] , p = 0.097 ) but was not different in obese insulin - sensitive subjects ( cvd : 0.76 [ 0.1973.11 ] , p = 0.71 ; cancer : 1.05 [ 0.333.56 ] , p = 0.94 ) and nonobese insulin - resistant subjects ( cvd : [ 1.09 [ 0.771.54 ] , p = 0.65 ; cancer : 1.01 [ 0.932.27 ] , p = 0.95 ) than nonobese insulin - sensitive subjects . finally , instead of the preselected homa - ir of 2.5 , we repeated the analysis using cutoff values ( top tertile and top quartile ) for homa - ir obtained from the present study . even in this case , the results did not change ( see the online appendix , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0665/dc1 ) . the 15-year follow - up of the cremona study demonstrates that obese insulin - sensitive individuals , also known as mho individuals 1 ) have a prevalence of 11% in the obese population and 2% in the entire population ; 2 ) have less features of the metabolic syndrome , when compared with obese insulin - resistant individuals ; and 3 ) do not have increased all - cause , cvd , and cancer mortality , when compared with nonobese insulin - sensitive ( reference group ) subjects . the prevalence of the obese insulin - sensitive phenotype ( 11% ) in our obese cohort was lower than reported by iacobellis et al . ( 3 ) ( 27.5% ) in a cohort of 681 obese individuals living in rome and the surrounding areas . the discrepancy may be related to the different regional habits of the italian cohorts but most likely to the different definition of mho . iacobellis et al . based their definition mainly on the metabolic syndrome ; meanwhile , our definition was centered on homa - ir , a surrogate index of insulin resistance , in order to compare our results with those recently published by kuk and ardern ( 7 ) , who analyzed the nhanes iii survey in u.s . our finding is in line with this report ( 7 ) ; therefore , we think that the frequency of this phenotype is lower than previously thought . the present study has also clearly shown that the obese insulin - sensitive phenotype carries less features of the metabolic syndrome . these subjects were characterized by lower waist circumference , blood pressure , circulating triglycerides , transaminases , gt ( as a surrogate markers of fatty liver ) , and fibrinogen ( as a surrogate marker of low - grade inflammation ) , when compared with the obese insulin - resistant subjects , in spite of similar bmis . not surprisingly , they had a lower prevalence of the metabolic syndrome ( 7% in comparison to the observed 41% in the obese insulin - resistant subjects ) and of diabetes ( 0 vs. 28% of the obese insulin - resistant subjects ) . we think , therefore , that the deleterious metabolic features associated with obesity are largely related to the presence of insulin resistance rather than obesity , per se . the present study has also shown that all - cause mortality is significantly higher in obese insulin - resistant subjects but not obese insulin - sensitive subjects , when compared with nonobese insulin - sensitive individuals ( considered as reference group ) . these findings were confirmed when the analysis was adjusted for ldl cholesterol and cigarette smoking ( risk factors not related to metabolic syndrome ) . population that suggest increased all - cause mortality in mho subjects ( defined according to the same bmi and homa - ir criteria we use here ) ( 7 ) . the potential explanations for this discrepancy are number of events , reference hr , and different ethnicity . even though our population was smaller , the number of events was higher ( 495 vs. 292 , or 25 vs. 5% ) . this was likely because of the longer observational period ( 15 vs. 8.7 years ) . it is important to point out that a 10- to 15-year follow - up may be the least to see the effects of metabolic risk factors on mortality ( 8,12 ) . regarding reference hrs , we studied nonobese insulin - sensitive individuals , which also includes overweight individuals with bmis ranging between 25 and 29.9 kg / m , whereas kuk et al . ( 7 ) studied normal - weight insulin - sensitive subjects with bmi < 25 kg / m . ( 8) in a scandinavian population in which overweight and obese individuals without the metabolic syndrome showed a higher mortality when compared with normal - weight and insulin - sensitive individuals . we believe that the reason for this discrepancy could be attributed to sex differences because our study included both male and female subjects , whereas only male subjects were included in the scandinavian study . this is worth mentioning because male sex was a significant risk factor for all - cause mortality in our study . we used all - cause mortality as primary outcome ( because this variable is less affected by errors in reporting ) , whereas cvd and cancer mortality were considered secondary outcomes . mortality for cvd and cancer , as for all - cause mortality , were also higher in obese insulin - resistant individuals but not in the mho subjects . the following are major strengths of the present study : 1 ) this was a population - based study including both male and female subjects , 2 ) there was careful and homogeneous acquisition of the anthropometric parameter of interest , 3 ) there was a robust end point ( all - cause mortality ) whose ascertainment was based on the regional health registry , and 4 ) there was a long follow - up period ( 15 years ) . the following are the limitations of this study : 1 ) there was a small sample size of the group of obese insulin - sensitive subjects ( n = 43 ) , and their low prevalence in the cohort ( 2% ) could represent a problem because of the consequent small number of events even if it was similar to previously reported data ( 8) ; 2 ) there was a lack of collection of intermediate data points about the parameters of interest during the 15-year observation period ; 3 ) the glucose clamp technique is the gold standard for the assessment of insulin sensitivity and homa is inferior ; nevertheless , it was suggested that homa appeared to be specifically suited to large - scale epidemiologic studies in which only fasting glucose and insulin concentrations were available ( 13 ) ; and 4 ) there was a lack of collection of the dietary habits and habitual physical activity , known to have a well recognized impact on insulin sensitivity . it was reported that a lower amount of visceral fat content may contribute to the favorable metabolic profile ( 1,6 ) . fitting this view , the waist circumference was lower in mho subjects than in the obese insulin - resistant subjects ; on the other hand , it was higher in comparison to the nonobese and insulin - sensitive group ( table 1 ) , in spite of a similar all - cause mortality . we speculate that visceral fat and insulin resistance may , in combination , explain the difference and the trends observed between groups in our study , and , in addition , an undetectable effect of ectopic fat accumulation in the skeletal muscle ( 14 ) and the liver ( 15 ) should be considered . in particular , the potential , but yet - to - be - demonstrated , role of the liver ( see the profile of surrogate markers of fatty liver ) in mediating the increased cvd mortality may be hypothesized based on the proinflammatory and proatherosclerotic profile of individuals with nonalcoholic fatty liver disease but also based on some initial epidemiological data ( 16 ) . all - cause mortality in obese insulin - resistant subjects but not in mho subjects is higher when compared with nonobese insulin - sensitive subjects . the effect of obesity on the increasing risk is strongly related with insulin resistance , and we therefore agree with bonora et al . ( 18 ) that it is important to not limit our risk evaluation to the identification of obesity alone but to put more effort into identifying those at higher risk , insulin - resistant obese individuals . the prevalence of the obese insulin - sensitive phenotype ( 11% ) in our obese cohort was lower than reported by iacobellis et al . ( 3 ) ( 27.5% ) in a cohort of 681 obese individuals living in rome and the surrounding areas . the discrepancy may be related to the different regional habits of the italian cohorts but most likely to the different definition of mho . iacobellis et al . based their definition mainly on the metabolic syndrome ; meanwhile , our definition was centered on homa - ir , a surrogate index of insulin resistance , in order to compare our results with those recently published by kuk and ardern ( 7 ) , who analyzed the nhanes iii survey in u.s . our finding is in line with this report ( 7 ) ; therefore , we think that the frequency of this phenotype is lower than previously thought . the present study has also clearly shown that the obese insulin - sensitive phenotype carries less features of the metabolic syndrome . these subjects were characterized by lower waist circumference , blood pressure , circulating triglycerides , transaminases , gt ( as a surrogate markers of fatty liver ) , and fibrinogen ( as a surrogate marker of low - grade inflammation ) , when compared with the obese insulin - resistant subjects , in spite of similar bmis . not surprisingly , they had a lower prevalence of the metabolic syndrome ( 7% in comparison to the observed 41% in the obese insulin - resistant subjects ) and of diabetes ( 0 vs. 28% of the obese insulin - resistant subjects ) . we think , therefore , that the deleterious metabolic features associated with obesity are largely related to the presence of insulin resistance rather than obesity , per se . the present study has also shown that all - cause mortality is significantly higher in obese insulin - resistant subjects but not obese insulin - sensitive subjects , when compared with nonobese insulin - sensitive individuals ( considered as reference group ) . these findings were confirmed when the analysis was adjusted for ldl cholesterol and cigarette smoking ( risk factors not related to metabolic syndrome ) . population that suggest increased all - cause mortality in mho subjects ( defined according to the same bmi and homa - ir criteria we use here ) ( 7 ) . the potential explanations for this discrepancy are number of events , reference hr , and different ethnicity . even though our population was smaller , the number of events was higher ( 495 vs. 292 , or 25 vs. 5% ) . this was likely because of the longer observational period ( 15 vs. 8.7 years ) . it is important to point out that a 10- to 15-year follow - up may be the least to see the effects of metabolic risk factors on mortality ( 8,12 ) . regarding reference hrs , we studied nonobese insulin - sensitive individuals , which also includes overweight individuals with bmis ranging between 25 and 29.9 kg / m , whereas kuk et al . ( 8) in a scandinavian population in which overweight and obese individuals without the metabolic syndrome showed a higher mortality when compared with normal - weight and insulin - sensitive individuals . we believe that the reason for this discrepancy could be attributed to sex differences because our study included both male and female subjects , whereas only male subjects were included in the scandinavian study . this is worth mentioning because male sex was a significant risk factor for all - cause mortality in our study . we used all - cause mortality as primary outcome ( because this variable is less affected by errors in reporting ) , whereas cvd and cancer mortality were considered secondary outcomes . mortality for cvd and cancer , as for all - cause mortality , were also higher in obese insulin - resistant individuals but not in the mho subjects . the following are major strengths of the present study : 1 ) this was a population - based study including both male and female subjects , 2 ) there was careful and homogeneous acquisition of the anthropometric parameter of interest , 3 ) there was a robust end point ( all - cause mortality ) whose ascertainment was based on the regional health registry , and 4 ) there was a long follow - up period ( 15 years ) . the following are the limitations of this study : 1 ) there was a small sample size of the group of obese insulin - sensitive subjects ( n = 43 ) , and their low prevalence in the cohort ( 2% ) could represent a problem because of the consequent small number of events even if it was similar to previously reported data ( 8) ; 2 ) there was a lack of collection of intermediate data points about the parameters of interest during the 15-year observation period ; 3 ) the glucose clamp technique is the gold standard for the assessment of insulin sensitivity and homa is inferior ; nevertheless , it was suggested that homa appeared to be specifically suited to large - scale epidemiologic studies in which only fasting glucose and insulin concentrations were available ( 13 ) ; and 4 ) there was a lack of collection of the dietary habits and habitual physical activity , known to have a well recognized impact on insulin sensitivity . it was reported that a lower amount of visceral fat content may contribute to the favorable metabolic profile ( 1,6 ) . fitting this view , the waist circumference was lower in mho subjects than in the obese insulin - resistant subjects ; on the other hand , it was higher in comparison to the nonobese and insulin - sensitive group ( table 1 ) , in spite of a similar all - cause mortality . we speculate that visceral fat and insulin resistance may , in combination , explain the difference and the trends observed between groups in our study , and , in addition , an undetectable effect of ectopic fat accumulation in the skeletal muscle ( 14 ) and the liver ( 15 ) should be considered . in particular , the potential , but yet - to - be - demonstrated , role of the liver ( see the profile of surrogate markers of fatty liver ) in mediating the increased cvd mortality may be hypothesized based on the proinflammatory and proatherosclerotic profile of individuals with nonalcoholic fatty liver disease but also based on some initial epidemiological data ( 16 ) . all - cause mortality in obese insulin - resistant subjects but not in mho subjects is higher when compared with nonobese insulin - sensitive subjects . the effect of obesity on the increasing risk is strongly related with insulin resistance , and we therefore agree with bonora et al . ( 18 ) that it is important to not limit our risk evaluation to the identification of obesity alone but to put more effort into identifying those at higher risk , insulin - resistant obese individuals .

objectivesome obese individuals have normal insulin sensitivity . it is controversial whether this phenotype is associated with increased all - cause mortality risk.research design and methodsfifteen - year all - cause mortality data were obtained through the regional health registry for 2,011 of 2,074 caucasian middle - aged individuals of the cremona study , a population study on the prevalence of diabetes in italy . individuals were divided in four categories according to bmi ( nonobese : < 30 kg / m2 ; obese : 30 kg / m2 ) and estimated insulin resistance ( insulin sensitive : homeostasis model assessment of insulin resistance < 2.5 ; insulin resistant 2.5).resultsobese insulin - sensitive subjects represented 11% ( 95% ci 8.114.5 ) of the obese population . this phenotype had similar bmi but lower waist circumference , blood pressure , fasting glucose , triglycerides , and fibrinogen and higher hdl cholesterol than obese insulin - resistant subjects . in the 15-year follow - up , 495 deaths ( cardiovascular disease [ cvd ] : n = 221 ; cancer : n = 180 ) occurred . all - cause mortality adjusted for age and sex was higher in the obese insulin - resistant subjects ( hazard ratio 1.40 [ 95% ci 1.081.81 ] , p = 0.01 ) but not in the obese insulin - sensitive subjects ( 0.99 [ 0.462.11 ] , p = 0.97 ) when compared with nonobese insulin - sensitive subjects . also , mortality for cvd and cancer was higher in the obese insulin - resistant subjects but not in the obese insulin - sensitive subjects when compared with nonobese insulin - sensitive subjects.conclusionsin contrast to obese insulin - resistant subjects , metabolically healthy obese individuals are less common than previously thought and do not show increased all - cause , cancer , and cvd mortality risks in a 15-year follow - up study .

RESEARCH DESIGN AND METHODS Study cohort and follow-up Definition of study groups Definition of diabetes, impaired glucose tolerance, and metabolic syndrome Analytical determinations Calculations Statistical analysis RESULTS Prevalence of the obese insulin-sensitive phenotype Anthropometric and metabolic features of obese insulin-sensitive individuals Mortality in the cohort CONCLUSIONS Major findings and comparison with the literature Strengths and limitations Pathogenic remarks

the cremona study is a population survey carried out in 19901991 in the health district of cremona ( lombardia , italy ) to determine the prevalence of diabetes according to the oral glucose tolerance test ( ogtt ) and world health organization criteria ( 8,9 ) . vital status and time of death were acquired from the regional health registry ( updated to 31 december 2005 ) , and causes of death were classified using the icd-9 ( death codes for cvd are from 401 to 448 and cancer from 140.0 to 208.9 ) . study subjects were divided in four categories based on bmi ( nonobese : < 30 kg / m ; obese : 30 kg / m ) and estimated insulin resistance ( insulin sensitive : homeostasis model assessment of insulin resistance [ homa - ir ] < 2.5 ; insulin resistant 2.5 ) . therefore , the four categories were 1 ) the nonobese subjects with normal insulin sensitivity , 2 ) the obese but insulin - sensitive subjects , 3 ) the nonobese but insulin - resistant subjects , and 4 ) the obese and insulin - resistant subjects . * p < 0.05 vs. obese insulin - resistant subjects ; p < 0.05 vs. nonobese insulin - resistant subjects ; p < 0.05 vs. nonobese insulin - sensitive subjects ; for categorical variables p < 0.05 vs. nonobese and obese insulin - sensitive subjects . the associations of each investigated risk factor with all - cause , cvd , and cancer mortality were estimated by the cox proportional hazard model , with adjustments for age and sex . the cremona study is a population survey carried out in 19901991 in the health district of cremona ( lombardia , italy ) to determine the prevalence of diabetes according to the oral glucose tolerance test ( ogtt ) and world health organization criteria ( 8,9 ) . study subjects were divided in four categories based on bmi ( nonobese : < 30 kg / m ; obese : 30 kg / m ) and estimated insulin resistance ( insulin sensitive : homeostasis model assessment of insulin resistance [ homa - ir ] < 2.5 ; insulin resistant 2.5 ) . therefore , the four categories were 1 ) the nonobese subjects with normal insulin sensitivity , 2 ) the obese but insulin - sensitive subjects , 3 ) the nonobese but insulin - resistant subjects , and 4 ) the obese and insulin - resistant subjects . the associations of each investigated risk factor with all - cause , cvd , and cancer mortality were estimated by the cox proportional hazard model , with adjustments for age and sex . there were a total of 43 obese insulin - sensitive individuals , representing 11.0% ( 95% ci 8.114.5 ) of the obese population and 2.1% ( 1.62.9 ) of the entire population . systolic and diastolic blood pressure , heart rate , plasma glucose , insulin , total cholesterol , hdl cholesterol , triglycerides , transaminases , -glutamyltransferase ( gt ) , alkaline phosphatase ( alp ) , and fibrinogen did not differ between the two insulin - sensitive groups . individuals in the insulin - sensitive groups were younger , had lower heart rates , had higher plasma hdl cholesterol , and had lower fibrinogen and triglycerides , as well as had a lower prevalence of diabetes and metabolic syndrome than insulin - resistant groups . waist circumference was higher in obese insulin - sensitive than nonobese insulin - resistant subjects but were lower than in obese insulin - resistant subjects . systolic and diastolic blood pressure , plasma transaminases , gt , and alp were higher in the two groups of insulin - resistant subjects than in nonobese insulin - sensitive subjects . during the 15-year observation period , age and sex were associated with higher all - cause mortality ( age : hr 1.11 [ 95% ci 1.101.12 ] , p < 0.0001 ; female sex : 0.42 [ 0.350.50 ] , p < 0.0001 ) , mortality for cvd ( age : 1.15 [ 1.131.17 ] , p < 0.0001 ; female sex : 0.40 [ 0.310.53 ] , p < 0.0001 ) , and mortality for cancer ( age : 1.07 [ 1.061.09 ] , p < 0.0001 ; female sex : 0.39 [ 0.290.52 ] , p < 0.0001 ) . all - cause mortality was higher in the obese insulin - resistant subjects ( 31% ) in comparison with the reference group of nonobese insulin - sensitive subjects ( 20% ) ( age- and sex - adjusted hr 1.4 , p = 0.01 ) ( table 2 ) ( fig . 1 ) but not in the obese insulin - sensitive subjects ( 12% ) ( 0.99 , p = 0.97 ) ( table 2 ) ( fig . 1 ) and in the nonobese insulin - resistant subjects ( 26% ) ( 1.11 , p = 0.35 ) ( table 2 ) ( fig . subjects were divided according to bmi ( nonobese : < 30 kg / m ; obese : 30 kg / m ) and estimated insulin resistance ( insulin sensitive : homa - ir < 2.5 ; insulin resistant : 2.5 ) . nob - ir , nonobese insulin - resistant subjects ; nob - is , nonobese insulin - sensitive subjects ( the reference ) ; ob - ir , obese insulin - resistant subjects ; ob - is , obese insulin - sensitive subjects . also , mortality for cvd ( 15% , p = 0.015 ) and cancer ( 12% , p = 0.04 ) ( table 2 ) was higher in the obese insulin - resistant subjects but not in the obese insulin - sensitive subjects ( cvd related : 5% , p = 0.66 , and cancer related : 7% , p = 0.95 ) and nonobese insulin - resistant subjects ( cvd related : 12% , p = 0.29 , and cancer related : 9% , p = 0.64 ) when compared with nonobese insulin - sensitive subjects ( cvd related : 8% and cancer related : 7% ) . because the prevalence of cigarette smoking and baseline plasma ldl cholesterol were different among the groups all - cause mortality remained higher in the obese insulin - resistant subjects ( hr 1.66 [ 95% ci 1.122.46 ] , p = 0.011 ) but not in obese insulin - sensitive subjects ( 0.79 [ 0.193.28 ] , p = 0.75 ) and in nonobese insulin - resistant subjects ( 1.22 [ 0.881.70 ] , p = 0.23 ) when compared with nonobese and insulin - sensitive subjects . when compared with nonobese insulin - sensitive ( reference group ) , all - cause mortality tended to be higher in obese insulin - resistant subjects ( hr 1.29 [ 95% ci 0.961.73 ] , p = 0.087 ) but was again not different in obese insulin - sensitive subjects ( 1.01 [ 0.472.17 ] , p = 0.97 ) and nonobese insulin - resistant subjects ( 1.00 [ 0.801.25 ] , p = 0.98 ) . similarly , mortality for cvd and cancer tended to be higher in obese insulin - resistant subjects ( cvd : hr 1.40 [ 95% ci 0.942.11 ] , p = 0.071 ; cancer : 1.46 [ 0.942.27 ] , p = 0.097 ) but was not different in obese insulin - sensitive subjects ( cvd : 0.76 [ 0.1973.11 ] , p = 0.71 ; cancer : 1.05 [ 0.333.56 ] , p = 0.94 ) and nonobese insulin - resistant subjects ( cvd : [ 1.09 [ 0.771.54 ] , p = 0.65 ; cancer : 1.01 [ 0.932.27 ] , p = 0.95 ) than nonobese insulin - sensitive subjects . there were a total of 43 obese insulin - sensitive individuals , representing 11.0% ( 95% ci 8.114.5 ) of the obese population and 2.1% ( 1.62.9 ) of the entire population . systolic and diastolic blood pressure , heart rate , plasma glucose , insulin , total cholesterol , hdl cholesterol , triglycerides , transaminases , -glutamyltransferase ( gt ) , alkaline phosphatase ( alp ) , and fibrinogen did not differ between the two insulin - sensitive groups . individuals in the insulin - sensitive groups were younger , had lower heart rates , had higher plasma hdl cholesterol , and had lower fibrinogen and triglycerides , as well as had a lower prevalence of diabetes and metabolic syndrome than insulin - resistant groups . waist circumference was higher in obese insulin - sensitive than nonobese insulin - resistant subjects but were lower than in obese insulin - resistant subjects . systolic and diastolic blood pressure , plasma transaminases , gt , and alp were higher in the two groups of insulin - resistant subjects than in nonobese insulin - sensitive subjects . age and sex were associated with higher all - cause mortality ( age : hr 1.11 [ 95% ci 1.101.12 ] , p < 0.0001 ; female sex : 0.42 [ 0.350.50 ] , p < 0.0001 ) , mortality for cvd ( age : 1.15 [ 1.131.17 ] , p < 0.0001 ; female sex : 0.40 [ 0.310.53 ] , p < 0.0001 ) , and mortality for cancer ( age : 1.07 [ 1.061.09 ] , p < 0.0001 ; female sex : 0.39 [ 0.290.52 ] , p < 0.0001 ) . all - cause mortality was higher in the obese insulin - resistant subjects ( 31% ) in comparison with the reference group of nonobese insulin - sensitive subjects ( 20% ) ( age- and sex - adjusted hr 1.4 , p = 0.01 ) ( table 2 ) ( fig . 1 ) but not in the obese insulin - sensitive subjects ( 12% ) ( 0.99 , p = 0.97 ) ( table 2 ) ( fig . 1 ) and in the nonobese insulin - resistant subjects ( 26% ) ( 1.11 , p = 0.35 ) ( table 2 ) ( fig . subjects were divided according to bmi ( nonobese : < 30 kg / m ; obese : 30 kg / m ) and estimated insulin resistance ( insulin sensitive : homa - ir < 2.5 ; insulin resistant : 2.5 ) . nob - ir , nonobese insulin - resistant subjects ; nob - is , nonobese insulin - sensitive subjects ( the reference ) ; ob - ir , obese insulin - resistant subjects ; ob - is , obese insulin - sensitive subjects . also , mortality for cvd ( 15% , p = 0.015 ) and cancer ( 12% , p = 0.04 ) ( table 2 ) was higher in the obese insulin - resistant subjects but not in the obese insulin - sensitive subjects ( cvd related : 5% , p = 0.66 , and cancer related : 7% , p = 0.95 ) and nonobese insulin - resistant subjects ( cvd related : 12% , p = 0.29 , and cancer related : 9% , p = 0.64 ) when compared with nonobese insulin - sensitive subjects ( cvd related : 8% and cancer related : 7% ) . all - cause mortality remained higher in the obese insulin - resistant subjects ( hr 1.66 [ 95% ci 1.122.46 ] , p = 0.011 ) but not in obese insulin - sensitive subjects ( 0.79 [ 0.193.28 ] , p = 0.75 ) and in nonobese insulin - resistant subjects ( 1.22 [ 0.881.70 ] , p = 0.23 ) when compared with nonobese and insulin - sensitive subjects . when compared with nonobese insulin - sensitive ( reference group ) , all - cause mortality tended to be higher in obese insulin - resistant subjects ( hr 1.29 [ 95% ci 0.961.73 ] , p = 0.087 ) but was again not different in obese insulin - sensitive subjects ( 1.01 [ 0.472.17 ] , p = 0.97 ) and nonobese insulin - resistant subjects ( 1.00 [ 0.801.25 ] , p = 0.98 ) . similarly , mortality for cvd and cancer tended to be higher in obese insulin - resistant subjects ( cvd : hr 1.40 [ 95% ci 0.942.11 ] , p = 0.071 ; cancer : 1.46 [ 0.942.27 ] , p = 0.097 ) but was not different in obese insulin - sensitive subjects ( cvd : 0.76 [ 0.1973.11 ] , p = 0.71 ; cancer : 1.05 [ 0.333.56 ] , p = 0.94 ) and nonobese insulin - resistant subjects ( cvd : [ 1.09 [ 0.771.54 ] , p = 0.65 ; cancer : 1.01 [ 0.932.27 ] , p = 0.95 ) than nonobese insulin - sensitive subjects . the 15-year follow - up of the cremona study demonstrates that obese insulin - sensitive individuals , also known as mho individuals 1 ) have a prevalence of 11% in the obese population and 2% in the entire population ; 2 ) have less features of the metabolic syndrome , when compared with obese insulin - resistant individuals ; and 3 ) do not have increased all - cause , cvd , and cancer mortality , when compared with nonobese insulin - sensitive ( reference group ) subjects . the prevalence of the obese insulin - sensitive phenotype ( 11% ) in our obese cohort was lower than reported by iacobellis et al . these subjects were characterized by lower waist circumference , blood pressure , circulating triglycerides , transaminases , gt ( as a surrogate markers of fatty liver ) , and fibrinogen ( as a surrogate marker of low - grade inflammation ) , when compared with the obese insulin - resistant subjects , in spite of similar bmis . not surprisingly , they had a lower prevalence of the metabolic syndrome ( 7% in comparison to the observed 41% in the obese insulin - resistant subjects ) and of diabetes ( 0 vs. 28% of the obese insulin - resistant subjects ) . the present study has also shown that all - cause mortality is significantly higher in obese insulin - resistant subjects but not obese insulin - sensitive subjects , when compared with nonobese insulin - sensitive individuals ( considered as reference group ) . mortality for cvd and cancer , as for all - cause mortality , were also higher in obese insulin - resistant individuals but not in the mho subjects . the following are major strengths of the present study : 1 ) this was a population - based study including both male and female subjects , 2 ) there was careful and homogeneous acquisition of the anthropometric parameter of interest , 3 ) there was a robust end point ( all - cause mortality ) whose ascertainment was based on the regional health registry , and 4 ) there was a long follow - up period ( 15 years ) . the following are the limitations of this study : 1 ) there was a small sample size of the group of obese insulin - sensitive subjects ( n = 43 ) , and their low prevalence in the cohort ( 2% ) could represent a problem because of the consequent small number of events even if it was similar to previously reported data ( 8) ; 2 ) there was a lack of collection of intermediate data points about the parameters of interest during the 15-year observation period ; 3 ) the glucose clamp technique is the gold standard for the assessment of insulin sensitivity and homa is inferior ; nevertheless , it was suggested that homa appeared to be specifically suited to large - scale epidemiologic studies in which only fasting glucose and insulin concentrations were available ( 13 ) ; and 4 ) there was a lack of collection of the dietary habits and habitual physical activity , known to have a well recognized impact on insulin sensitivity . fitting this view , the waist circumference was lower in mho subjects than in the obese insulin - resistant subjects ; on the other hand , it was higher in comparison to the nonobese and insulin - sensitive group ( table 1 ) , in spite of a similar all - cause mortality . all - cause mortality in obese insulin - resistant subjects but not in mho subjects is higher when compared with nonobese insulin - sensitive subjects . the prevalence of the obese insulin - sensitive phenotype ( 11% ) in our obese cohort was lower than reported by iacobellis et al . these subjects were characterized by lower waist circumference , blood pressure , circulating triglycerides , transaminases , gt ( as a surrogate markers of fatty liver ) , and fibrinogen ( as a surrogate marker of low - grade inflammation ) , when compared with the obese insulin - resistant subjects , in spite of similar bmis . not surprisingly , they had a lower prevalence of the metabolic syndrome ( 7% in comparison to the observed 41% in the obese insulin - resistant subjects ) and of diabetes ( 0 vs. 28% of the obese insulin - resistant subjects ) . the present study has also shown that all - cause mortality is significantly higher in obese insulin - resistant subjects but not obese insulin - sensitive subjects , when compared with nonobese insulin - sensitive individuals ( considered as reference group ) . population that suggest increased all - cause mortality in mho subjects ( defined according to the same bmi and homa - ir criteria we use here ) ( 7 ) . mortality for cvd and cancer , as for all - cause mortality , were also higher in obese insulin - resistant individuals but not in the mho subjects . the following are major strengths of the present study : 1 ) this was a population - based study including both male and female subjects , 2 ) there was careful and homogeneous acquisition of the anthropometric parameter of interest , 3 ) there was a robust end point ( all - cause mortality ) whose ascertainment was based on the regional health registry , and 4 ) there was a long follow - up period ( 15 years ) . the following are the limitations of this study : 1 ) there was a small sample size of the group of obese insulin - sensitive subjects ( n = 43 ) , and their low prevalence in the cohort ( 2% ) could represent a problem because of the consequent small number of events even if it was similar to previously reported data ( 8) ; 2 ) there was a lack of collection of intermediate data points about the parameters of interest during the 15-year observation period ; 3 ) the glucose clamp technique is the gold standard for the assessment of insulin sensitivity and homa is inferior ; nevertheless , it was suggested that homa appeared to be specifically suited to large - scale epidemiologic studies in which only fasting glucose and insulin concentrations were available ( 13 ) ; and 4 ) there was a lack of collection of the dietary habits and habitual physical activity , known to have a well recognized impact on insulin sensitivity . fitting this view , the waist circumference was lower in mho subjects than in the obese insulin - resistant subjects ; on the other hand , it was higher in comparison to the nonobese and insulin - sensitive group ( table 1 ) , in spite of a similar all - cause mortality . all - cause mortality in obese insulin - resistant subjects but not in mho subjects is higher when compared with nonobese insulin - sensitive subjects .

breast cancer is the most commonly diagnosed cancer in women and has the highest incidence of cancer in women . in 2012 the number of patients who were diagnosed with breast cancer was about 1.7 million worldwide . according to the american cancer society , about 246,660 women and 2,600 men will be diagnosed with invasive breast cancer in 2016 in the united states . however , if breast cancer is detected early enough , the five - year survival rate is over 90% [ 1 , 2 ] . current techniques for breast imaging are x - ray mammography , ultrasound , mri , and positron emission tomography ( pet ) . x - ray mammography uses low - energy x - rays to create images of the breast . the number of women in their 40s experiencing harm from starting regular screening mammography is larger than for older women . the results of mammography revealed 1,212 of 10,000 women screened turn out to be false positives in their 40s . harmful effects of mammography include overtreatment , unnecessary , and sometimes invasive follow - up testing and psychological harm associated with false positive test results . further , x - ray mammography needs compression of the breast to lie as flat as possible during the examination ( figure 1 ) , which causes patient pain . it is difficult to distinguish tumors in dense breast mammogram images [ 1 , 6 ] , because both dense tissue and cancer appear white in the mammogram image , unlike fatty tissue that appears black . in figure 2 , a small tumor is conspicuous in a fatty breast ; on the other hand , dense breast tissue in a heterogeneously dense breast obscures a 4 cm tumor . a transducer placed on the skin sends ultrasound pulses into the body and detects the echoes from inside the body , which are used to make ultrasound images . additionally , ultrasound is mostly used as a secondary technique , after a mammogram result shows a suspected mass . mri uses radio waves and strong magnetic fields to make images of the inside body . this method utilizes the amount of absorption energy of different type of tissues . when used with soft tissue like breast , contrast liquid moreover , mri is used for screening evaluation before surgical approach , for example , breast - conserving lumpectomy to mastectomy . pet is an imaging technique that observes how an injection mixture of radioactive materials with glucose or glycoprotein is used and synthesized in the human body . pet makes an image by detecting these positrons . unlike x - ray , ct , and mri , pet can diagnose cancer in the very early stages , because pet detects the movement of molecules in early disease cells . however , it has low resolution . such limitations of existing breast diagnostic imaging methods motivate the research to develop alternative imaging methods . microwave imaging has been researched as an alternative to x - ray mammography , because of its harmlessness to humans [ 3437 ] . microwave imaging uses the scattering wave or reflected wave that arises from the contrast in dielectric properties between normal and malignant breast tissues . in this paper microwave breast imaging could be possible due to the difference in dielectric properties between malignant tissue and normal breast tissue . in addition , we introduce our studies of a newly proposed cmos chip - based time - domain microwave imaging system . several groups have studied breast dielectric properties , and research results have already been published [ 1113 , 38 , 39 ] . the dielectric properties of malignant and normal breast tissues show large differences ; and these have been demonstrated by joines et al . 1980 , 1994 ; chaudhary et al . 1984 ; and surowiec et al . 1988 . figure 3 shows the dielectric properties of malignant and normal breast tissues from earlier studies . according to chaudhary et al . 1984 , malignant breast tissues at 1~2 ghz are 3~5 times greater than normal breast tissues . 1994 showed difference between malignant and normal breast tissues at 900 mhz of 200~500% , judging from 12 measurements of each normal and malignant breast tissue . the dielectric properties of malignant breast tissues are approximately ten times as large as normal breast tissues ( figure 3 ) . this is because malignant tissues contain much water and are more active than normal [ 40 , 41 ] . later , a collaborative research team from the university of wisconsin and the university of calgary in 2007 reported a large - scale study of the ultra - wideband microwave dielectric properties of normal breast tissue obtained from reduction surgeries and a large - scale study of the ultra - wideband microwave dielectric properties of normal , benign , and malignant breast tissues [ 10 , 15 ] . the total number of patients was 93 , and the total number of measurements was 488 . in the latter study , the total number of patients involved in the study was 196 , and the total number of samples was 319 . the study measured dielectric constant data in the 0.5~20 ghz frequency range and found that while malignant tissues were ten times larger than adipose - dominated tissues , the difference between malignant and normal glandular or fibroconnective tissues was no more than about 10% . dielectric properties of normal tissues span a very large range , from 030% adipose tissues to 85100% adipose tissues . figure 4 depicts the dielectric properties of 85100% adipose tissues as solid lines , 030% as dash - dot lines and 3184% adipose tissues as dashed lines . figure 5(a ) shows the dielectric properties of malignant tissue were similar to the results of previous works , unlike the dielectric properties of normal breast tissues in figure 5(b ) . whereas initial experiments found the differences of dielectric properties between normal breast tissue and malignant tissue to be close to 10 times , large - scale experiments found that the differences were much smaller . nevertheless , microwave imaging is still used to distinguish breast cancer [ 4244 ] . besides , breast cancer classification research in microwave imaging technique has been studied in the national university of ireland [ 4552 ] . the research of university of wisconsin and university of calgary [ 10 , 15 ] was conducted at the ex vivo states . dartmouth college published the correlation of in vivo and ex vivo tissue dielectric - c properties which were measured during mastectomy procedure in the operating room ( in vivo ) and after resection in the same location immediately from six women . the research was carried out to validate the range of properties obtained from the restored images image . overall , the properties between ex vivo and in vivo are similar , but the properties obtained ex vivo decrease when compared the properties measured in vivo ( figure 6 ) . in addition , the research teams from university calgary and university wisconsin have been studying dielectric properties estimation using measurement system [ 5357 ] . imaging techniques using electromagnetic waves have been used in specific fields , such as the nondestructive test of structures and detection of hidden objects . recently , with the technological development of algorithms , computation techniques , and hardware , many studies are being conducted to apply electromagnetic wave imaging technology to human diagnosis [ 17 , 59 , 60 ] . microwave imaging is a promising new method for early - stage breast cancer detection . this is based on the contrast of electric parameters between the tumor and the normal breast tissue within the microwave spectrum . the method reconstructs the breast image from the received signals that are scattered and reflected within the breast . while the resolution becomes higher as the frequency increases , tissue loss increases . thus , it is difficult to obtain a clear image , so a limit of upper frequency of the band is needed for acceptable penetration into the tissue . since the penetration loss of healthy fat tissue is less than 4 db / cm with microwave signal which is centered at 6 ghz , it is possible to reach low power signal to an antenna on the other side of an object . the specific absorption rate ( sar ) is normally used when setting the safety standards for the maximum allowed exposure to the human body . standard c95.1 - 1999 states that , in the case of devices operating at 100 khz to 6 ghz , it should not exceed a maximum 1.6 w / kg for human tissue of the average 1 g . as a guide , compared to a mobile phone using the same frequency band , microwave breast cancer imaging method should be free from health hazardous exposure to the patient , because of suffering less exposure than from a mobile phone . from the technical point of view , microwave breast cancer imaging method may detect a small size tumor and also has the advantage of low cost . in addition , microwave imaging can obtain an image quickly compared to other imaging methods . there are two approaches to the microwave imaging method : microwave tomography and radar - based . the systems transmit the microwave signals into the breast and then measure scattering signals reflected from the tumor . meaney from dartmouth college in the united states are one of the representative research groups . they proposed an iterative reconstruction algorithm that obtains dielectric properties in a 2d lossy medium in 1995 . the goal of microwave tomography is to recover the profile using the inverse problem of the dielectric properties of the breast . however , the inverse problem takes much time , because the calculation process is complicated . also , a nonlinear inverse scattering problem must be solved , and iterative image reconstruction algorithms are usually required to obtain a solution . in general , these ill - posed inverse scattering approaches suffer from nonuniqueness and require regularization in order to achieve convergence to a meaningful solution [ 66 , 90 ] . the dartmouth group has developed a clinical prototype for active microwave imaging of the breast for the first time . they developed a 32-channel data acquisition system operating at frequency range 500 mhz to 3 ghz to obtain data from a clinical prototype exam . the clinical prototype illuminates the breast with 16 monopole antennas that operate in the 300 mhz to 1 ghz frequency range . the clinical exam was conducted with 5 women , and the total acquisition time was 1015 minutes per breast . the data were obtained from seven different array heights for seven different frequencies at each array position . figure 8 shows the woman being screened using the clinical prototype and figure 9 shows the resultant images . they have also developed a prototype of breast cancer detection system based on 3d fem method microwave tomography [ 19 , 20 ] . they presented the first clinical 3d microwave tomographic images of the breast using the prototype . the study achieved improvements of both hardware and software aspects and achieved data acquisition time of less than 2 minutes by development of special hardware . this is very fast compared to ten of hours or even days for a single 3d microwave tomographic image . they used the 3d microwave breast imaging system for monitoring treatment response during neoadjuvant chemotherapy [ 20 , 92 ] . tomographic images were obtained eight times from eight patients undergoing neoadjuvant chemotherapy during the course of treatment . the results of the study indicate that the conductivity in locally advanced breast cancer matched well with overall neoadjuvant therapy treatment response . in korea , electronics and telecommunications research institute ( etri ) started research of the breast cancer diagnostic technology of microwave tomography in 2007 . etri mt breast cancer diagnostic techniques have been studied using a microwave signal in the 500 mhz to 3 ghz range [ 21 , 78 , 93 ] . experimental etri mt system consists of cylindrical breast phantom and cylindrical tumor phantoms ( figure 13 ) . the five thin - wall plastic pipes displayed here are filled with the liquid of complex permittivity similar to a real tumor . figure 14(a ) shows reconstructed images of a breast without a tumor , and figures 14(b ) and 14(c ) present images of a single cylindrical tumor at the central and shifted positions . king 's college in london has been developing antenna for microwave tomography [ 84 , 94 ] , and they applied an adaptive multithreshold iterative shrinkage algorithm to the linear inversion at each iteration of the distorted born iterative method ( dbim ) . some groups are researching on reducing high computational demands due to a number of iteration and discretization of the object image for accuracy . xu et al . in university of manitoba proposed an iterative process that involves two algorithms : finite - difference time - domain ( fdtd ) and genetic algorithm ( ga ) . they developed a parallel algorithm for microwave tomography on cpu - based homogeneous multicore distributed memory machines and the cell be processor [ 68 , 69 , 76 ] . the research group in national university of ireland galway in ireland presented a novel parallelization strategy to accelerate microwave tomography . irea , national research council of italy , has developed diagnostic and therapeutic strategies based on the use of electromagnetic fields at microwave frequencies for some years . they have researched magnetic nanoparticles ( mnp ) enhanced microwave imaging which is able to reduce the rate of false positive and negative [ 22 , 9698 ] . figure 15 shows the experimental setup for the mnp enhanced mwi technique which irea developed . microwave ablation monitoring via microwave tomography also has been researched in irea . in addition , the research has been progressed in therapeutic aspects with microwave ablation at irea and university of wisconsin - madison [ 100102 ] . it uses the reflection that arises due to difference in the electrical dielectric properties of normal and malignant breast tissues , when microwaves transmit the internal breast with tumor . this approach , which was first developed as a military ground - penetrating application , is applied to the human body . it was designed in the late 1990s by hagness in wisconsin university , and benjamin in bristol university [ 9 , 24 , 103106 ] . researchers in national university of ireland galway , tianjin university in china , and hiroshima university are also working on this subject [ 107123 ] . recently , goethe university of frankfurt and university of bristol have been developed experimental phantom based on 3d printing technology that was one step close to the development of standard breast phantoms . a 3d image of the received signals through the breast is obtained by focusing algorithms . before applying the focusing algorithm , preprocessing may contain the extracting tumor response , compensation tissue losses , or radial spread . among the preprocessing , extracting tumor response must be conducted , because the received signal includes not only the tumor response , but also unwanted signals such as antenna coupling , directly received signals from the transmit antenna , and reflections from the skin . differential calibration techniques use a reference signal obtained from a scan of a healthy breast . however , it is hard to use when previous knowledge of the cancer - free breast does not exist . in the array rotation calibration method , two sets of measured data are received from a physically rotating antenna array , in which unwanted signals are almost the same . a calibrated signal is obtained by subtracting one of the two sets of the signals from the other . we have proposed a new calibration method called in - place calibration to extract a tumor response [ 125 , 126 ] . the received signal and the reference signal eliminates unwanted signals using dual receivers , rx1 and rx1 , located an equal distance from the transmitter tx in figure 16 . antenna coupling and skin reflection received by the two antennae occur at an identical time , unlike tumor responses that transpire in a different time due to a slight delay . thus , the calibrated signal of a tumor response can be obtained by subtracting one received signal from the other . in - place calibration in addition , measurement errors that occur due to environmental changes during calibration , such as a mechanical error during antenna array rotation or an accidental error due to the unexpected movement of the patient , are minimized . we assumed that the breast shape is hemispherical while a real breast is not exactly rotational symmetric . a possible solution for this is to provide different sizes of hemispherical measurement bowls such that a breast can tightly fit into the bowl during measurement . focusing algorithms that are used for image reconstruction in radar - based microwave imaging include the delay and sum ( das ) [ 36 , 127129 ] , microwave imaging via space - time ( mist ) [ 108 , 128 , 130 ] , robust weighted capon beamforming ( rwcb ) , multistatic adaptive microwave imaging ( mami ) [ 36 , 110 ] , and generalized likelihood ratio test ( glrt ) [ 109 , 132 ] . das is a simple and robust method . also , the computation time is short . the signals are shifted by time ti calculated from their respective location relative to the antennae , to restore the image . the focal point is assumed to be p = ( x , y , z ) . the time delay tmn is assumed as the time delay of the signal from transmitting at the mth antenna to receiving at the nth antenna . the time delay tmn at the focal point p can be expressed as(1)tmnp=1vxmx2+ymy2+zmz21/2+xnx2+yny2+znz21/2 . if the number of antennae is n , the number of signals used by the focusing algorithm is m = n ( n 1)/2 . the time delay applies the received signal rsi differently for each transmit antenna and receive antenna . the intensity of a reconstruction image is calculated by moving the focal point from one position to another within the breast . normally , uses 50 percent longer than the input pulse width , due to the antenna effects and dispersion . the intensity ip of the restored image at the given focal point can be expressed as(2)ipx , y , z=0i=1mwix , y , zrsittix , y , z2dt , where wi is the location - dependent weight calculated during preprocessing , rsi is the received signal , and ti is the time delay . microwave imaging via space - time ( mist ) beamforming uses finite impulse response ( fir ) filters to compensate for the frequency - dependent time delay , such as dispersion and fractional time delay . microwave breast cancer imaging uses a generalized likelihood ratio test ( glrt ) , which is a hypothesis testing problem for each voxel , with the null hypothesis representing the tumor - free case . however , most of those methods have suffered from performance degradation used with dense breast . thus , in the national university of ireland galway they used preprocessing filter to compensate the path - dependent attenuation and phase effects . in addition , they investigated the focal quality metrics to estimate average dielectric properties to enhance tumor detection . university of bristol also presented a time - domain wideband adaptive beamforming to reduce clutter . the approach uses an adapted equalization filter that adapts a calculated estimation of averaging dielectric properties of the breast . radar - based microwave imaging can be done through two approaches : frequency domain analysis and time - domain analysis . there have been several microwave systems reported in the literature , most of which are based on frequency domain analysis [ 24 , 26 , 28 , 36 , 135 ] . however , frequency domain analysis requires equipment , such as a costly vector network analyzer ( vna ) . the process takes a lot of time , because it measures s - parameters by sweeping frequencies ; and measurement time is critical in medical imaging : patient movement during a scan may generate artifacts . university of bristol team performed experimental and clinical study using uwb microwave radar imaging method for the first time . figure 18 shows a schematic of the multistatic radar - based microwave breast imaging system . a switch matrix connected to the feed of the antennas changes the pair of antennae during measurement of the signals . the signals are measured , in turn , by all possible pairs of antennae that activate the transmit port and receive port . the measured signals with frequency domain the image is reconstructed by a focusing algorithm using the time - domain signals . figure 19 shows the microwave radar - based clinical setup for breast cancer detection with a patient . clinical verification was performed with real breast cancer patients at the bristol oncology center . figure 20 shows that the contribution compared images obtained using x - ray mammography and the radar - based microwave system . however , 90 seconds is a long time to scan patients , and artifacts can occur due to patient movement . subsequently , the team developed a 60-element antenna array with 8 port vna ( figure 21(b ) ) . figure 22 shows the electromechanical switching interface for the 60-way tx / rx . the scan time with the 60-element array achieved 10 seconds . figure 23 shows the radar - based breast imaging system of micrima which was founded in 2006 to develop and commercialize the technology of microwave radar breast imaging at the university of bristol in the uk . the micrima 's maria system has undergone clinical trials at the several breast cancer imaging centers in the uk and got european regulatory approval in 2015 . university of calgary have developed a tissue sensing adaptive radar ( tsar ) prototype of radar - based breast imaging system ( figure 24 ) . the patient lies face down on the table to be scanned and immerses the breast in a tank filled with immersion liquid . their system was different from the system of bristol university , using a monostatic method and filtering the reflection from the skin . the results were positive , since several restored images exhibited responses which were similar to clinical results . they also studied 3d surfaces acquisition of the breast for radar - based microwave breast imaging and investigated the impact of the breast model complexity on microwave imaging signals . several research groups have recently reported time - domain measurement microwave breast imaging systems [ 29 , 31 , 37 , 139 ] . time - domain measurement systems have the advantage of being cost - effective and requiring less scan time . mcgill university in canada is a leading research group in time - domain measurement microwave imaging systems for the detection of breast cancer . figure 25 shows the experimental system that is a multistatic radar - based microwave system with 16-element antenna array for breast cancer detection that their research team developed . figure 26 shows that the time system transmits input pulse using a pulse generator and receives the signal from antennae using an oscilloscope in real time [ 29 , 140 ] . further , the mcgill university research team have performed an early clinical study of time - domain microwave radar for breast health monitoring [ 30 , 141 ] . they collected a total of 342 breast scans over an eight - month period with 13 healthy volunteers for monthly scans . the purpose of the study was to investigate the impact of unavoidable multiple factors of human in monthly monitoring . figure 27 shows a scanning patient on the table with the clinical system at mcgill university . in addition , they have developed wearable bra prototype using microwaves for breast health monitoring [ 142 , 143 ] . however , the system still depends on a high - precision pulse generator and very high - speed oscilloscope and may require complicated switching circuits . additionally , a time - domain system requires a very fast sampling clock such that even a very small jitter in the sampling clock might blur the resulting images . figure 28 shows the system implementation using cmos circuits as an alternative to high - speed pulse generator and oscilloscope proposed by hiroshima university in japan . figure 29 shows the measurement setup with cmos gaussian monocycle pulse ( gmp ) transmitter developed by hiroshima university . also , figure 30 shows a gmp equivalent time sampling circuit for confocal imaging developed by a hiroshima university team . in figure 31 shows the configuration of the time - domain microwave breast imaging system that we previously presented . it consists of 16 uwb transceivers around a 3d hemisphere , with a master controller at the center . we used cmos transceiver chips instead of a high - precision pulse generator and high - speed oscilloscope ; also , a master controller performs the role of switching matrix . we used the equivalent time sampling technique to achieve a sampling rate of 28.2 gs / s by 1.76 ghz sampling clock . the transceivers transmit a uwb pulse one at a time , and the rest of them receive the signal simultaneously , such that the total scan time can be dramatically reduced to 1.32 sec at 1.76 ghz sampling clock , while a typical scan time with a frequency domain system is more than 10 seconds . as patient movements during measurement cause artifacts in the acquired image , and the master controller coordinates the operation of 16 uwb transceivers and distributes a global clock to transceivers . also , it collects all received signals from transceivers and samples them using equivalent time sampling . the time - domain measurement system suffers from a low signal to noise ratio , because high frequency rf signal attenuates rapidly within breast tissue . thus , the signals have to be measured repeatedly and averaged to improve snr , because snr is proportional to the square root of the number of measurements . figure 33 shows the restored image of 3d hemispherical breast model composed of a 2 mm thick skin , fat , and 3 mm radius tumor with 1,000 measurements when all possible noise sources are present , such as clock skew ( 1% + 5 ps ) , clock jitter ( 1.6 ps ) , and white noise . the diameter of the breast model is 84 mm and the tumor located at x = 70 , y = 60 , and z = 17 . 16 antennae which are placed along the breast model transmit a modulated gaussian pulse which is centered 6 ghz with pulse width 0.3 ns . several research groups have studied microwave breast imaging systems that are noninvasive and not harmful to human as alternative early diagnostic breast cancer imaging methods . unlike initial studies , a large - scale study published in 2007 found that the variation of dielectric properties of the normal breast is large . several groups have already conducted clinical trials with patients and the results are very promising . in particular , a cmos chip - based time - domain measurement microwave system can reduce the scan time to within 1 sec when the 1000 repeated signals are averaged with 16 antennae . in the time - domain measurement , it takes 1.32 sec as once measurement by equivalent time sampling with 1.76 ghz sampling clock . thus , it can diminish the artifacts caused by patient movements due to long measurement time . in addition , it can be implemented at low prices , because it does not require expensive equipment like vna , oscilloscope , and pulse generator . the prices of vna , oscilloscope , and pulse generator are about $ 10,000~100,000 while a transceiver chip is about $ 100 . thus , the production cost of our imaging system prototype may be less than $ 3,000 . the development of such a microwave breast imaging system will help young women with dense breast to receive regular breast cancer screening safely .

breast cancer is a disease that occurs most often in female cancer patients . early detection can significantly reduce the mortality rate . microwave breast imaging , which is noninvasive and harmless to human , offers a promising alternative method to mammography . this paper presents a review of recent advances in microwave imaging for breast cancer detection . we conclude by introducing new research on a microwave imaging system with time - domain measurement that achieves short measurement time and low system cost . in the time - domain measurement system , scan time would take less than 1 sec , and it does not require very expensive equipment such as vna .

1. Introduction 2. Breast Tissue Properties in the Microwave Region 3. Microwave Breast Imaging 4. Conclusions

breast cancer is the most commonly diagnosed cancer in women and has the highest incidence of cancer in women . in 2012 the number of patients who were diagnosed with breast cancer was about 1.7 million worldwide . according to the american cancer society , about 246,660 women and 2,600 men will be diagnosed with invasive breast cancer in 2016 in the united states . however , if breast cancer is detected early enough , the five - year survival rate is over 90% [ 1 , 2 ] . current techniques for breast imaging are x - ray mammography , ultrasound , mri , and positron emission tomography ( pet ) . the results of mammography revealed 1,212 of 10,000 women screened turn out to be false positives in their 40s . harmful effects of mammography include overtreatment , unnecessary , and sometimes invasive follow - up testing and psychological harm associated with false positive test results . further , x - ray mammography needs compression of the breast to lie as flat as possible during the examination ( figure 1 ) , which causes patient pain . it is difficult to distinguish tumors in dense breast mammogram images [ 1 , 6 ] , because both dense tissue and cancer appear white in the mammogram image , unlike fatty tissue that appears black . a transducer placed on the skin sends ultrasound pulses into the body and detects the echoes from inside the body , which are used to make ultrasound images . additionally , ultrasound is mostly used as a secondary technique , after a mammogram result shows a suspected mass . pet is an imaging technique that observes how an injection mixture of radioactive materials with glucose or glycoprotein is used and synthesized in the human body . pet makes an image by detecting these positrons . unlike x - ray , ct , and mri , pet can diagnose cancer in the very early stages , because pet detects the movement of molecules in early disease cells . however , it has low resolution . microwave imaging has been researched as an alternative to x - ray mammography , because of its harmlessness to humans [ 3437 ] . microwave imaging uses the scattering wave or reflected wave that arises from the contrast in dielectric properties between normal and malignant breast tissues . in this paper microwave breast imaging could be possible due to the difference in dielectric properties between malignant tissue and normal breast tissue . in addition , we introduce our studies of a newly proposed cmos chip - based time - domain microwave imaging system . several groups have studied breast dielectric properties , and research results have already been published [ 1113 , 38 , 39 ] . 1988 . according to chaudhary et al . 1984 , malignant breast tissues at 1~2 ghz are 3~5 times greater than normal breast tissues . 1994 showed difference between malignant and normal breast tissues at 900 mhz of 200~500% , judging from 12 measurements of each normal and malignant breast tissue . this is because malignant tissues contain much water and are more active than normal [ 40 , 41 ] . later , a collaborative research team from the university of wisconsin and the university of calgary in 2007 reported a large - scale study of the ultra - wideband microwave dielectric properties of normal breast tissue obtained from reduction surgeries and a large - scale study of the ultra - wideband microwave dielectric properties of normal , benign , and malignant breast tissues [ 10 , 15 ] . the total number of patients was 93 , and the total number of measurements was 488 . in the latter study , the total number of patients involved in the study was 196 , and the total number of samples was 319 . the study measured dielectric constant data in the 0.5~20 ghz frequency range and found that while malignant tissues were ten times larger than adipose - dominated tissues , the difference between malignant and normal glandular or fibroconnective tissues was no more than about 10% . figure 4 depicts the dielectric properties of 85100% adipose tissues as solid lines , 030% as dash - dot lines and 3184% adipose tissues as dashed lines . nevertheless , microwave imaging is still used to distinguish breast cancer [ 4244 ] . besides , breast cancer classification research in microwave imaging technique has been studied in the national university of ireland [ 4552 ] . the research of university of wisconsin and university of calgary [ 10 , 15 ] was conducted at the ex vivo states . dartmouth college published the correlation of in vivo and ex vivo tissue dielectric - c properties which were measured during mastectomy procedure in the operating room ( in vivo ) and after resection in the same location immediately from six women . in addition , the research teams from university calgary and university wisconsin have been studying dielectric properties estimation using measurement system [ 5357 ] . imaging techniques using electromagnetic waves have been used in specific fields , such as the nondestructive test of structures and detection of hidden objects . recently , with the technological development of algorithms , computation techniques , and hardware , many studies are being conducted to apply electromagnetic wave imaging technology to human diagnosis [ 17 , 59 , 60 ] . microwave imaging is a promising new method for early - stage breast cancer detection . the method reconstructs the breast image from the received signals that are scattered and reflected within the breast . since the penetration loss of healthy fat tissue is less than 4 db / cm with microwave signal which is centered at 6 ghz , it is possible to reach low power signal to an antenna on the other side of an object . the specific absorption rate ( sar ) is normally used when setting the safety standards for the maximum allowed exposure to the human body . standard c95.1 - 1999 states that , in the case of devices operating at 100 khz to 6 ghz , it should not exceed a maximum 1.6 w / kg for human tissue of the average 1 g . as a guide , compared to a mobile phone using the same frequency band , microwave breast cancer imaging method should be free from health hazardous exposure to the patient , because of suffering less exposure than from a mobile phone . from the technical point of view , microwave breast cancer imaging method may detect a small size tumor and also has the advantage of low cost . in addition , microwave imaging can obtain an image quickly compared to other imaging methods . there are two approaches to the microwave imaging method : microwave tomography and radar - based . the systems transmit the microwave signals into the breast and then measure scattering signals reflected from the tumor . meaney from dartmouth college in the united states are one of the representative research groups . however , the inverse problem takes much time , because the calculation process is complicated . also , a nonlinear inverse scattering problem must be solved , and iterative image reconstruction algorithms are usually required to obtain a solution . in general , these ill - posed inverse scattering approaches suffer from nonuniqueness and require regularization in order to achieve convergence to a meaningful solution [ 66 , 90 ] . the dartmouth group has developed a clinical prototype for active microwave imaging of the breast for the first time . they developed a 32-channel data acquisition system operating at frequency range 500 mhz to 3 ghz to obtain data from a clinical prototype exam . the clinical prototype illuminates the breast with 16 monopole antennas that operate in the 300 mhz to 1 ghz frequency range . the clinical exam was conducted with 5 women , and the total acquisition time was 1015 minutes per breast . they have also developed a prototype of breast cancer detection system based on 3d fem method microwave tomography [ 19 , 20 ] . they presented the first clinical 3d microwave tomographic images of the breast using the prototype . the study achieved improvements of both hardware and software aspects and achieved data acquisition time of less than 2 minutes by development of special hardware . they used the 3d microwave breast imaging system for monitoring treatment response during neoadjuvant chemotherapy [ 20 , 92 ] . tomographic images were obtained eight times from eight patients undergoing neoadjuvant chemotherapy during the course of treatment . the results of the study indicate that the conductivity in locally advanced breast cancer matched well with overall neoadjuvant therapy treatment response . in korea , electronics and telecommunications research institute ( etri ) started research of the breast cancer diagnostic technology of microwave tomography in 2007 . etri mt breast cancer diagnostic techniques have been studied using a microwave signal in the 500 mhz to 3 ghz range [ 21 , 78 , 93 ] . figure 14(a ) shows reconstructed images of a breast without a tumor , and figures 14(b ) and 14(c ) present images of a single cylindrical tumor at the central and shifted positions . king 's college in london has been developing antenna for microwave tomography [ 84 , 94 ] , and they applied an adaptive multithreshold iterative shrinkage algorithm to the linear inversion at each iteration of the distorted born iterative method ( dbim ) . in university of manitoba proposed an iterative process that involves two algorithms : finite - difference time - domain ( fdtd ) and genetic algorithm ( ga ) . they developed a parallel algorithm for microwave tomography on cpu - based homogeneous multicore distributed memory machines and the cell be processor [ 68 , 69 , 76 ] . they have researched magnetic nanoparticles ( mnp ) enhanced microwave imaging which is able to reduce the rate of false positive and negative [ 22 , 9698 ] . it uses the reflection that arises due to difference in the electrical dielectric properties of normal and malignant breast tissues , when microwaves transmit the internal breast with tumor . this approach , which was first developed as a military ground - penetrating application , is applied to the human body . it was designed in the late 1990s by hagness in wisconsin university , and benjamin in bristol university [ 9 , 24 , 103106 ] . researchers in national university of ireland galway , tianjin university in china , and hiroshima university are also working on this subject [ 107123 ] . before applying the focusing algorithm , preprocessing may contain the extracting tumor response , compensation tissue losses , or radial spread . among the preprocessing , extracting tumor response must be conducted , because the received signal includes not only the tumor response , but also unwanted signals such as antenna coupling , directly received signals from the transmit antenna , and reflections from the skin . differential calibration techniques use a reference signal obtained from a scan of a healthy breast . however , it is hard to use when previous knowledge of the cancer - free breast does not exist . in the array rotation calibration method , two sets of measured data are received from a physically rotating antenna array , in which unwanted signals are almost the same . a calibrated signal is obtained by subtracting one of the two sets of the signals from the other . the received signal and the reference signal eliminates unwanted signals using dual receivers , rx1 and rx1 , located an equal distance from the transmitter tx in figure 16 . thus , the calibrated signal of a tumor response can be obtained by subtracting one received signal from the other . in - place calibration in addition , measurement errors that occur due to environmental changes during calibration , such as a mechanical error during antenna array rotation or an accidental error due to the unexpected movement of the patient , are minimized . focusing algorithms that are used for image reconstruction in radar - based microwave imaging include the delay and sum ( das ) [ 36 , 127129 ] , microwave imaging via space - time ( mist ) [ 108 , 128 , 130 ] , robust weighted capon beamforming ( rwcb ) , multistatic adaptive microwave imaging ( mami ) [ 36 , 110 ] , and generalized likelihood ratio test ( glrt ) [ 109 , 132 ] . das is a simple and robust method . also , the computation time is short . the signals are shifted by time ti calculated from their respective location relative to the antennae , to restore the image . the focal point is assumed to be p = ( x , y , z ) . the time delay tmn is assumed as the time delay of the signal from transmitting at the mth antenna to receiving at the nth antenna . the time delay tmn at the focal point p can be expressed as(1)tmnp=1vxmx2+ymy2+zmz21/2+xnx2+yny2+znz21/2 . the time delay applies the received signal rsi differently for each transmit antenna and receive antenna . the intensity of a reconstruction image is calculated by moving the focal point from one position to another within the breast . the intensity ip of the restored image at the given focal point can be expressed as(2)ipx , y , z=0i=1mwix , y , zrsittix , y , z2dt , where wi is the location - dependent weight calculated during preprocessing , rsi is the received signal , and ti is the time delay . microwave imaging via space - time ( mist ) beamforming uses finite impulse response ( fir ) filters to compensate for the frequency - dependent time delay , such as dispersion and fractional time delay . microwave breast cancer imaging uses a generalized likelihood ratio test ( glrt ) , which is a hypothesis testing problem for each voxel , with the null hypothesis representing the tumor - free case . however , most of those methods have suffered from performance degradation used with dense breast . thus , in the national university of ireland galway they used preprocessing filter to compensate the path - dependent attenuation and phase effects . university of bristol also presented a time - domain wideband adaptive beamforming to reduce clutter . radar - based microwave imaging can be done through two approaches : frequency domain analysis and time - domain analysis . there have been several microwave systems reported in the literature , most of which are based on frequency domain analysis [ 24 , 26 , 28 , 36 , 135 ] . however , frequency domain analysis requires equipment , such as a costly vector network analyzer ( vna ) . the process takes a lot of time , because it measures s - parameters by sweeping frequencies ; and measurement time is critical in medical imaging : patient movement during a scan may generate artifacts . university of bristol team performed experimental and clinical study using uwb microwave radar imaging method for the first time . figure 18 shows a schematic of the multistatic radar - based microwave breast imaging system . a switch matrix connected to the feed of the antennas changes the pair of antennae during measurement of the signals . the signals are measured , in turn , by all possible pairs of antennae that activate the transmit port and receive port . the measured signals with frequency domain the image is reconstructed by a focusing algorithm using the time - domain signals . figure 19 shows the microwave radar - based clinical setup for breast cancer detection with a patient . clinical verification was performed with real breast cancer patients at the bristol oncology center . however , 90 seconds is a long time to scan patients , and artifacts can occur due to patient movement . subsequently , the team developed a 60-element antenna array with 8 port vna ( figure 21(b ) ) . the scan time with the 60-element array achieved 10 seconds . figure 23 shows the radar - based breast imaging system of micrima which was founded in 2006 to develop and commercialize the technology of microwave radar breast imaging at the university of bristol in the uk . the micrima 's maria system has undergone clinical trials at the several breast cancer imaging centers in the uk and got european regulatory approval in 2015 . university of calgary have developed a tissue sensing adaptive radar ( tsar ) prototype of radar - based breast imaging system ( figure 24 ) . their system was different from the system of bristol university , using a monostatic method and filtering the reflection from the skin . the results were positive , since several restored images exhibited responses which were similar to clinical results . they also studied 3d surfaces acquisition of the breast for radar - based microwave breast imaging and investigated the impact of the breast model complexity on microwave imaging signals . several research groups have recently reported time - domain measurement microwave breast imaging systems [ 29 , 31 , 37 , 139 ] . time - domain measurement systems have the advantage of being cost - effective and requiring less scan time . mcgill university in canada is a leading research group in time - domain measurement microwave imaging systems for the detection of breast cancer . figure 25 shows the experimental system that is a multistatic radar - based microwave system with 16-element antenna array for breast cancer detection that their research team developed . figure 26 shows that the time system transmits input pulse using a pulse generator and receives the signal from antennae using an oscilloscope in real time [ 29 , 140 ] . further , the mcgill university research team have performed an early clinical study of time - domain microwave radar for breast health monitoring [ 30 , 141 ] . figure 27 shows a scanning patient on the table with the clinical system at mcgill university . in addition , they have developed wearable bra prototype using microwaves for breast health monitoring [ 142 , 143 ] . however , the system still depends on a high - precision pulse generator and very high - speed oscilloscope and may require complicated switching circuits . additionally , a time - domain system requires a very fast sampling clock such that even a very small jitter in the sampling clock might blur the resulting images . in figure 31 shows the configuration of the time - domain microwave breast imaging system that we previously presented . the transceivers transmit a uwb pulse one at a time , and the rest of them receive the signal simultaneously , such that the total scan time can be dramatically reduced to 1.32 sec at 1.76 ghz sampling clock , while a typical scan time with a frequency domain system is more than 10 seconds . as patient movements during measurement cause artifacts in the acquired image , and the master controller coordinates the operation of 16 uwb transceivers and distributes a global clock to transceivers . the time - domain measurement system suffers from a low signal to noise ratio , because high frequency rf signal attenuates rapidly within breast tissue . figure 33 shows the restored image of 3d hemispherical breast model composed of a 2 mm thick skin , fat , and 3 mm radius tumor with 1,000 measurements when all possible noise sources are present , such as clock skew ( 1% + 5 ps ) , clock jitter ( 1.6 ps ) , and white noise . the diameter of the breast model is 84 mm and the tumor located at x = 70 , y = 60 , and z = 17 . 16 antennae which are placed along the breast model transmit a modulated gaussian pulse which is centered 6 ghz with pulse width 0.3 ns . several research groups have studied microwave breast imaging systems that are noninvasive and not harmful to human as alternative early diagnostic breast cancer imaging methods . several groups have already conducted clinical trials with patients and the results are very promising . in particular , a cmos chip - based time - domain measurement microwave system can reduce the scan time to within 1 sec when the 1000 repeated signals are averaged with 16 antennae . in the time - domain measurement , it takes 1.32 sec as once measurement by equivalent time sampling with 1.76 ghz sampling clock . thus , it can diminish the artifacts caused by patient movements due to long measurement time . in addition , it can be implemented at low prices , because it does not require expensive equipment like vna , oscilloscope , and pulse generator . the prices of vna , oscilloscope , and pulse generator are about $ 10,000~100,000 while a transceiver chip is about $ 100 . thus , the production cost of our imaging system prototype may be less than $ 3,000 . the development of such a microwave breast imaging system will help young women with dense breast to receive regular breast cancer screening safely .

from its origins in 1895 with dd palmer s original focus on magnetic healing , chiropractic identity has been beset with the challenging task of keeping up not only with clinical and scientific observation but with political trade winds involving public perception and the marketplace of health care . originally , dd palmer viewed the body from a more mechanical viewpoint , like a machine , for at the turn of the 20th century he indicated that:a human being is a human machine and , like a machine , would run smoothly , without any friction , if every part was in its proper place . if every bone , nerve , and all blood vessels , muscles , etc . a chiropractic looks the human machine over , and finds what parts are out of place , why the blood does not circulate freely to all parts , why the nerves cry out with pain . disease is the effect or result of some part of the body being disarranged . to put them in their proper place , would give the diseased person ease , and allow nature to rebuild without being obstructed " . a human being is a human machine and , like a machine , would run smoothly , without any friction , if every part was in its proper place . a chiropractic looks the human machine over , and finds what parts are out of place , why the blood does not circulate freely to all parts , why the nerves cry out with pain . disease is the effect or result of some part of the body being disarranged . to put them in their proper place , would give the diseased person ease , and allow nature to rebuild without being obstructed " . with that in mind , dd palmer paid particular attention to the nerves:the human body is a bundle of fine sensitive nerves , passing over , under , and between the two hundred bones and many muscles and ligaments . these nerves are liable to be pinched , strained , stretched , or pulled out of place by the displacement of any one of the bones , muscles or ligaments , causing any of the many nerve diseases . the human body is a bundle of fine sensitive nerves , passing over , under , and between the two hundred bones and many muscles and ligaments . these nerves are liable to be pinched , strained , stretched , or pulled out of place by the displacement of any one of the bones , muscles or ligaments , causing any of the many nerve diseases . it was from this origin that the popular but often misinterpreted concept grew that doctors of chiropractic dealt with wrestling with what would become a perennial question of chiropractic identity nearly a century later , the world federation of chiropractic ( wfc ) organized 2 years of workshops , driven in part by a survey conducted by manifest communications that emphasized what had become a disparity between what the public and doctors of chiropractic commonly perceived as chiropractic treatment . by a wide margin , the survey and various studies suggested that doctors of chiropractic were managing primarily musculoskeletal problems with emphasis upon back pain.2 , 3 , 4 , 5 taking these findings to the wfc congress in sydney , australia , in june of 2005 , the identity consultation task force concluded the following : ( 1 ) it is important for the profession to have an identity ; ( 2 ) most agree that the chiropractic profession suffers from an unclear identity and position within today s health care plans ; and ( 3 ) it is important to understand how doctors of chiropractic think that the profession should be viewed and how they believe that it is actually viewed . here one finds substantial discrepancies : ( 1 ) whether the profession offers primary or specialist health care ; ( 2 ) whether the profession is mainstream ( ie , core to the health care delivery system ) or if it is an alternative ; and ( 3 ) whether the profession offers wellness and nonsurgical , nondrug health care or simply manages back , neck , and spinal problems . the final identity consultation task force final report that emerged from the 2005 wfc conference concluded that a leading statement of identity was mandatory to be: established and maintained through the use of the following three linked concepts:1.a leading statement on identity , which must be clear , concise , and immediately relevant to both the public and the profession the pole ( brand platform).2.several important qualifying statements , which provide the necessary context and foundation for the pole ground ( brand pillars).3.a description of the qualities or essential personality of chiropractors the personality ( tone ) . established and maintained through the use of the following three linked concepts:1.a leading statement on identity , which must be clear , concise , and immediately relevant to both the public and the profession the pole ( brand platform).2.several important qualifying statements , which provide the necessary context and foundation for the pole ground ( brand pillars).3.a description of the qualities or essential personality of chiropractors the personality ( tone ) . a leading statement on identity , which must be clear , concise , and immediately relevant to both the public and the profession the pole ( brand platform ) . several important qualifying statements , which provide the necessary context and foundation for the pole ground ( brand pillars ) . a description of the qualities or essential personality of chiropractors the personality ( tone ) . the pole was that doctors of chiropractic should be regarded as the spinal health care experts in the health care system . the ground statements emphasized ( a ) a patient - centered approach to health care ; ( b ) wellness ; ( c ) the self - healing powers of the individual ; ( d ) avoidance of the use of drugs and surgery wherever possible ; ( e ) examination , diagnosis , and treatment based upon available research ; and perhaps the most critical element of all , ( f ) the relationship between the spine and the nervous system . in adopting these identity statements by consensus , the wfc conference clearly identified the spine as an element , without which most perceptions of chiropractic were assumed to wither away , wiping out chiropractic s identity in the process . this raised the question of whether the public would now regard the profession as a specialist form of health care delivery and fail to perceive the spine as a dynamic entity that is hard - wired into the nervous system , such that the latter network and actual scope of practice of the profession would be overlooked . in other words , there would be a repudiation of the framework with which chiropractic needed to be regarded as expressed by none other than dd palmer:life is the expression of tone . in that sentence tone is the expression in function by normal elasticity , activity , strength and excitability of the various organs as observed in a state of health . tone is the expression in function by normal elasticity , activity , strength and excitability of the various organs as observed in a state of health . as joseph brimhall , president of the council of chiropractic education and director of the council of chiropractic education international , explained , there was no wording in the accreditation standards of the council of chiropractic education ( us ) , the model standards of council of chiropractic education international , or other jurisdictions that restricted the chiropractic profession to the spine . adding fuel to these fires of unrest are 2 major questions , 1 philosophical and 1 as to whether the chiropractic profession is based upon the chiropractic subluxation ( including a meaningful definition of the latter ) and should offer limited prescriptions , given that the medical profession has shown in several instances to be deficient in understanding and especially diagnosing a variety of musculoskeletal conditions.8 , 9 , 10 , 11 chiropractic s identity thus remains , at best , a work in progress and , at worst , a matter of considerable controversy . therefore , the purpose of this article is to address this dilemma through discussion of the following 6 topics : ( 1 ) concepts of manual medicine ; ( 2 ) areas of interest beyond the spine ; ( 3 ) concepts of neurology ; ( 4 ) concepts of the chiropractic subluxation ; ( 5 ) mainstream or alternative ; and ( 6 ) primary care , first - contact provider , or specialist . chiropractic occupies 1 niche in the broad field of physical or manual medicine that spans soft tissue , mobilization , and manipulation techniques.12 , 13 , 14 , 15 , 16 included in the manual medicine portfolio are such interventions as osteopathic manipulative medicine , massage , physical therapy , mckenzie method , craniosacral therapy , myofascial release , rolfing structural integration , qigong , shiatsu , and even acupressure . although chiropractic has attempted to define its niche in spinal manipulation using short - lever , high - velocity , low - amplitude ( hvla ) techniques , those approaches are not fully circumscribed by doctors of chiropractic alone . the challenge becomes even greater when one attempts to reconcile the nearly 100 named chiropractic techniques that have been identified by bergmann . from such an expanded catalog , it is apparent that a vast array of low - velocity , soft - tissue , and even instrumental techniques are included , which speak to an extensive overlap with other branches of manual medicine . craniosacral therapy and myofascial release , for example , are commonly practiced by osteopathic manipulative therapists . and hvla manipulations , which traditionally have been associated with doctors of chiropractic , have also been applied by osteopaths , physical therapists , german medical therapists , and practitioners of chinese medicine.21 , 22 part of the identity problem is that doctors of chiropractic are often included with other practitioners . however , the literature shows an inferior safety record with the other professions and laypeople having had lesser training and/or displayed less competence in musculoskeletal medicine.23 , 24 , 25 the superior safety record of doctors of chiropractic compared with other manual therapists clearly stands out as a distinguishing characteristic of chiropractic . the safety of the hvla procedures delivered by doctors of chiropractic has been shown to be greater than what has been encountered in medical procedures.27 , 28 in addition , many of the soft tissue techniques applied by doctors of chiropractic could be conflated with approaches used by massage therapists . therefore , on the basis of overlap with such a broad array of techniques applied by all the above - named practitioners , it would be unproductive to define chiropractic on the basis of manual techniques alone . the historical record and majority of rigorous research as well as the aforementioned identity consultation task force has focused upon the spine as the most successful area of intervention with which doctors of chiropractic are identified . nevertheless , alternatives in addition to the spine in which the usefulness of chiropractic care has been documented include the following:1.extremities : the successful outcomes regarding chiropractic treatments of the extremities , supported by a body of literature which suggests effective outcomes in managing repetitive motion disorders.29 , 30 , 31 , 322.neck muscles : published reports of chiropractic effleurage of the sternocleidomastoid muscles , proposed to be an effective part of the chiropractic management of otitis media.3.skull : the successful applications of craniosacral therapy , reported by the national board of chiropractic examiners to have been practiced by 38.0% of doctors of chiropractic in 2003 and focusing upon the skull as well as the vertebral canal.4.neck muscles : the application of myofascial release , also known as ischemic compression , directed in at least 1 study to the active upper trapezius trigger points with a successful outcome.5.temporomandibular joint : chiropractic treatment of temporomandibular disorders , necessarily involving applications to areas of the jaw and sides of the neck , for which a fair level of supporting evidence of effectiveness has been reported.6.hip and knee : chiropractic management of osteoarthritis of the hip and knee , for which supportive evidence of efficacy has recently been emerging.7.muscles : chiropractic testing of muscle function in all parts of the body is 1 application ( applied kinesiology ) , reported by the national board of chiropractic examiners to have been practiced by 37.6% of doctors of chiropractic in 2003 and involving muscles throughout the entire body.8.nutrition : nutrition and wellness , major components of health maintenance and prevention , often advocated by doctors of chiropractic . in fact , the national board of chiropractic examiners found in 2003 that more than 96% of the chiropractic profession in america engaged in instructing their patients in health and wellness . extremities : the successful outcomes regarding chiropractic treatments of the extremities , supported by a body of literature which suggests effective outcomes in managing repetitive motion disorders.29 , 30 , 31 , 32 neck muscles : published reports of chiropractic effleurage of the sternocleidomastoid muscles , proposed to be an effective part of the chiropractic management of otitis media . skull : the successful applications of craniosacral therapy , reported by the national board of chiropractic examiners to have been practiced by 38.0% of doctors of chiropractic in 2003 and focusing upon the skull as well as the vertebral canal . neck muscles : the application of myofascial release , also known as ischemic compression , directed in at least 1 study to the active upper trapezius trigger points with a successful outcome . temporomandibular joint : chiropractic treatment of temporomandibular disorders , necessarily involving applications to areas of the jaw and sides of the neck , for which a fair level of supporting evidence of effectiveness has been reported . hip and knee : chiropractic management of osteoarthritis of the hip and knee , for which supportive evidence of efficacy has recently been emerging . muscles : chiropractic testing of muscle function in all parts of the body is 1 application ( applied kinesiology ) , reported by the national board of chiropractic examiners to have been practiced by 37.6% of doctors of chiropractic in 2003 and involving muscles throughout the entire body . nutrition : nutrition and wellness , major components of health maintenance and prevention , often advocated by doctors of chiropractic . in fact , the national board of chiropractic examiners found in 2003 that more than 96% of the chiropractic profession in america engaged in instructing their patients in health and wellness . thus , it is awkward to consider chiropractic interventions to be limited to the spine . the forgoing body of literature shows that treatment limited to the spine should not be considered to be the sole lens through which chiropractic is viewed . applications by doctors of chiropractic to other regions of the body have demonstrated clinical effectiveness and should not be categorically dismissed . identity of chiropractic has been intimately associated with the chiropractic subluxation40 , 41 , a topic of considerable debate within itself . as defined by the association of chiropractic colleges in july 1996 and the subject of texts that have been considered to be definitive , the chiropractic subluxation was regarded to be : a complex of functional and/or structural and/or pathological articular changes that compromise neural integrity and may influence organ system function and general health . a complex of functional and/or structural and/or pathological articular changes that compromise neural integrity and may influence organ system function and general health . historically , definitions of chiropractic subluxation that presented the term in 3 dimensions were beset by a number of challenges44 , 45 , 46:1.in terms of misalignments , it was clear that these could not be detectable by technological methods existing at that time.2.in terms of aberration of movement integrity , either deficient or excessive motion , reliable measurements of motion remained elusive.3.in terms of physiologic dysfunction , these could be present with or without pain and could facilitate one s understanding of chiropractic subluxations . but simply presenting a clinical complaint as a physiological dysfunction was essentially a circular argument , begging the question and demanding further clarification . in terms of misalignments , it was clear that these could not be detectable by technological methods existing at that time . in terms of aberration of movement integrity , either deficient or excessive motion , , these could be present with or without pain and could facilitate one s understanding of chiropractic subluxations . but simply presenting a clinical complaint as a physiological dysfunction was essentially a circular argument , begging the question and demanding further clarification . therefore , the question has remained whether the chiropractic subluxation could be shown as a clinical reality by objective measurements , in addition to what its attributes really were . capturing the proper definition of chiropractic subluxation fortuitously , it is the emergence of research with an eye toward systemic and arguably nonmuscular domains responding to spinal manipulation that could be credited with a new , progressive recasting of the chiropractic definition of chiropractic subluxation appearing in the fourth edition of the clinical practice guidelines ( 2013 ) issued by the council on chiropractic practice which reads:subluxation is a neurological imbalance or distortion in the body associated with adverse physiological responses and/or structural changes , which may become persistent or progressive . the most frequent site for the chiropractic correction of the subluxation is via the vertebral column . subluxation is a neurological imbalance or distortion in the body associated with adverse physiological responses and/or structural changes , which may become persistent or progressive . the most frequent site for the chiropractic correction of the subluxation is via the vertebral column . this groundbreaking concept meant that the term neurological imbalance had now emerged into public view , taking precedent over so many of the hidebound terms that used to be grounded in segmental , articular , or vertebral terms from the 1930s for at least 60 years . to what the change in the definition represents the board s analysis and research into the continued evidence supporting spinal adjustment of dysfunctional vertebra leading to brain metabolic and transient cortical plastic changes in the brain and nervous system . a few more outstanding chapters in research that have paved the way toward this dramatic revision of the chiropractic subluxation s definition are worth citing:1.the finding that spinal manipulation at the c5/c6 spinal segment is regionally related to the infraspinatus but not gluteus medius muscle , suggesting in the authors own words that the primary physiological effect of smt may be neurological rather than changed joint mechanics ( italics mine).2.the dramatic observation by karason and drysdale that an hvla thrust at the lumbosacral junction produced a significant increase in cutaneous blood flow over the l5 dermatome in nonsmokers but not smokers , suggesting the role of a nicotine - sensitive receptor or other trigger.3.the award - winning research of song and his coworkers at parker college , demonstrating a broad spectrum of anti - inflammatory , joint - specific effects of activator treatments in a rat model encompassing behavioral , cytological , and neurophysiological benchmarks.4.the demonstration that a bilateral hypothenar type adjustment accompanied by audible cavitation specifically produced a decrease in the production of the inflammatory cytokines tumor necrosis factor and interleukin-1 in human subjects.5.the revelation that 2 patients with cervicogenic headache after 4 weeks of manipulative therapy posted reductions of tumor necrosis factor exceeding 50%.6.continuing with observations regarding the inflammatory cytokines , the observation that the direction of strain in cultured fibroblasts determined the levels of specific cytokines produced , raising the practical concern that the effects of manipulation may vary in patients depending upon tissue strain directions.7.two of the outstanding colic studies , 1 of only 2 areas of pediatric chiropractic care supported by clinical trials and possibly involving nonmusculoskeletal mechanisms , demonstrating that manipulation rapidly produced significant reductions of this disorder.56 , 578.the finding that cervical spine manipulation not only altered cortical integration of dual somatosensory input but also changed the way the central nervous system responded to a subsequent motor training ( typing ) task . the finding that spinal manipulation at the c5/c6 spinal segment is regionally related to the infraspinatus but not gluteus medius muscle , suggesting in the authors own words that the primary physiological effect of smt may be neurological rather than changed joint mechanics ( italics mine ) . the dramatic observation by karason and drysdale that an hvla thrust at the lumbosacral junction produced a significant increase in cutaneous blood flow over the l5 dermatome in nonsmokers but not smokers , suggesting the role of a nicotine - sensitive receptor or other trigger . the award - winning research of song and his coworkers at parker college , demonstrating a broad spectrum of anti - inflammatory , joint - specific effects of activator treatments in a rat model encompassing behavioral , cytological , and neurophysiological benchmarks . the demonstration that a bilateral hypothenar type adjustment accompanied by audible cavitation specifically produced a decrease in the production of the inflammatory cytokines tumor necrosis factor and interleukin-1 in human subjects . the revelation that 2 patients with cervicogenic headache after 4 weeks of manipulative therapy posted reductions of tumor necrosis factor exceeding 50% . continuing with observations regarding the inflammatory cytokines , the observation that the direction of strain in cultured fibroblasts determined the levels of specific cytokines produced , raising the practical concern that the effects of manipulation may vary in patients depending upon tissue strain directions . two of the outstanding colic studies , 1 of only 2 areas of pediatric chiropractic care supported by clinical trials and possibly involving nonmusculoskeletal mechanisms , demonstrating that manipulation rapidly produced significant reductions of this disorder.56 , 57 the finding that cervical spine manipulation not only altered cortical integration of dual somatosensory input but also changed the way the central nervous system responded to a subsequent motor training ( typing ) task . all these findings defined objective physical attributes that could be attributed to what one referred to as the chiropractic subluxation and how chiropractic interventions attenuated this disorder . these discoveries and the resulting redefinition of the chiropractic subluxation did not exist at the turn of the century in 2000 . casting the chiropractic subluxation in light of these systemic changes brings this discussion back to the original concept of dd palmer , identifying chiropractic s primary target as the nervous system as quoted above . elaborating upon dd palmer s manifesto , the link between the systemic effects ( anti - inflammatory , somatosensory , behavioral , and cytological ) and spinal manipulation is best understood in terms of neurology . an appreciation of this approach begins with a consideration of numerous animal studies as shown in table 1 . these demonstrate that a variety of physiological effects ( a ) are distinct from pain and ( b ) extend far from the area of stimulation . with several of these investigations demonstrating that nerve conductivity is specifically affected,72 , 73 , 74 it can be construed that the nervous system provides an essential link between the experimentally produced aberrations and the physiological changes observed . thus , a wide range of stimuli are capable of producing physiological responses , providing a much broader canvas with which chiropractic subluxations and , for that matter , chiropractic identity can be represented in experimental research , again placing the nervous system at the center . with regard to changes in neural function in response to manipulation , however , a few observations can be brought to light : in their different approaches , all demonstrate inductions of activities within the nervous system that project responses to manipulation well beyond the area of contact.1.abnormal somatosensory evoked potentials from the paraspinal musculature were found to be correlating with decreased pain responses after lumbar manipulation , possibly due to a central effect of sensory processing.2.in a cohort of 12 subjects with a history of recurrent neck stiffness and/or neck pain but no acute symptoms at the time of study , a single session of cervical spine manipulation revealed a significant decrease in the amplitude of 2 components of somatosensory evoked potentials , lasting 20 minutes following the intervention . the implication was that cervical spine manipulation may alter cortical somatosensory processing and sensorimotor integration , shedding light upon the mechanisms for the relief of pain and restoration of functional mobility which are the most widely observed outcomes to treatment by spinal manipulation.3.in subjects subjected to side - posture manipulation , both hoffman reflex and m - wave responses displayed the greatest attenuation with actual manipulation as opposed to a positioning maneuver.4.following sacroiliac joint manipulation , there was a decreased inhibitory effect of knee joint pathology on quadriceps muscle activity , suggesting an interaction between spinal manipulation and the inhibition of voluntary activities produced by pain.5.power spectrum analyses of patient electrocardiograms suggested alterations of sympathetic and parasympathetic activity produced by spinal manipulation.79 , 80 , 816.more recently , in 36 subjects with identifiable myofascial pain syndrome in the infraspinatus and gluteus medius muscles , spinal manipulation at the c5/c6 spinal segment significantly reduced the pressure - pain threshold in the infraspinatus muscle but not the gluteus medius . the implication was that the primary physiological effect of spinal manipulation may be neurophysiological rather than changed joint mechanics , in which spinal manipulation produces inhibitory mechanisms in the myofascial tissues . abnormal somatosensory evoked potentials from the paraspinal musculature were found to be correlating with decreased pain responses after lumbar manipulation , possibly due to a central effect of sensory processing . in a cohort of 12 subjects with a history of recurrent neck stiffness and/or neck pain but no acute symptoms at the time of study , a single session of cervical spine manipulation revealed a significant decrease in the amplitude of 2 components of somatosensory evoked potentials , lasting 20 minutes following the intervention . the implication was that cervical spine manipulation may alter cortical somatosensory processing and sensorimotor integration , shedding light upon the mechanisms for the relief of pain and restoration of functional mobility which are the most widely observed outcomes to treatment by spinal manipulation . in subjects subjected to side - posture manipulation , both hoffman reflex and m - wave responses displayed the greatest attenuation with actual manipulation as opposed to a positioning maneuver . following sacroiliac joint manipulation , there was a decreased inhibitory effect of knee joint pathology on quadriceps muscle activity , suggesting an interaction between spinal manipulation and the inhibition of voluntary activities produced by pain . power spectrum analyses of patient electrocardiograms suggested alterations of sympathetic and parasympathetic activity produced by spinal manipulation.79 , 80 , 81 more recently , in 36 subjects with identifiable myofascial pain syndrome in the infraspinatus and gluteus medius muscles , spinal manipulation at the c5/c6 spinal segment significantly reduced the pressure - pain threshold in the infraspinatus muscle but not the gluteus medius . the implication was that the primary physiological effect of spinal manipulation may be neurophysiological rather than changed joint mechanics , in which spinal manipulation produces inhibitory mechanisms in the myofascial tissues . these observations point toward a model of physiological activity in which form follows function . in so doing , it is useful to frame chiropractic in terms of functional neurology , a term in keeping with the more progressive definition of chiropractic subluxation and thus more amenable to interpretation , discussion , and ultimate acceptance by both other health care professions and the public . the cachet and impact of functional neurology can be illustrated by the formation of such professional societies as the international association of functional neurology and rehabilitation and the massive society for neuroscience . i had the pleasure of presenting research at the society for neuroscience convention in new orleans in 2012 and found my discussions and presentation on functional neurology widely and warmly received . these are both organizations which have much to offer chiropractic and vice versa and which i believe would both solidify and promote evolving concepts of chiropractic identity . these examples lead to a vindication of dd palmer s original concept of the importance of tone , an affirmation of the significance of the nervous system as a core element of what chiropractic has attempted to address . neither the disorder nor the intervention is necessarily confined to a local site , but rather the consequences of the disorder and the therapeutic applications involve systemic effects that could be triggered by the nervous system . a final aspect of establishing an identity of chiropractic addresses the question of whether this discipline needs to be considered a mainstream or alternative branch of health care delivery . assumptions have been that mainstream status confers battle - tested , scientific validity as opposed to an alternative , the latter sometimes categorized as keeping bad company with , for example , purveyors of laetrile . at the same time , mainstream status may confine chiropractic to a low back or neck role by downplaying interventions outside of these regions . in addition , this recognition ignores the fact that , 35 years ago , little research existed to support what would now be considered mainstream status for chiropractic care for low back pain . in other words , those concluding that chiropractic should be considered only as a mainstream venue would be attaching such strings as back specialists or musculoskeletal pain relievers to this classification , overlooking the research and interventions that suggest the potential for chiropractic to transcend these boundaries . answers to address if chiropractic is mainstream or alternative are best generated by first examining what are varying concepts of alternative medicine . the german nicht - schulmedizine model considers alternative medicine as all health care practices not taught at university medical facilities or medical schools . more facetiously , health policy fanatics sometimes refer to alternative medicine as those health care practices that are not paid for by insurance and which a patient does not tell his or her primary doctor . this takes after the findings of david eisenberg which represent the emergence of alternative medicine into national consciousness . but perhaps the most telling is an opinion voiced by what was the forerunner of the national center for complementary and integrative health ; that is , alternative medicine represents any health care practice that is not politically dominant at the time . the debate over alternative medicine was best represented by the opposing viewpoints of andrew weil:patients in unprecedented numbers are going outside of conventional medicine to look for help . the therapies that we aim for are those that are reasonable , that are supported by what evidence is now available , that above all are not harmful , and that work from the premise that the body can heal itself , if you give it a chance.and arnold relman:most alternative systems of treatment are based on irrational or fanciful thinking it [ alternative medicine ] could not be woven in to the fabric of the medical curriculum without confusion , contradiction , and an undermining of the scientific foundation upon which modern medicine rests . patients in unprecedented numbers are going outside of conventional medicine to look for help . the therapies that we aim for are those that are reasonable , that are supported by what evidence is now available , that above all are not harmful , and that work from the premise that the body can heal itself , if you give it a chance . most alternative systems of treatment are based on irrational or fanciful thinking it [ alternative medicine ] could not be woven in to the fabric of the medical curriculum without confusion , contradiction , and an undermining of the scientific foundation upon which modern medicine rests . however , the authoritative health and medicine division of the national academies of sciences , engineering , and medicine ( formerly known as the institute of medicine ) has concluded in an exhaustive review that revered medical evidence is lacking and in need of substantial improvement . in fact , in 1 study comparing the extent to which different medical specialties were evidence based , chiropractic practice was found to have the highest proportion of care ( 68.3% ) supported by good quality , experimental evidence and was superior to several medical subspecialties . add to this a recent study of 2500 medical treatments in which only 15% were identified as being beneficial , 22% likely to be beneficial , and 47% unknown , and the question arises whether the mainstream medical foundation is as commanding as it has commonly been made out to be . in short , despite its clear prominence in terms of public perception and third - party payers , mainstream status is not without its flaws . chiropractic , in its rush to achieve singular distinction and to seize mainstream ground in which most of the training , research , and public awareness have taken place ( centering around the spine and musculoskeletal care ) , may have compromised its future . the profession runs the risk of mortgaging many of its traditional and hard - won attributes . these would include the very foundations of chiropractic , such as ( a ) the ability to diagnose ; ( b ) patient - centered care ; ( c ) primary wellness care in offering an alternative to the current medical paradigm ; and ( d ) numerous research findings already published representing the full range of basic research , randomized clinical trials , and case series all of which point toward a scope of practice and model of patient care which extend well beyond the specialist role of simply managing back and neck pain . in looking ahead and allowing room to expand into those areas in which there is merit , some of chiropractic s leading researchers have declared that there is reason to devote future research efforts beyond the spine and spinal conditions ( ie , low back and neck pain ) . for these reasons , alternative medicine offers the proper niche into which chiropractic may continue to evolve and express its full potential . yet there is no denying that , for managing low back and neck pain as well as headache , chiropractic has already achieved there are more 100 published clinical trials in scientific journals to support this claim , as well as the public perception that doctors of chiropractic do , in fact , know more than a little about the spine and spinal health.72 , 73 , 91 , 92 , 93 , 94 , 95 for these reasons , chiropractic needs to be considered a hybrid of both alternative and mainstream medicine : mainstream , based upon what it has attained , and alternative , in areas in which its documented growth has occurred and must continue to occur . like alternative medicine , multiple definitions have been provided for primary care that are worth reviewing:1.starfield : first - contact , longitudinal , coordinated , and comprehensive care ( ie , practitioner should take care of the majority of problems without referral).2.public health service act : services which require family medicine , internal medicine , pediatrics , obstetrics / gynecology , dentistry , or mental health as provided by physicians or other health care professionals.3.institute of medicine : emphasis upon accessibility , comprehensiveness , continuity , and coordination . starfield : first - contact , longitudinal , coordinated , and comprehensive care ( ie , practitioner should take care of the majority of problems without referral ) . public health service act : services which require family medicine , internal medicine , pediatrics , obstetrics / gynecology , dentistry , or mental health as provided by physicians or other health care professionals . primary care is a way of delivering health care , not a body of knowledge as such . a comprehensive community study by abt associates that convened 2 panels of medical experts ( 1 consisting solely of doctors of chiropractic ) concluded that , with respect to a list of 53 primary functions found to occur daily in medical offices , doctors of chiropractic were capable of making diagnoses in 92% of those activities and capable of making therapeutic contributions in more than 50% of them . on the other hand , in a competency examination comparing performances of chiropractic and medical students , chiropractic students scored higher than their medical counterparts only on the musculoskeletal portion of the examination and in several other areas scored substantially lower . and access to tertiary care facilities , such as hospitals , although growing for doctors of chiropractic , remains very limited . at the same time , health care delivery as well as competency in musculoskeletal areas by medical doctors has been shown in multiple settings to be inadequate.12 , 25 , 101 these findings would seem to position doctors of chiropractic in the united states on a spectrum somewhere between primary care providers and specialists upon referral . at the very least , first contact for a minimum of musculoskeletal conditions would be indicated for doctors of chiropractic , buttressed by such findings by sarnat et al102 , 103 that demonstrated major reductions of hospital days , outpatient surgical procedures , and health care costs when doctors of chiropractic were given first - contact privileges to patients within an independent physicians association . beyond managing only musculoskeletal conditions , doctors of chiropractic in 4 medical underserved communities ( rural and urban underserved ) tended to deliver primary care services . the same pattern was found to be true in rural communities of ontario , with doctors of chiropractic delivering primary health care , bridged care , and interim care . it would appear that the scope of chiropractic extends beyond specialist to a first - contact provider . this scope would certainly be for musculoskeletal conditions with the potential to extend into primary care should adequate research be able to justify such an expansion . indeed , given the shortages of primary care providers in some communities and the cost - effectiveness and satisfaction with doctors of chiropractic serving in a de facto primary care capacity , it would therefore seem appropriate to consider doctors of chiropractic as having successfully vacated the specialist s role . chiropractic occupies 1 niche in the broad field of physical or manual medicine that spans soft tissue , mobilization , and manipulation techniques.12 , 13 , 14 , 15 , 16 included in the manual medicine portfolio are such interventions as osteopathic manipulative medicine , massage , physical therapy , mckenzie method , craniosacral therapy , myofascial release , rolfing structural integration , qigong , shiatsu , and even acupressure . although chiropractic has attempted to define its niche in spinal manipulation using short - lever , high - velocity , low - amplitude ( hvla ) techniques , those approaches are not fully circumscribed by doctors of chiropractic alone . the challenge becomes even greater when one attempts to reconcile the nearly 100 named chiropractic techniques that have been identified by bergmann . from such an expanded catalog , it is apparent that a vast array of low - velocity , soft - tissue , and even instrumental techniques are included , which speak to an extensive overlap with other branches of manual medicine . craniosacral therapy and myofascial release , for example , are commonly practiced by osteopathic manipulative therapists . and hvla manipulations , which traditionally have been associated with doctors of chiropractic , have also been applied by osteopaths , physical therapists , german medical therapists , and practitioners of chinese medicine.21 , 22 part of the identity problem is that doctors of chiropractic are often included with other practitioners . however , the literature shows an inferior safety record with the other professions and laypeople having had lesser training and/or displayed less competence in musculoskeletal medicine.23 , 24 , 25 the superior safety record of doctors of chiropractic compared with other manual therapists clearly stands out as a distinguishing characteristic of chiropractic . the safety of the hvla procedures delivered by doctors of chiropractic has been shown to be greater than what has been encountered in medical procedures.27 , 28 in addition , many of the soft tissue techniques applied by doctors of chiropractic could be conflated with approaches used by massage therapists . therefore , on the basis of overlap with such a broad array of techniques applied by all the above - named practitioners , it would be unproductive to define chiropractic on the basis of manual techniques alone . the historical record and majority of rigorous research as well as the aforementioned identity consultation task force has focused upon the spine as the most successful area of intervention with which doctors of chiropractic are identified . nevertheless , alternatives in addition to the spine in which the usefulness of chiropractic care has been documented include the following:1.extremities : the successful outcomes regarding chiropractic treatments of the extremities , supported by a body of literature which suggests effective outcomes in managing repetitive motion disorders.29 , 30 , 31 , 322.neck muscles : published reports of chiropractic effleurage of the sternocleidomastoid muscles , proposed to be an effective part of the chiropractic management of otitis media.3.skull : the successful applications of craniosacral therapy , reported by the national board of chiropractic examiners to have been practiced by 38.0% of doctors of chiropractic in 2003 and focusing upon the skull as well as the vertebral canal.4.neck muscles : the application of myofascial release , also known as ischemic compression , directed in at least 1 study to the active upper trapezius trigger points with a successful outcome.5.temporomandibular joint : chiropractic treatment of temporomandibular disorders , necessarily involving applications to areas of the jaw and sides of the neck , for which a fair level of supporting evidence of effectiveness has been reported.6.hip and knee : chiropractic management of osteoarthritis of the hip and knee , for which supportive evidence of efficacy has recently been emerging.7.muscles : chiropractic testing of muscle function in all parts of the body is 1 application ( applied kinesiology ) , reported by the national board of chiropractic examiners to have been practiced by 37.6% of doctors of chiropractic in 2003 and involving muscles throughout the entire body.8.nutrition : nutrition and wellness , major components of health maintenance and prevention , often advocated by doctors of chiropractic . in fact , the national board of chiropractic examiners found in 2003 that more than 96% of the chiropractic profession in america engaged in instructing their patients in health and wellness . extremities : the successful outcomes regarding chiropractic treatments of the extremities , supported by a body of literature which suggests effective outcomes in managing repetitive motion disorders.29 , 30 , 31 , 32 neck muscles : published reports of chiropractic effleurage of the sternocleidomastoid muscles , proposed to be an effective part of the chiropractic management of otitis media . skull : the successful applications of craniosacral therapy , reported by the national board of chiropractic examiners to have been practiced by 38.0% of doctors of chiropractic in 2003 and focusing upon the skull as well as the vertebral canal . neck muscles : the application of myofascial release , also known as ischemic compression , directed in at least 1 study to the active upper trapezius trigger points with a successful outcome . temporomandibular joint : chiropractic treatment of temporomandibular disorders , necessarily involving applications to areas of the jaw and sides of the neck , for which a fair level of supporting evidence of effectiveness has been reported . hip and knee : chiropractic management of osteoarthritis of the hip and knee , for which supportive evidence of efficacy has recently been emerging . muscles : chiropractic testing of muscle function in all parts of the body is 1 application ( applied kinesiology ) , reported by the national board of chiropractic examiners to have been practiced by 37.6% of doctors of chiropractic in 2003 and involving muscles throughout the entire body . nutrition : nutrition and wellness , major components of health maintenance and prevention , often advocated by doctors of chiropractic . in fact , the national board of chiropractic examiners found in 2003 that more than 96% of the chiropractic profession in america engaged in instructing their patients in health and wellness . thus , it is awkward to consider chiropractic interventions to be limited to the spine . the forgoing body of literature shows that treatment limited to the spine should not be considered to be the sole lens through which chiropractic is viewed . applications by doctors of chiropractic to other regions of the body have demonstrated clinical effectiveness and should not be categorically dismissed . identity of chiropractic has been intimately associated with the chiropractic subluxation40 , 41 , a topic of considerable debate within itself . as defined by the association of chiropractic colleges in july 1996 and the subject of texts that have been considered to be definitive , the chiropractic subluxation was regarded to be : a complex of functional and/or structural and/or pathological articular changes that compromise neural integrity and may influence organ system function and general health . a complex of functional and/or structural and/or pathological articular changes that compromise neural integrity and may influence organ system function and general health . historically , definitions of chiropractic subluxation that presented the term in 3 dimensions were beset by a number of challenges44 , 45 , 46:1.in terms of misalignments , it was clear that these could not be detectable by technological methods existing at that time.2.in terms of aberration of movement integrity , either deficient or excessive motion , reliable measurements of motion remained elusive.3.in terms of physiologic dysfunction , these could be present with or without pain and could facilitate one s understanding of chiropractic subluxations . but simply presenting a clinical complaint as a physiological dysfunction was essentially a circular argument , begging the question and demanding further clarification . in terms of misalignments , it was clear that these could not be detectable by technological methods existing at that time . in terms of aberration of movement integrity , either deficient or excessive motion , , these could be present with or without pain and could facilitate one s understanding of chiropractic subluxations . but simply presenting a clinical complaint as a physiological dysfunction was essentially a circular argument , begging the question and demanding further clarification . therefore , the question has remained whether the chiropractic subluxation could be shown as a clinical reality by objective measurements , in addition to what its attributes really were . capturing the proper definition of chiropractic subluxation fortuitously , it is the emergence of research with an eye toward systemic and arguably nonmuscular domains responding to spinal manipulation that could be credited with a new , progressive recasting of the chiropractic definition of chiropractic subluxation appearing in the fourth edition of the clinical practice guidelines ( 2013 ) issued by the council on chiropractic practice which reads:subluxation is a neurological imbalance or distortion in the body associated with adverse physiological responses and/or structural changes , which may become persistent or progressive . the most frequent site for the chiropractic correction of the subluxation is via the vertebral column . subluxation is a neurological imbalance or distortion in the body associated with adverse physiological responses and/or structural changes , which may become persistent or progressive . the most frequent site for the chiropractic correction of the subluxation is via the vertebral column . this groundbreaking concept meant that the term neurological imbalance had now emerged into public view , taking precedent over so many of the hidebound terms that used to be grounded in segmental , articular , or vertebral terms from the 1930s for at least 60 years . to what the change in the definition represents the board s analysis and research into the continued evidence supporting spinal adjustment of dysfunctional vertebra leading to brain metabolic and transient cortical plastic changes in the brain and nervous system . a few more outstanding chapters in research that have paved the way toward this dramatic revision of the chiropractic subluxation s definition are worth citing:1.the finding that spinal manipulation at the c5/c6 spinal segment is regionally related to the infraspinatus but not gluteus medius muscle , suggesting in the authors own words that the primary physiological effect of smt may be neurological rather than changed joint mechanics ( italics mine).2.the dramatic observation by karason and drysdale that an hvla thrust at the lumbosacral junction produced a significant increase in cutaneous blood flow over the l5 dermatome in nonsmokers but not smokers , suggesting the role of a nicotine - sensitive receptor or other trigger.3.the award - winning research of song and his coworkers at parker college , demonstrating a broad spectrum of anti - inflammatory , joint - specific effects of activator treatments in a rat model encompassing behavioral , cytological , and neurophysiological benchmarks.4.the demonstration that a bilateral hypothenar type adjustment accompanied by audible cavitation specifically produced a decrease in the production of the inflammatory cytokines tumor necrosis factor and interleukin-1 in human subjects.5.the revelation that 2 patients with cervicogenic headache after 4 weeks of manipulative therapy posted reductions of tumor necrosis factor exceeding 50%.6.continuing with observations regarding the inflammatory cytokines , the observation that the direction of strain in cultured fibroblasts determined the levels of specific cytokines produced , raising the practical concern that the effects of manipulation may vary in patients depending upon tissue strain directions.7.two of the outstanding colic studies , 1 of only 2 areas of pediatric chiropractic care supported by clinical trials and possibly involving nonmusculoskeletal mechanisms , demonstrating that manipulation rapidly produced significant reductions of this disorder.56 , 578.the finding that cervical spine manipulation not only altered cortical integration of dual somatosensory input but also changed the way the central nervous system responded to a subsequent motor training ( typing ) task . the finding that spinal manipulation at the c5/c6 spinal segment is regionally related to the infraspinatus but not gluteus medius muscle , suggesting in the authors own words that the primary physiological effect of smt may be neurological rather than changed joint mechanics ( italics mine ) . the dramatic observation by karason and drysdale that an hvla thrust at the lumbosacral junction produced a significant increase in cutaneous blood flow over the l5 dermatome in nonsmokers but not smokers , suggesting the role of a nicotine - sensitive receptor or other trigger . the award - winning research of song and his coworkers at parker college , demonstrating a broad spectrum of anti - inflammatory , joint - specific effects of activator treatments in a rat model encompassing behavioral , cytological , and neurophysiological benchmarks . the demonstration that a bilateral hypothenar type adjustment accompanied by audible cavitation specifically produced a decrease in the production of the inflammatory cytokines tumor necrosis factor and interleukin-1 in human subjects . the revelation that 2 patients with cervicogenic headache after 4 weeks of manipulative therapy posted reductions of tumor necrosis factor exceeding 50% . continuing with observations regarding the inflammatory cytokines , the observation that the direction of strain in cultured fibroblasts determined the levels of specific cytokines produced , raising the practical concern that the effects of manipulation may vary in patients depending upon tissue strain directions . two of the outstanding colic studies , 1 of only 2 areas of pediatric chiropractic care supported by clinical trials and possibly involving nonmusculoskeletal mechanisms , demonstrating that manipulation rapidly produced significant reductions of this disorder.56 , 57 the finding that cervical spine manipulation not only altered cortical integration of dual somatosensory input but also changed the way the central nervous system responded to a subsequent motor training ( typing ) task . all these findings defined objective physical attributes that could be attributed to what one referred to as the chiropractic subluxation and how chiropractic interventions attenuated this disorder . these discoveries and the resulting redefinition of the chiropractic subluxation did not exist at the turn of the century in 2000 . casting the chiropractic subluxation in light of these systemic changes brings this discussion back to the original concept of dd palmer , identifying chiropractic s primary target as the nervous system as quoted above . elaborating upon dd palmer s manifesto , the link between the systemic effects ( anti - inflammatory , somatosensory , behavioral , and cytological ) and spinal manipulation is best understood in terms of neurology . an appreciation of this approach begins with a consideration of numerous animal studies as shown in table 1 . these demonstrate that a variety of physiological effects ( a ) are distinct from pain and ( b ) extend far from the area of stimulation . with several of these investigations demonstrating that nerve conductivity is specifically affected,72 , 73 , 74 it can be construed that the nervous system provides an essential link between the experimentally produced aberrations and the physiological changes observed . thus , a wide range of stimuli are capable of producing physiological responses , providing a much broader canvas with which chiropractic subluxations and , for that matter , chiropractic identity can be represented in experimental research , again placing the nervous system at the center . with regard to changes in neural function in response to manipulation , however , a few observations can be brought to light : in their different approaches , all demonstrate inductions of activities within the nervous system that project responses to manipulation well beyond the area of contact.1.abnormal somatosensory evoked potentials from the paraspinal musculature were found to be correlating with decreased pain responses after lumbar manipulation , possibly due to a central effect of sensory processing.2.in a cohort of 12 subjects with a history of recurrent neck stiffness and/or neck pain but no acute symptoms at the time of study , a single session of cervical spine manipulation revealed a significant decrease in the amplitude of 2 components of somatosensory evoked potentials , lasting 20 minutes following the intervention . the implication was that cervical spine manipulation may alter cortical somatosensory processing and sensorimotor integration , shedding light upon the mechanisms for the relief of pain and restoration of functional mobility which are the most widely observed outcomes to treatment by spinal manipulation.3.in subjects subjected to side - posture manipulation , both hoffman reflex and m - wave responses displayed the greatest attenuation with actual manipulation as opposed to a positioning maneuver.4.following sacroiliac joint manipulation , there was a decreased inhibitory effect of knee joint pathology on quadriceps muscle activity , suggesting an interaction between spinal manipulation and the inhibition of voluntary activities produced by pain.5.power spectrum analyses of patient electrocardiograms suggested alterations of sympathetic and parasympathetic activity produced by spinal manipulation.79 , 80 , 816.more recently , in 36 subjects with identifiable myofascial pain syndrome in the infraspinatus and gluteus medius muscles , spinal manipulation at the c5/c6 spinal segment significantly reduced the pressure - pain threshold in the infraspinatus muscle but not the gluteus medius . the implication was that the primary physiological effect of spinal manipulation may be neurophysiological rather than changed joint mechanics , in which spinal manipulation produces inhibitory mechanisms in the myofascial tissues . abnormal somatosensory evoked potentials from the paraspinal musculature were found to be correlating with decreased pain responses after lumbar manipulation , possibly due to a central effect of sensory processing . in a cohort of 12 subjects with a history of recurrent neck stiffness and/or neck pain but no acute symptoms at the time of study , a single session of cervical spine manipulation revealed a significant decrease in the amplitude of 2 components of somatosensory evoked potentials , lasting 20 minutes following the intervention . the implication was that cervical spine manipulation may alter cortical somatosensory processing and sensorimotor integration , shedding light upon the mechanisms for the relief of pain and restoration of functional mobility which are the most widely observed outcomes to treatment by spinal manipulation . in subjects subjected to side - posture manipulation , both hoffman reflex and m - wave responses displayed the greatest attenuation with actual manipulation as opposed to a positioning maneuver . following sacroiliac joint manipulation , there was a decreased inhibitory effect of knee joint pathology on quadriceps muscle activity , suggesting an interaction between spinal manipulation and the inhibition of voluntary activities produced by pain . power spectrum analyses of patient electrocardiograms suggested alterations of sympathetic and parasympathetic activity produced by spinal manipulation.79 , 80 , 81 more recently , in 36 subjects with identifiable myofascial pain syndrome in the infraspinatus and gluteus medius muscles , spinal manipulation at the c5/c6 spinal segment significantly reduced the pressure - pain threshold in the infraspinatus muscle but not the gluteus medius . there was no decrease in either muscle in the sham - treated group . the implication was that the primary physiological effect of spinal manipulation may be neurophysiological rather than changed joint mechanics , in which spinal manipulation produces inhibitory mechanisms in the myofascial tissues . in so doing , it is useful to frame chiropractic in terms of functional neurology , a term in keeping with the more progressive definition of chiropractic subluxation and thus more amenable to interpretation , discussion , and ultimate acceptance by both other health care professions and the public . the cachet and impact of functional neurology can be illustrated by the formation of such professional societies as the international association of functional neurology and rehabilitation and the massive society for neuroscience . i had the pleasure of presenting research at the society for neuroscience convention in new orleans in 2012 and found my discussions and presentation on functional neurology widely and warmly received . these are both organizations which have much to offer chiropractic and vice versa and which i believe would both solidify and promote evolving concepts of chiropractic identity . these examples lead to a vindication of dd palmer s original concept of the importance of tone , an affirmation of the significance of the nervous system as a core element of what chiropractic has attempted to address . neither the disorder nor the intervention is necessarily confined to a local site , but rather the consequences of the disorder and the therapeutic applications involve systemic effects that could be triggered by the nervous system . a final aspect of establishing an identity of chiropractic addresses the question of whether this discipline needs to be considered a mainstream or alternative branch of health care delivery . assumptions have been that mainstream status confers battle - tested , scientific validity as opposed to an alternative , the latter sometimes categorized as keeping bad company with , for example , purveyors of laetrile . at the same time , mainstream status may confine chiropractic to a low back or neck role by downplaying interventions outside of these regions . in addition , this recognition ignores the fact that , 35 years ago , little research existed to support what would now be considered mainstream status for chiropractic care for low back pain . in other words , those concluding that chiropractic should be considered only as a mainstream venue would be attaching such strings as back specialists or musculoskeletal pain relievers to this classification , overlooking the research and interventions that suggest the potential for chiropractic to transcend these boundaries . answers to address if chiropractic is mainstream or alternative are best generated by first examining what are varying concepts of alternative medicine . the german nicht - schulmedizine model considers alternative medicine as all health care practices not taught at university medical facilities or medical schools . more facetiously , health policy fanatics sometimes refer to alternative medicine as those health care practices that are not paid for by insurance and which a patient does not tell his or her primary doctor . this takes after the findings of david eisenberg which represent the emergence of alternative medicine into national consciousness . but perhaps the most telling is an opinion voiced by what was the forerunner of the national center for complementary and integrative health ; that is , alternative medicine represents any health care practice that is not politically dominant at the time . the debate over alternative medicine was best represented by the opposing viewpoints of andrew weil:patients in unprecedented numbers are going outside of conventional medicine to look for help . why are people doing this ? clearly , there is dissatisfaction with conventional medicine . the therapies that we aim for are those that are reasonable , that are supported by what evidence is now available , that above all are not harmful , and that work from the premise that the body can heal itself , if you give it a chance.and arnold relman:most alternative systems of treatment are based on irrational or fanciful thinking it [ alternative medicine ] could not be woven in to the fabric of the medical curriculum without confusion , contradiction , and an undermining of the scientific foundation upon which modern medicine rests . patients in unprecedented numbers are going outside of conventional medicine to look for help . the therapies that we aim for are those that are reasonable , that are supported by what evidence is now available , that above all are not harmful , and that work from the premise that the body can heal itself , if you give it a chance . most alternative systems of treatment are based on irrational or fanciful thinking it [ alternative medicine ] could not be woven in to the fabric of the medical curriculum without confusion , contradiction , and an undermining of the scientific foundation upon which modern medicine rests . however , the authoritative health and medicine division of the national academies of sciences , engineering , and medicine ( formerly known as the institute of medicine ) has concluded in an exhaustive review that revered medical evidence is lacking and in need of substantial improvement . in fact , in 1 study comparing the extent to which different medical specialties were evidence based , chiropractic practice was found to have the highest proportion of care ( 68.3% ) supported by good quality , experimental evidence and was superior to several medical subspecialties . add to this a recent study of 2500 medical treatments in which only 15% were identified as being beneficial , 22% likely to be beneficial , and 47% unknown , and the question arises whether the mainstream medical foundation is as commanding as it has commonly been made out to be . in short , despite its clear prominence in terms of public perception and third - party payers , mainstream status is not without its flaws . chiropractic , in its rush to achieve singular distinction and to seize mainstream ground in which most of the training , research , and public awareness have taken place ( centering around the spine and musculoskeletal care ) , may have compromised its future . the profession runs the risk of mortgaging many of its traditional and hard - won attributes . these would include the very foundations of chiropractic , such as ( a ) the ability to diagnose ; ( b ) patient - centered care ; ( c ) primary wellness care in offering an alternative to the current medical paradigm ; and ( d ) numerous research findings already published representing the full range of basic research , randomized clinical trials , and case series all of which point toward a scope of practice and model of patient care which extend well beyond the specialist role of simply managing back and neck pain . in looking ahead and allowing room to expand into those areas in which there is merit , some of chiropractic s leading researchers have declared that there is reason to devote future research efforts beyond the spine and spinal conditions ( ie , low back and neck pain ) . for these reasons , alternative medicine offers the proper niche into which chiropractic may continue to evolve and express its full potential . yet there is no denying that , for managing low back and neck pain as well as headache , chiropractic has already achieved there are more 100 published clinical trials in scientific journals to support this claim , as well as the public perception that doctors of chiropractic do , in fact , know more than a little about the spine and spinal health.72 , 73 , 91 , 92 , 93 , 94 , 95 for these reasons , chiropractic needs to be considered a hybrid of both alternative and mainstream medicine : mainstream , based upon what it has attained , and alternative , in areas in which its documented growth has occurred and must continue to occur . like alternative medicine , multiple definitions have been provided for primary care that are worth reviewing:1.starfield : first - contact , longitudinal , coordinated , and comprehensive care ( ie , practitioner should take care of the majority of problems without referral).2.public health service act : services which require family medicine , internal medicine , pediatrics , obstetrics / gynecology , dentistry , or mental health as provided by physicians or other health care professionals.3.institute of medicine : emphasis upon accessibility , comprehensiveness , continuity , and coordination . starfield : first - contact , longitudinal , coordinated , and comprehensive care ( ie , practitioner should take care of the majority of problems without referral ) . public health service act : services which require family medicine , internal medicine , pediatrics , obstetrics / gynecology , dentistry , or mental health as provided by physicians or other health care professionals . primary care is a way of delivering health care , not a body of knowledge as such . a comprehensive community study by abt associates that convened 2 panels of medical experts ( 1 consisting solely of doctors of chiropractic ) concluded that , with respect to a list of 53 primary functions found to occur daily in medical offices , doctors of chiropractic were capable of making diagnoses in 92% of those activities and capable of making therapeutic contributions in more than 50% of them . on the other hand , in a competency examination comparing performances of chiropractic and medical students , chiropractic students scored higher than their medical counterparts only on the musculoskeletal portion of the examination and in several other areas scored substantially lower . and access to tertiary care facilities , such as hospitals , although growing for doctors of chiropractic , remains very limited . at the same time , health care delivery as well as competency in musculoskeletal areas by medical doctors has been shown in multiple settings to be inadequate.12 , 25 , 101 these findings would seem to position doctors of chiropractic in the united states on a spectrum somewhere between primary care providers and specialists upon referral . at the very least , first contact for a minimum of musculoskeletal conditions would be indicated for doctors of chiropractic , buttressed by such findings by sarnat et al102 , 103 that demonstrated major reductions of hospital days , outpatient surgical procedures , and health care costs when doctors of chiropractic were given first - contact privileges to patients within an independent physicians association . beyond managing only musculoskeletal conditions , doctors of chiropractic in 4 medical underserved communities ( rural and urban underserved ) tended to deliver primary care services . the same pattern was found to be true in rural communities of ontario , with doctors of chiropractic delivering primary health care , bridged care , and interim care . it would appear that the scope of chiropractic extends beyond specialist to a first - contact provider . this scope would certainly be for musculoskeletal conditions with the potential to extend into primary care should adequate research be able to justify such an expansion . indeed , given the shortages of primary care providers in some communities and the cost - effectiveness and satisfaction with doctors of chiropractic serving in a de facto primary care capacity , it would therefore seem appropriate to consider doctors of chiropractic as having successfully vacated the specialist s role . primary care , first - contact , or specialists upon referral ? debates on these topics may still linger but hopefully have been nourished by the topics discussed in this article . nevertheless , it is still possible to attach a number of distinguishing characteristics of doctors of chiropractic ( fig 1 ) . although there are numerous areas that present diverging viewpoints of chiropractic , at the same time , there are unambiguous , distinguishing characteristics of chiropractic . even with the recognition of the benefits of spinal manipulation in various government guidelines,107 , 108 , 109 , 110 , 111 the rise of the alternative health care movement , advances in the us military and veterans administration , and persistently high levels of patient satisfaction , the chiropractic profession remains underrepresented in most discussions of health care delivery . hopefully , a clarification of chiropractic s identity and a greater unity of practitioners in delivering this message will help to rectify this problem . this is a narrative review by a single author of multiple points of view of chiropractic , taking into consideration its relationships to other health care professions in manual therapy as well as its positioning in the 2 spectra of specialist / primary care and alternative / mainstream medicine . the main purpose of this assessment was to clarify , and in some instances refute , different viewpoints of chiropractic while providing several unambiguous characteristics to more clearly define chiropractic . this discussion provides a roadmap for understanding the struggle for identity that has beset chiropractic since its inception . it has provided 6 perspectives : ( 1 ) concepts of manual medicine ; ( 2 ) areas of interest beyond the spine ; ( 3 ) concepts of neurology ; ( 4 ) concepts of the chiropractic subluxation ; ( 5 ) mainstream or alternative health care ; and ( 6 ) primary care , first - contact provider , or specialist . numerous distinguishing characteristics , including providing a model of holistic , preventive medicine and embracing a concept of neurological imbalance , have prevailed . at present , the chiropractic profession continues to be underrepresented in most discussions of health care delivery , a situation in which the greater clarification of chiropractic s identity and more practitioner consensus may help to alleviate this problem . concept development ( provided idea for the research ) : a.l.r.design ( planned the methods to generate the results ) : a.l.r.supervision ( provided oversight , responsible for organization and implementation , writing of the manuscript ) : a.l.r.data collection / processing ( responsible for experiments , patient management , organization , or reporting data ) : a.l.r.analysis/interpretation ( responsible for statistical analysis , evaluation , and presentation of the results ) : a.l.r.literature search ( performed the literature search ) : a.l.r.writing ( responsible for writing a substantive part of the manuscript ) : a.l.r.critical review ( revised manuscript for intellectual content ; this does not relate to spelling and grammar checking ) : a.l.r . supervision ( provided oversight , responsible for organization and implementation , writing of the manuscript ) : a.l.r . data collection / processing ( responsible for experiments , patient management , organization , or reporting data ) : a.l.r . analysis / interpretation ( responsible for statistical analysis , evaluation , and presentation of the results ) : a.l.r . critical review ( revised manuscript for intellectual content ; this does not relate to spelling and grammar checking ) : a.l.r . practical applicationschiropractic s distinguishing characteristics may provide a useful framework to achieve a greater consensus in resolving the lack of unified identity.the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function . chiropractic s distinguishing characteristics may provide a useful framework to achieve a greater consensus in resolving the lack of unified identity.the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function . chiropractic s distinguishing characteristics may provide a useful framework to achieve a greater consensus in resolving the lack of unified identity . the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function . chiropractic s distinguishing characteristics may provide a useful framework to achieve a greater consensus in resolving the lack of unified identity.the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function . chiropractic s distinguishing characteristics may provide a useful framework to achieve a greater consensus in resolving the lack of unified identity . the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function .

objectivethe purpose of this article is to propose a focused assessment of the identity of chiropractic and its profession , triangulating multiple viewpoints converging upon various aspects and definitions of neurology , manual medicine , and alternative or mainstream medicine.discussionover 120 years since its inception , chiropractic has struggled to achieve an identity for which its foundations could provide optimal health care . despite recognition of the benefits of spinal manipulation in various government guidelines , advances in us military and veterans administration , and persistently high levels of patient satisfaction , the chiropractic profession remains underrepresented in most discussions of health care delivery . distinguishing characteristics of doctors of chiropractic include the following : ( 1 ) they embrace a model of holistic , preventive medicine ( wellness ) ; ( 2 ) they embrace a concept of neurological imbalance in which form follows function , disease follows disturbed biochemistry , and phenomenology follows physiology ; ( 3 ) they diagnose , and their institutions of training are accredited by a body recognized by the us department of education ; ( 4 ) they manage patients on a first - contact basis , often as primary care providers in geographical areas that are underserved ; ( 5 ) the spine is their primary but not exclusive area of interaction ; ( 6 ) they deliver high - velocity , low - amplitude adjustments with a superior safety record compared with other professions ; and ( 7 ) they use a network of institutions worldwide that have shown increasing commitments to research.conclusionthis article provides an overview of chiropractic identity from 6 points of view : ( 1 ) concepts of manual medicine ; ( 2 ) areas of interest beyond the spine ; ( 3 ) concepts of the chiropractic subluxation ; ( 4 ) concepts of neurology ; ( 5 ) concepts of mainstream or alternative health care ; and ( 6 ) concepts of primary care , first - contact provider , or specialist .

Introduction Concepts of Manual Medicine Areas of Interest Beyond the Spine Concepts of Chiropractic Subluxation Concepts of Neurology Mainstream or Alternative Primary Care, First-Contact Provider, or Specialist Discussion Limitations Conclusion Funding sources and potential conflicts of interest Contributorship information Practical Applications

by a wide margin , the survey and various studies suggested that doctors of chiropractic were managing primarily musculoskeletal problems with emphasis upon back pain.2 , 3 , 4 , 5 taking these findings to the wfc congress in sydney , australia , in june of 2005 , the identity consultation task force concluded the following : ( 1 ) it is important for the profession to have an identity ; ( 2 ) most agree that the chiropractic profession suffers from an unclear identity and position within today s health care plans ; and ( 3 ) it is important to understand how doctors of chiropractic think that the profession should be viewed and how they believe that it is actually viewed . here one finds substantial discrepancies : ( 1 ) whether the profession offers primary or specialist health care ; ( 2 ) whether the profession is mainstream ( ie , core to the health care delivery system ) or if it is an alternative ; and ( 3 ) whether the profession offers wellness and nonsurgical , nondrug health care or simply manages back , neck , and spinal problems . the ground statements emphasized ( a ) a patient - centered approach to health care ; ( b ) wellness ; ( c ) the self - healing powers of the individual ; ( d ) avoidance of the use of drugs and surgery wherever possible ; ( e ) examination , diagnosis , and treatment based upon available research ; and perhaps the most critical element of all , ( f ) the relationship between the spine and the nervous system . as joseph brimhall , president of the council of chiropractic education and director of the council of chiropractic education international , explained , there was no wording in the accreditation standards of the council of chiropractic education ( us ) , the model standards of council of chiropractic education international , or other jurisdictions that restricted the chiropractic profession to the spine . therefore , the purpose of this article is to address this dilemma through discussion of the following 6 topics : ( 1 ) concepts of manual medicine ; ( 2 ) areas of interest beyond the spine ; ( 3 ) concepts of neurology ; ( 4 ) concepts of the chiropractic subluxation ; ( 5 ) mainstream or alternative ; and ( 6 ) primary care , first - contact provider , or specialist . although chiropractic has attempted to define its niche in spinal manipulation using short - lever , high - velocity , low - amplitude ( hvla ) techniques , those approaches are not fully circumscribed by doctors of chiropractic alone . from such an expanded catalog , it is apparent that a vast array of low - velocity , soft - tissue , and even instrumental techniques are included , which speak to an extensive overlap with other branches of manual medicine . however , the literature shows an inferior safety record with the other professions and laypeople having had lesser training and/or displayed less competence in musculoskeletal medicine.23 , 24 , 25 the superior safety record of doctors of chiropractic compared with other manual therapists clearly stands out as a distinguishing characteristic of chiropractic . nevertheless , alternatives in addition to the spine in which the usefulness of chiropractic care has been documented include the following:1.extremities : the successful outcomes regarding chiropractic treatments of the extremities , supported by a body of literature which suggests effective outcomes in managing repetitive motion disorders.29 , 30 , 31 , 322.neck muscles : published reports of chiropractic effleurage of the sternocleidomastoid muscles , proposed to be an effective part of the chiropractic management of otitis media.3.skull : the successful applications of craniosacral therapy , reported by the national board of chiropractic examiners to have been practiced by 38.0% of doctors of chiropractic in 2003 and focusing upon the skull as well as the vertebral canal.4.neck muscles : the application of myofascial release , also known as ischemic compression , directed in at least 1 study to the active upper trapezius trigger points with a successful outcome.5.temporomandibular joint : chiropractic treatment of temporomandibular disorders , necessarily involving applications to areas of the jaw and sides of the neck , for which a fair level of supporting evidence of effectiveness has been reported.6.hip and knee : chiropractic management of osteoarthritis of the hip and knee , for which supportive evidence of efficacy has recently been emerging.7.muscles : chiropractic testing of muscle function in all parts of the body is 1 application ( applied kinesiology ) , reported by the national board of chiropractic examiners to have been practiced by 37.6% of doctors of chiropractic in 2003 and involving muscles throughout the entire body.8.nutrition : nutrition and wellness , major components of health maintenance and prevention , often advocated by doctors of chiropractic . capturing the proper definition of chiropractic subluxation fortuitously , it is the emergence of research with an eye toward systemic and arguably nonmuscular domains responding to spinal manipulation that could be credited with a new , progressive recasting of the chiropractic definition of chiropractic subluxation appearing in the fourth edition of the clinical practice guidelines ( 2013 ) issued by the council on chiropractic practice which reads:subluxation is a neurological imbalance or distortion in the body associated with adverse physiological responses and/or structural changes , which may become persistent or progressive . a few more outstanding chapters in research that have paved the way toward this dramatic revision of the chiropractic subluxation s definition are worth citing:1.the finding that spinal manipulation at the c5/c6 spinal segment is regionally related to the infraspinatus but not gluteus medius muscle , suggesting in the authors own words that the primary physiological effect of smt may be neurological rather than changed joint mechanics ( italics mine).2.the dramatic observation by karason and drysdale that an hvla thrust at the lumbosacral junction produced a significant increase in cutaneous blood flow over the l5 dermatome in nonsmokers but not smokers , suggesting the role of a nicotine - sensitive receptor or other trigger.3.the award - winning research of song and his coworkers at parker college , demonstrating a broad spectrum of anti - inflammatory , joint - specific effects of activator treatments in a rat model encompassing behavioral , cytological , and neurophysiological benchmarks.4.the demonstration that a bilateral hypothenar type adjustment accompanied by audible cavitation specifically produced a decrease in the production of the inflammatory cytokines tumor necrosis factor and interleukin-1 in human subjects.5.the revelation that 2 patients with cervicogenic headache after 4 weeks of manipulative therapy posted reductions of tumor necrosis factor exceeding 50%.6.continuing with observations regarding the inflammatory cytokines , the observation that the direction of strain in cultured fibroblasts determined the levels of specific cytokines produced , raising the practical concern that the effects of manipulation may vary in patients depending upon tissue strain directions.7.two of the outstanding colic studies , 1 of only 2 areas of pediatric chiropractic care supported by clinical trials and possibly involving nonmusculoskeletal mechanisms , demonstrating that manipulation rapidly produced significant reductions of this disorder.56 , 578.the finding that cervical spine manipulation not only altered cortical integration of dual somatosensory input but also changed the way the central nervous system responded to a subsequent motor training ( typing ) task . a final aspect of establishing an identity of chiropractic addresses the question of whether this discipline needs to be considered a mainstream or alternative branch of health care delivery . these would include the very foundations of chiropractic , such as ( a ) the ability to diagnose ; ( b ) patient - centered care ; ( c ) primary wellness care in offering an alternative to the current medical paradigm ; and ( d ) numerous research findings already published representing the full range of basic research , randomized clinical trials , and case series all of which point toward a scope of practice and model of patient care which extend well beyond the specialist role of simply managing back and neck pain . yet there is no denying that , for managing low back and neck pain as well as headache , chiropractic has already achieved there are more 100 published clinical trials in scientific journals to support this claim , as well as the public perception that doctors of chiropractic do , in fact , know more than a little about the spine and spinal health.72 , 73 , 91 , 92 , 93 , 94 , 95 for these reasons , chiropractic needs to be considered a hybrid of both alternative and mainstream medicine : mainstream , based upon what it has attained , and alternative , in areas in which its documented growth has occurred and must continue to occur . like alternative medicine , multiple definitions have been provided for primary care that are worth reviewing:1.starfield : first - contact , longitudinal , coordinated , and comprehensive care ( ie , practitioner should take care of the majority of problems without referral).2.public health service act : services which require family medicine , internal medicine , pediatrics , obstetrics / gynecology , dentistry , or mental health as provided by physicians or other health care professionals.3.institute of medicine : emphasis upon accessibility , comprehensiveness , continuity , and coordination . at the same time , health care delivery as well as competency in musculoskeletal areas by medical doctors has been shown in multiple settings to be inadequate.12 , 25 , 101 these findings would seem to position doctors of chiropractic in the united states on a spectrum somewhere between primary care providers and specialists upon referral . at the very least , first contact for a minimum of musculoskeletal conditions would be indicated for doctors of chiropractic , buttressed by such findings by sarnat et al102 , 103 that demonstrated major reductions of hospital days , outpatient surgical procedures , and health care costs when doctors of chiropractic were given first - contact privileges to patients within an independent physicians association . although chiropractic has attempted to define its niche in spinal manipulation using short - lever , high - velocity , low - amplitude ( hvla ) techniques , those approaches are not fully circumscribed by doctors of chiropractic alone . from such an expanded catalog , it is apparent that a vast array of low - velocity , soft - tissue , and even instrumental techniques are included , which speak to an extensive overlap with other branches of manual medicine . however , the literature shows an inferior safety record with the other professions and laypeople having had lesser training and/or displayed less competence in musculoskeletal medicine.23 , 24 , 25 the superior safety record of doctors of chiropractic compared with other manual therapists clearly stands out as a distinguishing characteristic of chiropractic . nevertheless , alternatives in addition to the spine in which the usefulness of chiropractic care has been documented include the following:1.extremities : the successful outcomes regarding chiropractic treatments of the extremities , supported by a body of literature which suggests effective outcomes in managing repetitive motion disorders.29 , 30 , 31 , 322.neck muscles : published reports of chiropractic effleurage of the sternocleidomastoid muscles , proposed to be an effective part of the chiropractic management of otitis media.3.skull : the successful applications of craniosacral therapy , reported by the national board of chiropractic examiners to have been practiced by 38.0% of doctors of chiropractic in 2003 and focusing upon the skull as well as the vertebral canal.4.neck muscles : the application of myofascial release , also known as ischemic compression , directed in at least 1 study to the active upper trapezius trigger points with a successful outcome.5.temporomandibular joint : chiropractic treatment of temporomandibular disorders , necessarily involving applications to areas of the jaw and sides of the neck , for which a fair level of supporting evidence of effectiveness has been reported.6.hip and knee : chiropractic management of osteoarthritis of the hip and knee , for which supportive evidence of efficacy has recently been emerging.7.muscles : chiropractic testing of muscle function in all parts of the body is 1 application ( applied kinesiology ) , reported by the national board of chiropractic examiners to have been practiced by 37.6% of doctors of chiropractic in 2003 and involving muscles throughout the entire body.8.nutrition : nutrition and wellness , major components of health maintenance and prevention , often advocated by doctors of chiropractic . capturing the proper definition of chiropractic subluxation fortuitously , it is the emergence of research with an eye toward systemic and arguably nonmuscular domains responding to spinal manipulation that could be credited with a new , progressive recasting of the chiropractic definition of chiropractic subluxation appearing in the fourth edition of the clinical practice guidelines ( 2013 ) issued by the council on chiropractic practice which reads:subluxation is a neurological imbalance or distortion in the body associated with adverse physiological responses and/or structural changes , which may become persistent or progressive . a few more outstanding chapters in research that have paved the way toward this dramatic revision of the chiropractic subluxation s definition are worth citing:1.the finding that spinal manipulation at the c5/c6 spinal segment is regionally related to the infraspinatus but not gluteus medius muscle , suggesting in the authors own words that the primary physiological effect of smt may be neurological rather than changed joint mechanics ( italics mine).2.the dramatic observation by karason and drysdale that an hvla thrust at the lumbosacral junction produced a significant increase in cutaneous blood flow over the l5 dermatome in nonsmokers but not smokers , suggesting the role of a nicotine - sensitive receptor or other trigger.3.the award - winning research of song and his coworkers at parker college , demonstrating a broad spectrum of anti - inflammatory , joint - specific effects of activator treatments in a rat model encompassing behavioral , cytological , and neurophysiological benchmarks.4.the demonstration that a bilateral hypothenar type adjustment accompanied by audible cavitation specifically produced a decrease in the production of the inflammatory cytokines tumor necrosis factor and interleukin-1 in human subjects.5.the revelation that 2 patients with cervicogenic headache after 4 weeks of manipulative therapy posted reductions of tumor necrosis factor exceeding 50%.6.continuing with observations regarding the inflammatory cytokines , the observation that the direction of strain in cultured fibroblasts determined the levels of specific cytokines produced , raising the practical concern that the effects of manipulation may vary in patients depending upon tissue strain directions.7.two of the outstanding colic studies , 1 of only 2 areas of pediatric chiropractic care supported by clinical trials and possibly involving nonmusculoskeletal mechanisms , demonstrating that manipulation rapidly produced significant reductions of this disorder.56 , 578.the finding that cervical spine manipulation not only altered cortical integration of dual somatosensory input but also changed the way the central nervous system responded to a subsequent motor training ( typing ) task . a final aspect of establishing an identity of chiropractic addresses the question of whether this discipline needs to be considered a mainstream or alternative branch of health care delivery . these would include the very foundations of chiropractic , such as ( a ) the ability to diagnose ; ( b ) patient - centered care ; ( c ) primary wellness care in offering an alternative to the current medical paradigm ; and ( d ) numerous research findings already published representing the full range of basic research , randomized clinical trials , and case series all of which point toward a scope of practice and model of patient care which extend well beyond the specialist role of simply managing back and neck pain . yet there is no denying that , for managing low back and neck pain as well as headache , chiropractic has already achieved there are more 100 published clinical trials in scientific journals to support this claim , as well as the public perception that doctors of chiropractic do , in fact , know more than a little about the spine and spinal health.72 , 73 , 91 , 92 , 93 , 94 , 95 for these reasons , chiropractic needs to be considered a hybrid of both alternative and mainstream medicine : mainstream , based upon what it has attained , and alternative , in areas in which its documented growth has occurred and must continue to occur . like alternative medicine , multiple definitions have been provided for primary care that are worth reviewing:1.starfield : first - contact , longitudinal , coordinated , and comprehensive care ( ie , practitioner should take care of the majority of problems without referral).2.public health service act : services which require family medicine , internal medicine , pediatrics , obstetrics / gynecology , dentistry , or mental health as provided by physicians or other health care professionals.3.institute of medicine : emphasis upon accessibility , comprehensiveness , continuity , and coordination . at the same time , health care delivery as well as competency in musculoskeletal areas by medical doctors has been shown in multiple settings to be inadequate.12 , 25 , 101 these findings would seem to position doctors of chiropractic in the united states on a spectrum somewhere between primary care providers and specialists upon referral . at the very least , first contact for a minimum of musculoskeletal conditions would be indicated for doctors of chiropractic , buttressed by such findings by sarnat et al102 , 103 that demonstrated major reductions of hospital days , outpatient surgical procedures , and health care costs when doctors of chiropractic were given first - contact privileges to patients within an independent physicians association . even with the recognition of the benefits of spinal manipulation in various government guidelines,107 , 108 , 109 , 110 , 111 the rise of the alternative health care movement , advances in the us military and veterans administration , and persistently high levels of patient satisfaction , the chiropractic profession remains underrepresented in most discussions of health care delivery . this is a narrative review by a single author of multiple points of view of chiropractic , taking into consideration its relationships to other health care professions in manual therapy as well as its positioning in the 2 spectra of specialist / primary care and alternative / mainstream medicine . it has provided 6 perspectives : ( 1 ) concepts of manual medicine ; ( 2 ) areas of interest beyond the spine ; ( 3 ) concepts of neurology ; ( 4 ) concepts of the chiropractic subluxation ; ( 5 ) mainstream or alternative health care ; and ( 6 ) primary care , first - contact provider , or specialist . numerous distinguishing characteristics , including providing a model of holistic , preventive medicine and embracing a concept of neurological imbalance , have prevailed . at present , the chiropractic profession continues to be underrepresented in most discussions of health care delivery , a situation in which the greater clarification of chiropractic s identity and more practitioner consensus may help to alleviate this problem . practical applicationschiropractic s distinguishing characteristics may provide a useful framework to achieve a greater consensus in resolving the lack of unified identity.the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function . chiropractic s distinguishing characteristics may provide a useful framework to achieve a greater consensus in resolving the lack of unified identity.the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function . the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function . chiropractic s distinguishing characteristics may provide a useful framework to achieve a greater consensus in resolving the lack of unified identity.the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function . the strongest assets of chiropractic include that it embraces a model of holistic , preventive medicine by embracing a model of neurological imbalance in which form follows function .

however , the role played by each of several acceleration and loss mechanisms is not yet established observationally [ millan and thorne , 2007 ] . precipitation into the atmosphere is considered to be one of the major electron loss mechanisms , which can completely deplete the radiation belts of electrons during the main phase of some geomagnetic storms [ o'brien et al . , 2004 ; selesnick , 2006 ] . using data from balloon - borne x - ray detectors , foat et al . , lorentzen et al . , and millan et al . have reported precipitating relativistic electrons in the dusk sector . these duskside precipitation events occur over a variety of magnetic activity levels [ lorentzen et al . , 2000 ; kokorowski et al . , 2008 ] and a broad radial distribution ranging from l=38 [ millan et al . , 2013 ] . the energy spectrum has been found to be well fit by an exponential distribution with an e - folding energy ranging from 0.5 to 3.6 mev [ millan et al . , 2002 ] . based primarily on the fact that all these events were found at dusk , the cause has been suggested to be the gyroresonant scattering by electromagnetic ion cyclotron ( emic ) waves . emic waves are observed throughout the inner magnetosphere but predominantly on the duskside and dayside [ anderson et al . , 1992a , 1992b ; meredith , 2003 ; erlandson and ukhorskiy , 2001 ; fraser et al . , 1996 , 2006 ; usanova et al . , 2012 ] . they are excited by anisotropic ring current ions injected into the inner magnetosphere [ e.g. , jordanova et al . , 2008 ] or by compressions of the magnetopause [ e.g. , anderson and hamilton , 1993 ] . the linear growth rate of emic waves maximizes in high - density regions such as the duskside plasmapause or plasmaspheric drainage plume due to reduced resonant energies [ cornwall et al . , 1970 ; horne and thorne , 1993 ] and wave guiding by steep density gradients near the plasmapause [ horne and thorne , 1993 ; jordanova et al . field - aligned emic waves interact with relativistic electrons through the gyroresonance condition 1where e is electron gyrofrequency , is the relativistic factor , k|| and v|| are components of the wave propagation vector particle velocity along the direction of the ambient magnetic field . emic waves are expected to effectively scatter relativistic electrons of geophysically interesting energies , preferably in duskside high - density regions , where the minimum energy limit is relatively low and the diffusion rate is close to the strong diffusion limit [ thorne and kennel , 1971 ; albert , 2003 ; summers , 2003 ] . duskside simultaneous proton and relativistic electron precipitation has also been observed , supporting the theory of precipitation caused by emic wave scattering [ e.g. , bortnik et al . , quasi - linear theory [ kennel and petschek , 1966 ] has been the dominant treatment ; however , recent work shows that nonlinear effects can be very significant and even reverse the conclusions [ albert and bortnik , 2009 ] . therefore , it is important to establish when and where each approach is applicable and to test these models with observations . existing theoretical discussions on emic waves as a possible loss mechanism mainly focus on the evolution of the trapped electron flux and its timescale . however , directly measured by balloon detectors is the energy spectrum of the bremsstrahlung x - rays produced by the precipitating electrons , and many satellites also measure the energy of the precipitating electrons [ millan et al . , 2007 ] . it is very important to understand the energy dependence of the precipitating flux driven by emic waves , as well as the time evolution of this dependence in order to test the theory with observations . this paper uses the quasi - linear formulation to evaluate the energy and time dependence of rep , and investigates how different parameters affect the diffusion coefficients and the energy spectrum . when applied with input wave and particle data from satellites ( e.g. , van allen probes , goes ) , the results from our model can be directly compared with balloon ( e.g. , balloon array for rbsp ( radiation belt storm probes ) relativistic electron losses ) and low - altitude satellite ( e.g. , solar anomalous and magnetospheric particle explorer ( sampex ) and polar - orbiting operational environmental satellites ( poes ) ) measurements to investigate the role of emic waves in causing rep as well as the effectiveness of the adopted theoretical model . we use quasi - linear diffusion theory to model the evolution of the distribution of electrons due to interactions with emic waves . radial and energy diffusion are ignorable because the frequency of the emic waves is well above the drift frequency and well below the gyrofrequency of the resonant particles [ kennel , 1966 ] . the bounce - averaged diffusion equation for pure pitch angle scattering can be written as [ davidson and walt , 1977 ; lyons , 1973 ; lyons and williams , 1984 ; shao et al . , 2009 ] 2where t(0)=1.38020.3198(sin(0)+ sin1/2(0 ) ) is the normalized bounce time , f0 is the trapped electron phase space density , 0 is the equatorial pitch angle , d(0,e) is the bounce - averaged pitch angle diffusion coefficient , and atm is the timescale for losses to the atmosphere . assuming that losses occur only from within the loss cone and that the loss cone is emptied twice per bounce period , we take atm to be half of the bounce period inside the loss cone and infinite outside the loss cone [ lyons , 1973 ; davidson and walt , 1977 ; lyons and williams , 1984 ; shao et al . , the differential flux is related to the phase space density through the electron momentum p as j0=pf0 . to capture the feature of the isotropic flux distribution in the loss cone in the many strong diffusion cases in our parameter studies , we set the boundary conditions to be . for weak diffusion cases , although does not have to be zero , the loss cone is essentially empty and a small number of electrons in the loss cone does not significantly affect the diffusion [ shprits et al . , , 2009 ] 3with an arbitrary scaling outside the loss cone and j0(t=0)=0 inside the loss cone . here e is the particle energy in mev and 0l is the equatorial loss cone angle given by sin2(0l)=(4l3l ) at a particular l shell . we calculate the bounce - averaged diffusion coefficient using the same method as summers and summers et al . for parallel - propagating emic waves in a multi - ion ( h , he , and o ) plasma . the diffusion coefficient is corrected with a factor of 2 [ albert , 2007 ] . the earth 's magnetic field is assumed dipolar , the wave amplitude is taken to be 1 nt , and the wave frequency spectrum is assumed to be a truncated gaussian , namely , 4where m is the center frequency , with lower frequency limit 1=m and upper frequency limit 2=m+. is a measure of the bandwidth . the emic waves are assumed to be confined to 15% in latitude and 10% of the electron drift orbit , propagating in a cold plasma of a storm time ion composition 70% h , 20% he , and 10% o [ meredith , 2003 ] . the wave dispersion relation is 5where k is the wave number k1<k < k2 . the suffix j denotes the ion species ; the values j=1,2 , and 3 refer to h , he , and o , respectively . the minimum resonant energy can be approximated as [ summers , 2003 ] 6 figure 1 shows the bounce - averaged diffusion coefficient as a function of equatorial pitch angles we calculated for 0.15 mev electrons with four sets of equatorial background magnetic field strength b0 , cold plasma density n0 , and wave frequency . each diffusion coefficient spans from 0 to an upper resonant pitch angle limit 0u and maximizes to a value d(0,e)m at a pitch angle 0 m . bounce - averaged pitch angle diffusion coefficients for emic waves interacting with electrons of energies 0.15 mev with an increment of 0.2 mev and four different sets of wave and plasma parameters . we next investigate how the diffusion coefficient varies with energy and pitch angles for a full range of parameters b0 , n0 , and . the varying diffusion coefficients will determine how the energy spectrum evolves ( section 4 ) . we choose b0 , n0 , and to vary from 75 to 1150 nt ( corresponding to l38 ) , 1 to 1000 cm ( assumed to be constant over the entire interaction region ) , and 1.9 to 3.9 o+ ( covering roughly the entire helium band , where the spectral intensity of emic waves is enhanced [ thorne et al . , 2006 ; anderson et al . , 1992a ; hu et al . , 2010 ] and pitch angle diffusion of geophysically interesting relativistic electrons is more likely to occur [ li et al . , 2007 ] ) , respectively . since emic waves are often observed near the geosynchronous orbit where n0100 cm [ halford et al . , 2010 ] , we set the control b0 and n0 to be 100 nt and 100 cm , respectively . we also choose the control wave frequency to be =2.43.4 o+ , estimated from wave observations [ meredith , 2003 ; ukhorskiy et al . , 2010 ] . in figure 2 ( first to fourth rows ) ( the fifth row will be discussed in section 4 ) , we plot d(00,e),d(0,e)m , 0 m , and 0u as a function of e to characterize the diffusion coefficient when one parameter is varied while the others are held fixed . satisfying the gyroresonance condition equation 1 , the dispersion relation equation 5 , and the gaussian wave frequency distribution equation 4 , the plots show that for all b0 , n0 , 1 , and 2 , the pitch angles 0u and 0 m increase monotonically with energy , whereas d(00,e) and d(0,e)m increase from the minimum resonant energy up to a certain peak energy and then decrease . the value of the peak energy decreases with decreasing b0 or increasing n0 , 1 , or 2 . the width of the peak significantly narrows with increasing 1 and 2 , meaning that at high frequencies , the diffusion coefficient sharply increases at the peak energy from surrounding energies . keeping energy fixed , as b0 decreases or n0 , 1 , or 2 increases , 0 m increases monotonically ; the diffusion coefficient increases at low energies yet decreases at high energies ( variations at > 5 mev are not shown in the figure ) ; d(0,e)m increases at low energies and decreases at high energies only with increasing 2 while increases at low energies and approaches the same values at high energies with decreasing b0 or increasing n0 or 1 ( variations at > 5 mev are not shown in the figure ) . lastly , the minimum resonant energy emin decreases while 0u increases with decreasing b0 or increasing n0 or 2 , but emin and 0u are unaffected by 1 . ( first to fourth rows ) d(00,e) , d(0,e)m , 0 m , and 0u are shown , respectively . ( fifth row ) precipitating flux at t=1 s derived from initial distribution equation 9 . ( left column ) varying b0 , n0=100 cm , 1=2.4 o+ , 2=3.4 o+ ; ( middle column ) varying n0 , b0=100 nt , 1=2.4 o+ , 2=3.4 o+ ; and ( right column ) varying , b0=100 nt , n0=100 cm are shown . applying the results for the diffusion coefficients from section 3 , we solve equation 2 for the trapped equatorial electron flux j0(0,e , t ) . as we can see , the loss cone fills up quickly ( e.g. , the first 60 s in figure 3 ) before gradually being depleted as the total flux drops due to loss to the loss cone . evolution of the pitch angle distribution of the trapped flux of 4 mev electrons in the simulation time 0 to 5 min . b0 , n0 , 1 , and 2 are the same as figure 1(c ) . at b0=200 nt ( l6.7 ) , the loss cone is 2.3. at 0 min , the trapped flux distribution is the initial maxwellian flux distribution equation 3 . the omnidirectional flux at any latitude on a dipole field line can be expressed in terms of equatorial flux and pitch angles as [ lyons and williams , 1984 ] , 7where . if we take to be the latitude where the field line intersects the atmosphere boundary , then b/b0=(4l3l ) , =0lc and no particle bounces back to the equator . the omnidirectional relativistic electron precipitation ( rep ) flux measured at the atmosphere boundary will then be 8jrep plotted versus e is then the energy spectrum of the precipitating electrons . to study how the jrep(e , t ) energy spectra are affected by the diffusion coefficients , we first apply an energy - independent initial distribution 9and examine the precipitation spectrum in the first second , i.e. , jrep(e , t=1s ) ( figure 2 , fifth row ) . these plots strongly resemble those of d(00,e) ( figure 2 , first row ) , indicating that the precipitation at the outset is largely controlled by the diffusion coefficients at small pitch angles . next , we again use the maxwellian initial distribution equation 3 and study how the spectrum is affected by time . we found two types of spectra ( figure 4 ) : in most cases , the spectrum is singly peaked ( figure 4a ) , but occasionally , increasing wave frequency can induce another peak ( figure 4b ) . as shown in the figures , initially , both spectra resemble maxwellian distributions with a lower cutoff at the minimum resonant energy . the precipitation quickly builds up from zero within the first second as the loss cone is being filled . shortly after , the rep flux near the minimum resonant energies largely drops and the rep flux starts to gradually decrease as the loss cone is being depleted . this rapid increase and slow decrease of the precipitation is consistent with many balloon observations [ millan et al . , 2007 ] . however , in figure 4a , the peak of the rep flux keeps moving toward higher energies and the curves flatten out . in figure 4b , the peak stays at a roughly constant energy , followed by another peak forming at a later time at a higher energy and moving toward even higher energies . the hardening of both spectra with time is due to the fact that 1 with increasing energy , diffusion becomes increasingly dominated by particles with larger pitch angles ( 0 m and 0u both increase with energy ) and they are scattered into the loss cone more slowly than the ones with smaller pitch angles ; 2 over time , with fewer particles left to interact with the waves , the precipitation is largely reduced at lower energies associated with shorter lifetimes ( larger diffusion coefficients ) and therefore the spectrum becomes harder and more isotropic ( flatter ) in energy . furthermore , as we discussed in the previous section , the increase of the diffusion coefficient at the peak energy from surrounding energies is significantly enhanced with frequency . when the increase is sharp enough , the precipitation of the particles with the highest diffusion coefficients can drastically reduce the flux and a trough can form in the middle of the spectrum . in figure 4b , no rep flux is produced above 4 mev since the diffusion coefficients in the loss cone are zero ( figure 1d ) . the energy distribution of the precipitating electrons at time 1 s to 50 min and the time evolution of the energies of the rep peaks assuming maxwellian initial distribution equation 3 . ( a and b ) b0 , n0 , 1 , and 2 are the same as figures 1c and 1d , respectively . note the x axis left end in figure 4a is higher than 100 kev for better visualization . in figure 5 , we show the variation of the spectra of rep at 30 min for the full range of parameters . in the first few seconds ( not shown ) , similar to the case of an initial distribution uniform in energy , with decreasing b0 or increasing n0 , 1 , or 2 , jrep increases at low energies but decreases at high energies . but at 30 min , in the case of lower b0 or higher n0 , 1 , or 2 , the rep flux curves become fairly flat or doubly peaked , and sometimes intersect and fall below the curves of higher b0 or lower n0 , 1 , or 2 ( e.g. , the precipitation with b0=125 nt is lower than that of 175 nt at 2.6 mev in figure 5a ; the precipitation with =3.4 - 3.9 o+ is lower than any other precipitation curve of 2=3.9 o+ at below 1 mev in figure 5c ) . when this happens , the role played by the changing parameter at these energy ranges is different than at early times . our simulation also shows that increasing n0 , or decreasing b0 lowers the energies of the peaks in both single- or double - peak spectra . energy spectra of precipitating electron flux at t=30 min with maxwellian initial distribution , plotted with the same parameters and color codes adopted in figure 2 . ( c ) the rep flux of 3.43.9 o+ is zero above 3.6 mev where diffusion coefficients are zero in the loss cone . in figure 6 , we integrate jrep in the energy ranges 0.10.3 , 0.31 , and 15 mev and vary two of the three parameters b0 , n0 , and simultaneously . these spectra can be compared with satellites with similar energy channels ( e.g. , poes and sampex ) [ yando et al . , the curves of the same color ( for the same fixed parameter ) get shorter and shorter , and can completely disappear when the energy of the channel is below the minimum resonant energies . with an increase in n0 or or a decrease in b0 , the rep flux increases , except occasionally at late times when the roles played by the parameters reverse ( explained in the previous paragraph ) , in which case strong initial scattering results in the significant decrease of the precipitation as the loss cone is depleted . precipitating electron flux in energy channels ( a ) 1=2.4 o+ , 2=3.4 o+ , b0=751150 nt , color - coded in n0 ranging from 25 to 1000 cm . ( b ) n0=100 cm , b0=751150 nt , color - coded in ranging from 1.93.9 o+ . ( c ) b0=100 nt , n0=11000 cm , color - coded in ranging from 1.9 to 3.9 o+ . the goal of this paper is to investigate the shape of the energy spectrum of relativistic electron precipitation ( rep ) due to quasi - linear interactions with emic waves , and how it varies with time and changing parameters such as background magnetic field strength b0 , cold plasma density n0 , and wave frequency . the key results can be summarized as follows : 1 . the rep energy spectrum is generally peaked , with a lower cutoff at the minimum resonant energy . over time the second peak appears later at a higher energy and moves toward even higher energies.3 . in both single- and double - peak cases , the precipitating flux first rapidly increases as the loss cone is being filled , and then slowly decreases as the loss cone is being depleted.4 . increasing n0 , , or decreasing b0 lowers the minimum resonant energy and the energies of the peak(s ) . it causes the precipitation to increase at low energies and decrease at high energies . over time , when strong scattering slows down , the role played by the changing parameter is altered.5 . we study how it is affected by changing parameters through varying two parameters at the same time . the integrated flux monotonically increases with increasing n0 and and decreases with increasing b0 , except at the energies where the role of the parameter reverses when strong scattering causes a large reduction of the particles interacting with the waves ( figure 6 ) . the rep energy spectrum is generally peaked , with a lower cutoff at the minimum resonant energy . over time , the peak moves toward higher energies and the spectrum flattens ( gets harder ) . the second peak appears later at a higher energy and moves toward even higher energies . the precipitating flux first rapidly increases as the loss cone is being filled , and then slowly decreases as the loss cone is being depleted . 4 . increasing n0 , , or decreasing b0 lowers the minimum resonant energy and the energies of the peak(s ) . it causes the precipitation to increase at low energies and decrease at high energies . over time , when strong scattering slows down , the role played by the changing parameter is altered . we study how it is affected by changing parameters through varying two parameters at the same time . the integrated flux monotonically increases with increasing n0 and and decreases with increasing b0 , except at the energies where the role of the parameter reverses when strong scattering causes a large reduction of the particles interacting with the waves ( figure 6 ) . to better explain the variation of the precipitation energy spectrum , we show how the spectrum is affected by three deterministic factors the diffusion coefficient , the initial trapped flux , and time . the diffusion coefficient is determined by the gyroresonance condition , the wave dispersion relation , and the wave frequency distribution and is a function of pitch angle . we calculate the diffusion coefficient for a range of input parameters b0 , n0 , and and characterize the pitch angle dependence of the diffusion coefficient with d(00,e) , the upper pitch angle limit 0u , and the maximum diffusion coefficient d(0,e)m along with its corresponding pitch angle 0 m . we show that the diffusion coefficient maximizes at increasing pitch angles with increasing energy and the upper limit 0u also increases . we also show that low b0 , high n0 and high lower the minimum resonant energy , increase the diffusion coefficients of low energy particles and reduce the diffusion coefficients of high energy particles , and further increase the precipitation at low energies and decrease the precipitation at high energies at the beginning . the number of particles scattered into the loss cone per unit of time increases with the number of initially trapped particles . therefore , when we switch the initial trapped flux energy distribution from uniform to maxwellian , the spectrum becomes slanted with an enhancement at the lower energies , and the peak of the spectrum shifts to the left . therefore , in the beginning , the precipitation is strongly affected by the diffusion coefficients at low pitch angles . since the diffusion coefficients tend to extend out and maximize at higher pitch angles with increasing energy , the precipitating flux of high energies has a growing relative significance over time , and the precipitation energy spectrum gets harder . in addition , if the scattering at a certain energy is initially strong , the particles at that energy are quickly lost and the precipitation will be largely reduced over time , and this also results in the rapid decrease of the precipitation at lower energies and the formation of the trough regions in the doubly peaked spectra . it is worth noting that thermal heating [ anderson and fuselier , 1994 ; thorne et al . , 2006 ] and the high plasma beta in the outer edge of the ring current during storm times [ lui et al . , 1987 ] may render the cold dispersion relation impractical . several studies [ isenberg , 1984 ; chen et al . , 2011 ; silin et al . , 2011 ; chen et al . , 2013 ] in which the hot dispersion relation is adopted suggest that at the vicinity of the ion cyclotron frequencies , the finite - beta effect may lead to the damping of the emic waves and the increase of the minimum resonant energies of relativistic electron scattering . based on their conclusions , in the situations where the warm / hot ions are abundant , we should expect to see a higher minimum resonant energy cutoff in our rep energy spectrum if the wave frequencies are just below the helium gyrofrequency . whether the doubly peaked spectra will still exist is uncertain because in our simulations , they usually only happen at high frequencies close to the helium gyrofrequency where the diffusion coefficient is significantly modified by the finite - beta effect . these model energy spectra show what we should expect to observe given various wave and geomagnetic environmental conditions if the precipitation is caused by emic wave scattering and can be simulated by the adopted diffusion model . further work will include event studies in which we will take the input data from satellites ( e.g. , van allen probes and goes ) and compare the simulated precipitating flux spectra with those detected by conjugate balloons ( e.g. , barrel ) and low altitude satellites ( e.g. , sampex and poes ) .

[ 1]previous studies on electromagnetic ion cyclotron ( emic ) waves as a possible cause of relativistic electron precipitation ( rep ) mainly focus on the time evolution of the trapped electron flux . however , directly measured by balloons and many satellites is the precipitating flux as well as its dependence on both time and energy . therefore , to better understand whether pitch angle scattering by emic waves is an important radiation belt electron loss mechanism and whether quasi - linear theory is a sufficient theoretical treatment , we simulate the quasi - linear wave - particle interactions for a range of parameters and generate energy spectra , laying the foundation for modeling specific events that can be compared with balloon and spacecraft observations . we show that the rep energy spectrum has a peaked structure , with a lower cutoff at the minimum resonant energy . the peak moves with time toward higher energies and the spectrum flattens . the precipitating flux , on the other hand , first rapidly increases and then gradually decreases . we also show that increasing wave frequency can lead to the occurrence of a second peak . in both single- and double - peak cases , increasing wave frequency , cold plasma density or decreasing background magnetic field strength lowers the energies of the peak(s ) and causes the precipitation to increase at low energies and decrease at high energies at the start of the precipitation .

1. Introduction 2. Diffusion Equation 3. Diffusion Coefficients 4. Time Evolution of Trapped and Precipitating Electron Fluxes 5. Summary and Discussion

however , the role played by each of several acceleration and loss mechanisms is not yet established observationally [ millan and thorne , 2007 ] . precipitation into the atmosphere is considered to be one of the major electron loss mechanisms , which can completely deplete the radiation belts of electrons during the main phase of some geomagnetic storms [ o'brien et al . the energy spectrum has been found to be well fit by an exponential distribution with an e - folding energy ranging from 0.5 to 3.6 mev [ millan et al . based primarily on the fact that all these events were found at dusk , the cause has been suggested to be the gyroresonant scattering by electromagnetic ion cyclotron ( emic ) waves . emic waves are observed throughout the inner magnetosphere but predominantly on the duskside and dayside [ anderson et al . , 2008 ] or by compressions of the magnetopause [ e.g. field - aligned emic waves interact with relativistic electrons through the gyroresonance condition 1where e is electron gyrofrequency , is the relativistic factor , k|| and v|| are components of the wave propagation vector particle velocity along the direction of the ambient magnetic field . emic waves are expected to effectively scatter relativistic electrons of geophysically interesting energies , preferably in duskside high - density regions , where the minimum energy limit is relatively low and the diffusion rate is close to the strong diffusion limit [ thorne and kennel , 1971 ; albert , 2003 ; summers , 2003 ] . duskside simultaneous proton and relativistic electron precipitation has also been observed , supporting the theory of precipitation caused by emic wave scattering [ e.g. , quasi - linear theory [ kennel and petschek , 1966 ] has been the dominant treatment ; however , recent work shows that nonlinear effects can be very significant and even reverse the conclusions [ albert and bortnik , 2009 ] . therefore , it is important to establish when and where each approach is applicable and to test these models with observations . existing theoretical discussions on emic waves as a possible loss mechanism mainly focus on the evolution of the trapped electron flux and its timescale . however , directly measured by balloon detectors is the energy spectrum of the bremsstrahlung x - rays produced by the precipitating electrons , and many satellites also measure the energy of the precipitating electrons [ millan et al . it is very important to understand the energy dependence of the precipitating flux driven by emic waves , as well as the time evolution of this dependence in order to test the theory with observations . this paper uses the quasi - linear formulation to evaluate the energy and time dependence of rep , and investigates how different parameters affect the diffusion coefficients and the energy spectrum . , van allen probes , goes ) , the results from our model can be directly compared with balloon ( e.g. , balloon array for rbsp ( radiation belt storm probes ) relativistic electron losses ) and low - altitude satellite ( e.g. , solar anomalous and magnetospheric particle explorer ( sampex ) and polar - orbiting operational environmental satellites ( poes ) ) measurements to investigate the role of emic waves in causing rep as well as the effectiveness of the adopted theoretical model . we use quasi - linear diffusion theory to model the evolution of the distribution of electrons due to interactions with emic waves . radial and energy diffusion are ignorable because the frequency of the emic waves is well above the drift frequency and well below the gyrofrequency of the resonant particles [ kennel , 1966 ] . the bounce - averaged diffusion equation for pure pitch angle scattering can be written as [ davidson and walt , 1977 ; lyons , 1973 ; lyons and williams , 1984 ; shao et al . , 2009 ] 2where t(0)=1.38020.3198(sin(0)+ sin1/2(0 ) ) is the normalized bounce time , f0 is the trapped electron phase space density , 0 is the equatorial pitch angle , d(0,e) is the bounce - averaged pitch angle diffusion coefficient , and atm is the timescale for losses to the atmosphere . assuming that losses occur only from within the loss cone and that the loss cone is emptied twice per bounce period , we take atm to be half of the bounce period inside the loss cone and infinite outside the loss cone [ lyons , 1973 ; davidson and walt , 1977 ; lyons and williams , 1984 ; shao et al . , the differential flux is related to the phase space density through the electron momentum p as j0=pf0 . to capture the feature of the isotropic flux distribution in the loss cone in the many strong diffusion cases in our parameter studies , we set the boundary conditions to be . here e is the particle energy in mev and 0l is the equatorial loss cone angle given by sin2(0l)=(4l3l ) at a particular l shell . the earth 's magnetic field is assumed dipolar , the wave amplitude is taken to be 1 nt , and the wave frequency spectrum is assumed to be a truncated gaussian , namely , 4where m is the center frequency , with lower frequency limit 1=m and upper frequency limit 2=m+. is a measure of the bandwidth . the emic waves are assumed to be confined to 15% in latitude and 10% of the electron drift orbit , propagating in a cold plasma of a storm time ion composition 70% h , 20% he , and 10% o [ meredith , 2003 ] . the minimum resonant energy can be approximated as [ summers , 2003 ] 6 figure 1 shows the bounce - averaged diffusion coefficient as a function of equatorial pitch angles we calculated for 0.15 mev electrons with four sets of equatorial background magnetic field strength b0 , cold plasma density n0 , and wave frequency . each diffusion coefficient spans from 0 to an upper resonant pitch angle limit 0u and maximizes to a value d(0,e)m at a pitch angle 0 m . bounce - averaged pitch angle diffusion coefficients for emic waves interacting with electrons of energies 0.15 mev with an increment of 0.2 mev and four different sets of wave and plasma parameters . we next investigate how the diffusion coefficient varies with energy and pitch angles for a full range of parameters b0 , n0 , and . the varying diffusion coefficients will determine how the energy spectrum evolves ( section 4 ) . we choose b0 , n0 , and to vary from 75 to 1150 nt ( corresponding to l38 ) , 1 to 1000 cm ( assumed to be constant over the entire interaction region ) , and 1.9 to 3.9 o+ ( covering roughly the entire helium band , where the spectral intensity of emic waves is enhanced [ thorne et al . , 2010 ] , we set the control b0 and n0 to be 100 nt and 100 cm , respectively . we also choose the control wave frequency to be =2.43.4 o+ , estimated from wave observations [ meredith , 2003 ; ukhorskiy et al . in figure 2 ( first to fourth rows ) ( the fifth row will be discussed in section 4 ) , we plot d(00,e),d(0,e)m , 0 m , and 0u as a function of e to characterize the diffusion coefficient when one parameter is varied while the others are held fixed . satisfying the gyroresonance condition equation 1 , the dispersion relation equation 5 , and the gaussian wave frequency distribution equation 4 , the plots show that for all b0 , n0 , 1 , and 2 , the pitch angles 0u and 0 m increase monotonically with energy , whereas d(00,e) and d(0,e)m increase from the minimum resonant energy up to a certain peak energy and then decrease . the value of the peak energy decreases with decreasing b0 or increasing n0 , 1 , or 2 . the width of the peak significantly narrows with increasing 1 and 2 , meaning that at high frequencies , the diffusion coefficient sharply increases at the peak energy from surrounding energies . keeping energy fixed , as b0 decreases or n0 , 1 , or 2 increases , 0 m increases monotonically ; the diffusion coefficient increases at low energies yet decreases at high energies ( variations at > 5 mev are not shown in the figure ) ; d(0,e)m increases at low energies and decreases at high energies only with increasing 2 while increases at low energies and approaches the same values at high energies with decreasing b0 or increasing n0 or 1 ( variations at > 5 mev are not shown in the figure ) . lastly , the minimum resonant energy emin decreases while 0u increases with decreasing b0 or increasing n0 or 2 , but emin and 0u are unaffected by 1 . ( fifth row ) precipitating flux at t=1 s derived from initial distribution equation 9 . applying the results for the diffusion coefficients from section 3 , we solve equation 2 for the trapped equatorial electron flux j0(0,e , t ) . evolution of the pitch angle distribution of the trapped flux of 4 mev electrons in the simulation time 0 to 5 min . at b0=200 nt ( l6.7 ) , the loss cone is 2.3. at 0 min , the trapped flux distribution is the initial maxwellian flux distribution equation 3 . if we take to be the latitude where the field line intersects the atmosphere boundary , then b/b0=(4l3l ) , =0lc and no particle bounces back to the equator . the omnidirectional relativistic electron precipitation ( rep ) flux measured at the atmosphere boundary will then be 8jrep plotted versus e is then the energy spectrum of the precipitating electrons . to study how the jrep(e , t ) energy spectra are affected by the diffusion coefficients , we first apply an energy - independent initial distribution 9and examine the precipitation spectrum in the first second , i.e. these plots strongly resemble those of d(00,e) ( figure 2 , first row ) , indicating that the precipitation at the outset is largely controlled by the diffusion coefficients at small pitch angles . next , we again use the maxwellian initial distribution equation 3 and study how the spectrum is affected by time . we found two types of spectra ( figure 4 ) : in most cases , the spectrum is singly peaked ( figure 4a ) , but occasionally , increasing wave frequency can induce another peak ( figure 4b ) . as shown in the figures , initially , both spectra resemble maxwellian distributions with a lower cutoff at the minimum resonant energy . shortly after , the rep flux near the minimum resonant energies largely drops and the rep flux starts to gradually decrease as the loss cone is being depleted . this rapid increase and slow decrease of the precipitation is consistent with many balloon observations [ millan et al . however , in figure 4a , the peak of the rep flux keeps moving toward higher energies and the curves flatten out . in figure 4b , the peak stays at a roughly constant energy , followed by another peak forming at a later time at a higher energy and moving toward even higher energies . the hardening of both spectra with time is due to the fact that 1 with increasing energy , diffusion becomes increasingly dominated by particles with larger pitch angles ( 0 m and 0u both increase with energy ) and they are scattered into the loss cone more slowly than the ones with smaller pitch angles ; 2 over time , with fewer particles left to interact with the waves , the precipitation is largely reduced at lower energies associated with shorter lifetimes ( larger diffusion coefficients ) and therefore the spectrum becomes harder and more isotropic ( flatter ) in energy . furthermore , as we discussed in the previous section , the increase of the diffusion coefficient at the peak energy from surrounding energies is significantly enhanced with frequency . when the increase is sharp enough , the precipitation of the particles with the highest diffusion coefficients can drastically reduce the flux and a trough can form in the middle of the spectrum . the energy distribution of the precipitating electrons at time 1 s to 50 min and the time evolution of the energies of the rep peaks assuming maxwellian initial distribution equation 3 . in figure 5 , we show the variation of the spectra of rep at 30 min for the full range of parameters . in the first few seconds ( not shown ) , similar to the case of an initial distribution uniform in energy , with decreasing b0 or increasing n0 , 1 , or 2 , jrep increases at low energies but decreases at high energies . but at 30 min , in the case of lower b0 or higher n0 , 1 , or 2 , the rep flux curves become fairly flat or doubly peaked , and sometimes intersect and fall below the curves of higher b0 or lower n0 , 1 , or 2 ( e.g. , the precipitation with b0=125 nt is lower than that of 175 nt at 2.6 mev in figure 5a ; the precipitation with =3.4 - 3.9 o+ is lower than any other precipitation curve of 2=3.9 o+ at below 1 mev in figure 5c ) . our simulation also shows that increasing n0 , or decreasing b0 lowers the energies of the peaks in both single- or double - peak spectra . energy spectra of precipitating electron flux at t=30 min with maxwellian initial distribution , plotted with the same parameters and color codes adopted in figure 2 . in figure 6 , we integrate jrep in the energy ranges 0.10.3 , 0.31 , and 15 mev and vary two of the three parameters b0 , n0 , and simultaneously . these spectra can be compared with satellites with similar energy channels ( e.g. , the curves of the same color ( for the same fixed parameter ) get shorter and shorter , and can completely disappear when the energy of the channel is below the minimum resonant energies . with an increase in n0 or or a decrease in b0 , the rep flux increases , except occasionally at late times when the roles played by the parameters reverse ( explained in the previous paragraph ) , in which case strong initial scattering results in the significant decrease of the precipitation as the loss cone is depleted . the goal of this paper is to investigate the shape of the energy spectrum of relativistic electron precipitation ( rep ) due to quasi - linear interactions with emic waves , and how it varies with time and changing parameters such as background magnetic field strength b0 , cold plasma density n0 , and wave frequency . the key results can be summarized as follows : 1 . the rep energy spectrum is generally peaked , with a lower cutoff at the minimum resonant energy . over time the second peak appears later at a higher energy and moves toward even higher energies.3 . in both single- and double - peak cases , the precipitating flux first rapidly increases as the loss cone is being filled , and then slowly decreases as the loss cone is being depleted.4 . increasing n0 , , or decreasing b0 lowers the minimum resonant energy and the energies of the peak(s ) . it causes the precipitation to increase at low energies and decrease at high energies . we study how it is affected by changing parameters through varying two parameters at the same time . the integrated flux monotonically increases with increasing n0 and and decreases with increasing b0 , except at the energies where the role of the parameter reverses when strong scattering causes a large reduction of the particles interacting with the waves ( figure 6 ) . the rep energy spectrum is generally peaked , with a lower cutoff at the minimum resonant energy . over time , the peak moves toward higher energies and the spectrum flattens ( gets harder ) . the second peak appears later at a higher energy and moves toward even higher energies . the precipitating flux first rapidly increases as the loss cone is being filled , and then slowly decreases as the loss cone is being depleted . increasing n0 , , or decreasing b0 lowers the minimum resonant energy and the energies of the peak(s ) . it causes the precipitation to increase at low energies and decrease at high energies . we study how it is affected by changing parameters through varying two parameters at the same time . the integrated flux monotonically increases with increasing n0 and and decreases with increasing b0 , except at the energies where the role of the parameter reverses when strong scattering causes a large reduction of the particles interacting with the waves ( figure 6 ) . to better explain the variation of the precipitation energy spectrum , we show how the spectrum is affected by three deterministic factors the diffusion coefficient , the initial trapped flux , and time . the diffusion coefficient is determined by the gyroresonance condition , the wave dispersion relation , and the wave frequency distribution and is a function of pitch angle . we calculate the diffusion coefficient for a range of input parameters b0 , n0 , and and characterize the pitch angle dependence of the diffusion coefficient with d(00,e) , the upper pitch angle limit 0u , and the maximum diffusion coefficient d(0,e)m along with its corresponding pitch angle 0 m . we show that the diffusion coefficient maximizes at increasing pitch angles with increasing energy and the upper limit 0u also increases . we also show that low b0 , high n0 and high lower the minimum resonant energy , increase the diffusion coefficients of low energy particles and reduce the diffusion coefficients of high energy particles , and further increase the precipitation at low energies and decrease the precipitation at high energies at the beginning . therefore , when we switch the initial trapped flux energy distribution from uniform to maxwellian , the spectrum becomes slanted with an enhancement at the lower energies , and the peak of the spectrum shifts to the left . therefore , in the beginning , the precipitation is strongly affected by the diffusion coefficients at low pitch angles . since the diffusion coefficients tend to extend out and maximize at higher pitch angles with increasing energy , the precipitating flux of high energies has a growing relative significance over time , and the precipitation energy spectrum gets harder . in addition , if the scattering at a certain energy is initially strong , the particles at that energy are quickly lost and the precipitation will be largely reduced over time , and this also results in the rapid decrease of the precipitation at lower energies and the formation of the trough regions in the doubly peaked spectra . , 2006 ] and the high plasma beta in the outer edge of the ring current during storm times [ lui et al . , 2013 ] in which the hot dispersion relation is adopted suggest that at the vicinity of the ion cyclotron frequencies , the finite - beta effect may lead to the damping of the emic waves and the increase of the minimum resonant energies of relativistic electron scattering . based on their conclusions , in the situations where the warm / hot ions are abundant , we should expect to see a higher minimum resonant energy cutoff in our rep energy spectrum if the wave frequencies are just below the helium gyrofrequency . whether the doubly peaked spectra will still exist is uncertain because in our simulations , they usually only happen at high frequencies close to the helium gyrofrequency where the diffusion coefficient is significantly modified by the finite - beta effect . these model energy spectra show what we should expect to observe given various wave and geomagnetic environmental conditions if the precipitation is caused by emic wave scattering and can be simulated by the adopted diffusion model . , van allen probes and goes ) and compare the simulated precipitating flux spectra with those detected by conjugate balloons ( e.g.

as advances in laparoscopic skills and instrumentation evolve , increasingly complex procedures are being performed , yet most complications are associated with primary access . gaining access into the abdomen access is associated with injuries to the gastrointestinal tracts and major blood vessels , and at least 50% of these occur prior to commencement of the intended surgery . bowel injuries have occurred in 0.7/1000 laparoscopies and major vascular injuries in 0.4/1000 laparoscopies . despite considerable advances in endoscopic techniques and instrumentation , routinely , primary trocar is placed at umbilical level , but an alternate entry site is recommended when umbilical placement of a primary trocar is deemed hazardous , like in cases with previous abdominal surgery . the present study aims to introduce a new access point as a safe primary port in previous abdominal surgery cases . further , in the text , we would refer to this new point as the jain point , a nomenclature fondly coined by fellows and trainees in our center . the jain point is located in the left paraumbilical region at the level of umbilicus , in a straight line drawn vertically upward from a point 2.5 cm medial to anterior superior iliac spine ( asis ) . the universally preferred point of entry in previous surgery cases is the palmer 's point developed by palmer . palmer 's point lies 3 cm below the left subcostal margin in the mid - clavicular line . many authors have described it as arguably the safest alternative insertion site for peritoneal access in women having undergone earlier abdominal surgeries . palmer 's point is a safe access port , but it is not used much during the later course of surgery due to its anatomically higher location . while jain point being lower and lateral in position , can be used as the main operating port throughout the surgery . moreover , jain point can be used for safe access even in patients with previous splenic or gastric surgery , hepatosplenomegaly , portal hypertension , and gastropancreatic masses which have to be excluded when using palmer 's point . the area between the anterior axillary line and the mid - clavicular line , between the palmer 's point and up to an inch beneath the umbilicus is an area in which the 5-mm trocar can be placed safely without the risk of vascular injury [ figure 1 ] . data of patients with a history of previous abdominal or pelvic surgery who underwent laparoscopic surgery at our center from january 2011 to december 2014 were collected . the history and previous operative notes were carefully recorded . patients ' age , body mass index ( bmi ) , indication of previous surgery ( gynecologic or nongynecologic ) , mode of previous surgery ( open or laparoscopic ) , and incision of previous surgery were tabulated . other incisions included open appendectomy incision ( mcburney 's or gridiron or lanz incision ) , open cholycystectomy incision ( upper right subcostal and kocher 's incision ) , incisions for renal surgeries , and nonclassical incisions such as previous drain sites , colostomy sites , bizarre vertical incisions extending from epigastrium up to pubic symphysis in prior surgery for intestinal obstruction , septicemia , and other complex pathologies . two patients had suffered burst abdomen in previous surgery and very thick multiple scars over the abdomen due to delayed , prolonged healing . cases with multiple surgeries were counted only once when tabulating data for indication of surgery and type of incision . for instance , if patient had undergone laparotomy with right paramedian incision and a cesarean section with pfannenstiel incision , only right paramedian incision was considered , as probability of having paraumbilical adhesions are more with this type of surgery and incision . complications were defined as events that significantly prolonged or altered the planned procedure , delayed discharge , or lead to a prolongation of convalescence . complications directly related to the entry technique that occurred intraoperatively , and discovered either immediately or up to 2 weeks postoperatively , were recorded . the preoperative preparation comprised low residual diet for 48 h prior to surgery and mechanical bowel preparation in all previous open surgery cases . the stomach was emptied of secretions and air by the use of orogastric tube by anesthetist , after endotracheal intubation . first and foremost , the whole abdomen is inspected , and all incision sites are carefully noted . jain point is marked at the level of umbilicus ( paraumbilical ) on the left side where usually the operating surgeon stands . jain point is placed in a straight line drawn vertically upward from a lower point which is 2.5 cm medial to asis [ figure 2 ] . palmer 's point ( p ) , jain point ( j ) , and anterior superior iliac spine . jain point lies in the left paraumbilical region , in a straight line drawn vertically upward from a point 2.5 cm medial and 1 cm above anterior superior iliac spine veress needle entry at jain point . patient has previous transverse incision we keep the patient in supine position and have the table height adjusted for the surgeon . place the left hand over the left middle quadrant around the jain point , make the skin taut between the index and middle finger , and enter the veress needle in a straight direction . it is important to hold the veress needle in such a manner to control the length of the needle going inside the abdominal wall in the first go . this length is estimated by patient 's bmi and abdominal wall fat as assessed on inspection of the abdomen . all safety checks for veress needle entry such as aspiration , saline injection , reaspiration , and drop test are done . the most important test is the low initial veress needle intra - peritoneal pressure , which should not be more than 8 mmhg for initial 10 seconds , as also supported by vilos . after confirming safe entry by above all tests of veress needle , the intra - peritoneal pressure is transiently raised to 25 mmhg and 5 mm trocar is inserted through the jain point when the total volume of co2 insufflation is around 34 l [ figure 4 ] . this transient high - pressure pneumoperitoneum in horizontal position produces greater splinting of abdominal wall , so allows a safer trocar insertion and does not compromise patient 's cardiopulmonary functions . once the 5 mm trocar is safely in , we introduce a 5 mm , 0 telescope , then we routinely do a 360 inspection of abdominal cavity to check for adhesions . the basic aim of making entry from jain point is to avoid abdominal and umbilical adhesions . after confirming safe entry , the entry for the 10 mm 30 telescope is optimized according to the mandate of the case . this entry can be anywhere between the umbilicus and xiphisternum according to the pelvic pathology , size of masses , and adhesion sites . it is safe to introduce the 10 mm port under the direct visual guidance of 5 mm telescope introduced through jain point . primary port at jain point after introducing the 10 mm port , safe abdominal entry of the 5 mm trocar through jain point is also confirmed . then lower right and left ports are inserted 2.5 cm medial and 1 cm above the asis under vision . the distance of the left lower port from the jain point is around 1012 cm . this distance of 1012 cm gives a good ergonomic working , and these become the main operating ipsilateral ports . the preoperative preparation comprised low residual diet for 48 h prior to surgery and mechanical bowel preparation in all previous open surgery cases . the stomach was emptied of secretions and air by the use of orogastric tube by anesthetist , after endotracheal intubation . first and foremost , the whole abdomen is inspected , and all incision sites are carefully noted . jain point is marked at the level of umbilicus ( paraumbilical ) on the left side where usually the operating surgeon stands . jain point is placed in a straight line drawn vertically upward from a lower point which is 2.5 cm medial to asis [ figure 2 ] . palmer 's point ( p ) , jain point ( j ) , and anterior superior iliac spine . jain point lies in the left paraumbilical region , in a straight line drawn vertically upward from a point 2.5 cm medial and 1 cm above anterior superior iliac spine veress needle entry at jain point . patient has previous transverse incision we keep the patient in supine position and have the table height adjusted for the surgeon . place the left hand over the left middle quadrant around the jain point , make the skin taut between the index and middle finger , and enter the veress needle in a straight direction . it is important to hold the veress needle in such a manner to control the length of the needle going inside the abdominal wall in the first go . this length is estimated by patient 's bmi and abdominal wall fat as assessed on inspection of the abdomen . all safety checks for veress needle entry such as aspiration , saline injection , reaspiration , and drop test are done . the most important test is the low initial veress needle intra - peritoneal pressure , which should not be more than 8 mmhg for initial 10 seconds , as also supported by vilos . after confirming safe entry by above all tests of veress needle , the intra - peritoneal pressure is transiently raised to 25 mmhg and 5 mm trocar is inserted through the jain point when the total volume of co2 insufflation is around 34 l [ figure 4 ] . this transient high - pressure pneumoperitoneum in horizontal position produces greater splinting of abdominal wall , so allows a safer trocar insertion and does not compromise patient 's cardiopulmonary functions . once the 5 mm trocar is safely in , we introduce a 5 mm , 0 telescope , then we routinely do a 360 inspection of abdominal cavity to check for adhesions . the basic aim of making entry from jain point is to avoid abdominal and umbilical adhesions . after confirming safe entry , the entry for the 10 mm 30 telescope is optimized according to the mandate of the case . this entry can be anywhere between the umbilicus and xiphisternum according to the pelvic pathology , size of masses , and adhesion sites . it is safe to introduce the 10 mm port under the direct visual guidance of 5 mm telescope introduced through jain point . primary port at jain point after introducing the 10 mm port , safe abdominal entry of the 5 mm trocar through jain point is also confirmed . then lower right and left ports are inserted 2.5 cm medial and 1 cm above the asis under vision . the distance of the left lower port from the jain point is around 1012 cm . this distance of 1012 cm gives a good ergonomic working , and these become the main operating ipsilateral ports . the preoperative preparation comprised low residual diet for 48 h prior to surgery and mechanical bowel preparation in all previous open surgery cases . the stomach was emptied of secretions and air by the use of orogastric tube by anesthetist , after endotracheal intubation . first and foremost , the whole abdomen is inspected , and all incision sites are carefully noted . jain point is marked at the level of umbilicus ( paraumbilical ) on the left side where usually the operating surgeon stands . jain point is placed in a straight line drawn vertically upward from a lower point which is 2.5 cm medial to asis [ figure 2 ] . palmer 's point ( p ) , jain point ( j ) , and anterior superior iliac spine . jain point lies in the left paraumbilical region , in a straight line drawn vertically upward from a point 2.5 cm medial and 1 cm above anterior superior iliac spine veress needle entry at jain point . we keep the patient in supine position and have the table height adjusted for the surgeon . place the left hand over the left middle quadrant around the jain point , make the skin taut between the index and middle finger , and enter the veress needle in a straight direction . it is important to hold the veress needle in such a manner to control the length of the needle going inside the abdominal wall in the first go . this length is estimated by patient 's bmi and abdominal wall fat as assessed on inspection of the abdomen . all safety checks for veress needle entry such as aspiration , saline injection , reaspiration , and drop test are done . the most important test is the low initial veress needle intra - peritoneal pressure , which should not be more than 8 mmhg for initial 10 seconds , as also supported by vilos . after confirming safe entry by above all tests of veress needle , the intra - peritoneal pressure is transiently raised to 25 mmhg and 5 mm trocar is inserted through the jain point when the total volume of co2 insufflation is around 34 l [ figure 4 ] . this transient high - pressure pneumoperitoneum in horizontal position produces greater splinting of abdominal wall , so allows a safer trocar insertion and does not compromise patient 's cardiopulmonary functions . once the 5 mm trocar is safely in , we introduce a 5 mm , 0 telescope , then we routinely do a 360 inspection of abdominal cavity to check for adhesions . the basic aim of making entry from jain point is to avoid abdominal and umbilical adhesions . after confirming safe entry , the entry for the 10 mm 30 telescope is optimized according to the mandate of the case . this entry can be anywhere between the umbilicus and xiphisternum according to the pelvic pathology , size of masses , and adhesion sites . it is safe to introduce the 10 mm port under the direct visual guidance of 5 mm telescope introduced through jain point . primary port at jain point after introducing the 10 mm port , safe abdominal entry of the 5 mm trocar through jain point is also confirmed . then lower right and left ports are inserted 2.5 cm medial and 1 cm above the asis under vision . the distance of the left lower port from the jain point is around 1012 cm . this distance of 1012 cm gives a good ergonomic working , and these become the main operating ipsilateral ports . of the total 2834 laparoscopic cases done between january 2011 and december 2014 , 624 cases had previous one or multiple surgeries . the mean patients ' age was 33.5 years ( range : 1470 years ) and mean bmi was 24.48 kg / m ( range : 12.946.9 kg / m ) . the previous surgery cases , which we have dealt , include wide variety of cases with 86.37% cases ( 539 out of 624 ) of gynae indication and 13.63% cases ( 85 out of 624 ) of general surgery . in the gynae group , 54.4% ( 293 out of 539 ) had previous laparotomy , and 45.6% ( 246 out of 539 ) had previous laparoscopic procedures . in the laparotomic approach , cesarean sections were the most common , i.e. , 53.5% ( 157 out of 293 ) . these previous cesarean sections were encountered in 40.7% ( 64 out of 157 ) cases for secondary infertility and 35.03% cases ( 55 out of 157 ) for total laparoscopic hysterectomy . previous cesarean cases showed an alarming rate of adhesions , abdominal wall adhesions were present in 89% cases , and total adherence of the uterus itself to the anterior abdominal wall was noted in 11% cases . the advent of the technique of single layer closure and leaving the visceral and parietal peritoneum unstitched could have led to greater adhesions . next most common indication was previous ectopic pregnancy either by laparotomy ( 13.65% , i.e. , 40 out of 293 ) or by laparoscopy ( 19.9% , i.e. , 49 out of 246 ) . this was a formidable group for adhesions as laparotomy was usually done in ruptured ectopic cases with intraperitoneal hemorrhage . in this indication , endometriosis , as is shown in the table , with very extensive obliteration of pouch of douglas and endometriomas has been a very frequent surgery with the previous open approach as well as laparoscopy bizarre adhesions have been noted , in repeat laparoscopy . cases with previous surgery for infectious pathologies such as genital koch 's and septic abortion were also encountered with lots of intra - abdominal and pelvic adhesions . the other indications where we did laparoscopy were in cases of previous pelvic floor repairs and microsurgical tubo tubal reanastomosis . hence , we employed the jain point in wide variety of cases with different and practically all types of scars in upper and lower abdomen and multiple scars . in nongynae cases , 67.1% cases ( 57 out of 85 ) cases had previous surgery by open route and 37.3% ( 28 out of 75 ) cases by laparoscopic route . maximum were gall bladder removals , appendectomy , and few with previous mesh hernia repairs . mesh hernia repairs pose specific challenge , and previous notes need to be studied carefully . many cases of intestinal obstruction which were done in childhood and due to abdominal koch 's have also been dealt . these have long vertical incisions extending from high up in abdomen going down up to pubic symphysis . many of the cases had bowel perforations , and multiple drain sites noted over the abdomen . we have also experienced two cases of hirschsprung disease and two cases of high imperforate anus which were managed in infancy by colostomy and then colon pull through procedure . hence , in nongynae group , we dealt extensive pathologies with high potential of intra - abdominal adhesions [ table 1 and pie diagrams 14 ] . patients profile depending on type and indication of previous abdominal surgery patient profile based on type and indications of previous open gynaecological surgery patient profile based on type and indications of previous laproscopic gynaecological surgery patient profile based on type and indications of previous open non gynaecological surgery patient profile based on type and indications of previous laproscopic non gynaecological surgery a total of 209 ( 33.5% ) patients had midline or paramedian incisions , 120 ( 19.2% ) had pfannenstiel incisions , 242 ( 38.8% ) had laparoscopic ports , and 53 ( 8.5% ) had other incisions table 2 . of 624 subjects , 177 ( 28.4% ) had more than one previous surgery , either open or laparoscopic or both table 3 . as we earlier stated that these cases were counted only once for the surgery , which had more chance of adhesions . patients profile depending on type of previous surgical incision patients profile depending on number of previous abdominal surgeries intra - abdominal adhesions were found in a significant majority , i.e. , 487 ( 78.0% ) of previous surgery cases . adhesions in the umbilical region were found in 404 ( 64.7% ) patients with previous abdominal surgeries [ figure 5 ] . no significant entry - related complications were reported in the present study with the use of jain point as primary port . the most important and potentially dangerous first step in laparoscopy is the safe and successful insertion of a primary port since half the complications with laparoscopy occur even before the start of the intended procedure . injuries may occur by advancing the instruments toward the posterior abdominal wall and encountering anatomically normal but distended bowel ( type 1 injuries ) or due to visceral adhesions to the anterior abdominal wall ( type 2 injuries ) . the reported incidence of intra - abdominal adhesions after laparotomy ranges between 30% and 90% . even patients with one previous laparoscopic procedure have been found to have both omentum and bowel adhesions beneath the umbilicus and port sites due to valsalva or accidental suturing . royal college of obstetricians and gynecologists guideline on preventing entry - related gynecological laparoscopic injuries states : the umbilicus may not , therefore , be the most appropriate site for primary trocar insertion following previous abdominal surgery . for this reason , alternative primary approaches to laparoscopic surgery have been introduced for use in patients with a history of laparotomy . to avoid injuries , we can either use alternate entry site avoiding umbilicus or can use modified techniques such as open technique , direct trocar entry , disposable shielded trocar , and optical trocar access through umbilicus . probably the safest initial entry site in high - risk patients is the left upper quadrant ( luq ) known as palmer 's point , first described in 1931 by palmer , which lies 3 cm below the left costal border in midclavicular line . this technique is recommended for obese as well as very thin patients and in patients where intra - peritoneal adhesions are being suspected it is essential at this site , to decompress the stomach using nasogastric suction and then introduction of the veress needle perpendicularly to the skin without lifting the abdominal wall . after the establishment of the pneumoperitoneum , trocars of 5 mm may be introduced at the same site as the veress , followed by additional trocar / cannula systems inserted under direct vision , as per the requirement of the case . a clear advantage of veress needle and trocar insertion at this location is that the abdominal wall is consistently thin , even in obese patients . another advantage is that no major retroperitoneal vessels lie directly below palmer 's point when instruments are placed perpendicular to the skin surface in the axial plane . but various authors , giannios and howard et al . , they have cautioned regarding the angle of entry of long laparoscopic instruments during the course of surgery due to proximity of retroperitoneal structure like left kidney , which we found commonly located below palmer 's point . so they proposed to enter veress / trocar at 45 angle to horizontal plane , especially in thin patients . comparing the vertebral position of abdominal organs also , we are in a safe zone at jain point at around l4 vertebral level , where stomach ( t10 to l1 ) , left kidney ( t12 to l3 ) , and spleen ( t10 to l1 ) lie out of the way . palmer 's point should be avoided in patients known to have hepatospleenomegaly , portal hypertension , history of previous gastric or spleen surgery , or probable gastropancreatic mass . here , the jain point makes a difference that initial entry through jain point can be performed in above cases too . the other limitation of palmer 's point is that we can not use it as an operative port during further course of surgery due to its higher location while jain point is used as a working port during the whole surgery . moreover , at palmer 's point , it can be more difficult to penetrate the peritoneum with the veress needle than it is at the umbilicus . to overcome this problem , some surgeons advocate using a subcostal approach or even going through the lowest ( 9 ) intercostal space . luq is invariably free of adhesions irrespective of previous incisions , but there is a risk of creating an iatrogenic pneumothorax and also injuring neurovascular bundle and stomach . in one retrospective study , left ninth intercostal space was used for veress needle entry and left subcostal space for placement of primary trocar . out of 504 cases , one case had bowel injury by veress insertion , and no other significant entry - related injury was encountered . in another study , the middle upper abdomen was used for insufflation and primary cannula insertion in patients with a history of gynecologic cancer or at least , one laparotomy . the reported complications ranged from superficial serosal damage to complete perforation of stomach and small intestine and injury to blood vessels . an open umbilical method hasson technique first described in 1991 by hasson to avoid bowel and vascular injuries was supposed to be beneficial in obese patients , very thin patients , and in cases who had previous one or more laparotomy with midline vertical incision where chances of adhesion are much more . the technique involves incising the skin , subcutaneous tissue , fascial layer then holding this fascial layer with sutures to stabilize the cannula followed by entry into the abdomen and preventing gas leak with cone - shaped sleeve . as this is not a blind procedure like veress needle entry , it prevents the vascular injury but the risk of entering the lumen of adherent bowel is still not eliminated . the other benefits include avoiding prepertioneal insufflation , gas embolism , and correct anatomical repair of abdominal wall incision after the surgery . however , the major limiting factor is greater time needed for port placement and maintenance of pneumoperitoneum . in even experienced hands , it takes about 310 min . however , still , long - standing controversy remains about safety profile and there is conflicting evidence between studies penfield , noted a 0.06% incidence of bowel injury mostly partial thickness . hasson reported his experience on 5284 women where 21 patients had minor wound infection , 4 had minor hematoma , 1 developed umbilical hernia , and 1 had an injury to small bowel . moreover , hasson technique does not give full safety against bowel injuries though it obviates vascular injuries . if bowel is so densely adherent to the umbilicus then it is exposed to the risk of inadvertent injury while making the primary 10 mm umbilical entry ; hence , jain point technique moves the primary entry site away from major vessel or bowel , as it is in the left paraumbilical area . direct trocar entry technique was introduced by dingfelder in 1978 , which eliminates veress needle complications such as failed pneumoperitoneum , preperitoneal insufflation , and gas embolism . however , being a blind procedure , it does not eliminate the risk of bowel and vascular injuries . trocar were introduced to the market , the manufacturer claimed that this trocar system works in a way that when it enters the abdominal cavity , the sharp edge retracts and the shield springs so that it avoids contact of the tip of the trocar with intra - abdominal content . however , there will be a moment when this trocar enters into the peritoneal cavity and before its retraction , it will be in contact with abdominal contents . this brief moment is sufficient to produce injury , especially with its very sharp end . a retropective study documented 30% of serious injuries caused by laparoscopic entry , and two deaths out of seven deaths caused by laparoscopic entry injury . this led the food and drug administration to directly write to the manufacturers to eliminate safety claims from the label of shielded trocars . as the complications during laparoscopic entry continued to occur , to improve primary access safety , disposable optical trocars ( endopath optiview trocar and visiport optical trocars ) were introduced in 1994 . these instruments have a 0 laparoscope loaded in hollow trocar with the distal crystal tip which transects abdominal tissue layers and transmits entry images during insertion . these visual entry instruments retain a push through trocar and cannula design where the insertion requires considerably more penetration force with no mechanism to offset overshoot . to overcome this problem , dr . ternamian in 1997 , introduced a reusable endotip visual cannula which had less requirement of penetration force with controlled visual entry . optical veress needle is a 2.1 mm visual veress needle , which has a 1.2 mm micro laparoscope , is also available to make the initial veress needle guided entry in previous surgery cases . however , in this visual access system , it is essential for the operator to clearly recognize tissue plane transition on the monitor during entry and appreciate the importance of not applying perpendicular force otherwise , you have to face unintended injury . optical trocar system can still result in serious injuries such as vascular injuries to aorta , vena cava or iliac vessels , significant bleeding from other sites , bowel perforations , liver laceration and stomach perforation , and mortality also . it is true that we can visualize , the bowel loops through peritoneum but to avoid injury of very densely adherent bowel loop is still a challenge , and we have repeatedly found that jain point is free of bowel loops . overall visual entry trocars are nonsuperior to other trocars since they do not avoid visceral and vascular injuries . hence , we have selected a point of entry where chances of bowel adhesion are least and also does not require expensive and sophisticated trocar and veress needle . it is not only the instrument but also it is a point of the abdomen where we have not seen adherent bowel loops . the present study explores the possibility of an ergonomically lower point which is safe in previous surgery cases and further on usable as a working port for pelvic surgeries . furthermore , it is a normal routine reusable trocar that does not require any sophisticated optical trocar or veress needle or an endotip cannula or a micro laparoscope . no significant entry - related injury was encountered during the use of jain point in the present study . jain point , due to its more lateral location , it avoids intra - abdominal adhesions of the midline vertical and paramedian incision sites . moreover , being lower than palmer 's point , it avoids the stomach , spleen , left kidney , and pancreas injury risk . being on the left side , it avoids all other adhesions which could be due to appendiceal , ileocecal , gall bladder , liver pathologies , etc . , which are routinely encountered in general surgery cases . we propose jain point due to its simplicity of access , no need of any special technique or instrument . we did not find bowel adhesions at this site , so gives benefit for the avoidance of hollow viscous injury ( stomach and bowel ) . it continues to be used all through the surgery right from gaining entry in the abdominal cavity to being used as major operating port . moreover , jain point is amenable to minor variations when the mandate of the case so demands . a mirror reflection of jain point can be used from the right side , like in microsurgery cases , where left ports are generally 3 mm ; or in cases where previous left paramedian incisions are extensively placed ; or in cases where colostomy or drain sites , burns and keloids preclude its use on left side . it is important for surgeons to be well versed in more than one safe laparoscopic access method and be knowledgeable in different entry techniques , as our patients have different medical or surgical needs . careful access selection , thorough knowledge of abdominal and pelvic anatomy , and meticulous attention to surgical technique during laparoscopic port entry can significantly reduce inadvertent injury and improve patient safety . with our continued entry through jain point in the various type of cases as we have described , it has been found that despite previous infectious pathologies such as koch 's , septicemia , previous multiple bowel , and pelvic surgery , jain point has been found to be free of bowel adhesions . there was no major vessel injury . due to the abdominal wall blood supply , bleeding at the site by veress needle or trocar entry the jain point derives its safety from the fact that it is more lateral ; hence , it avoids intra - abdominal adhesions at previous incision sites . being lower down at paraumbilical position , it is ergonomic to be used as operating port later on in the surgery . since it is much lower than the subcostal margin , it can very well be used in cases of hepatosplenomegaly , portal hypertension , and upper abdomen vertical or kocher 's incisions . jain point promises to be a safe portal for laparoscopic entry in cases with a history of previous surgery . further use by other high volume endoscopy units will give a critical appraisal of its safe profile .

introduction : the present study was performed to assess the safety and feasibility of a new laparoscopic entry site in cases suspected of adhesions due to previous surgery.materials and methods : it is a retrospective study undertaken at a tertiary care referral center for advanced gynecological laparoscopic surgery from january 2011 to december 2014.results:in 624 patients with a history of previous abdominal surgeries , the laparoscopic entry site was through a newly devised point . it is a point in the left paraumbilical region at the level of umbilicus , in a straight line drawn vertically upward from a point 2.5 cm medial to anterior superior iliac spine . intra - abdominal adhesions were found in 487 ( 78.0% ) patients , and umbilical adhesions in 404 ( 64.7% ) patients with past abdominal surgeries.conclusion:there were no significant entry - related , intra - operative , or postoperative complications with the use of this entry point . it is also suitable as a main working port during the course of surgery .

INTRODUCTION MATERIALS AND METHODS Technique Preoperative preparation Surgical technique Veress needle insertion RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

despite considerable advances in endoscopic techniques and instrumentation , routinely , primary trocar is placed at umbilical level , but an alternate entry site is recommended when umbilical placement of a primary trocar is deemed hazardous , like in cases with previous abdominal surgery . the present study aims to introduce a new access point as a safe primary port in previous abdominal surgery cases . further , in the text , we would refer to this new point as the jain point , a nomenclature fondly coined by fellows and trainees in our center . the jain point is located in the left paraumbilical region at the level of umbilicus , in a straight line drawn vertically upward from a point 2.5 cm medial to anterior superior iliac spine ( asis ) . palmer 's point lies 3 cm below the left subcostal margin in the mid - clavicular line . palmer 's point is a safe access port , but it is not used much during the later course of surgery due to its anatomically higher location . moreover , jain point can be used for safe access even in patients with previous splenic or gastric surgery , hepatosplenomegaly , portal hypertension , and gastropancreatic masses which have to be excluded when using palmer 's point . data of patients with a history of previous abdominal or pelvic surgery who underwent laparoscopic surgery at our center from january 2011 to december 2014 were collected . patients ' age , body mass index ( bmi ) , indication of previous surgery ( gynecologic or nongynecologic ) , mode of previous surgery ( open or laparoscopic ) , and incision of previous surgery were tabulated . other incisions included open appendectomy incision ( mcburney 's or gridiron or lanz incision ) , open cholycystectomy incision ( upper right subcostal and kocher 's incision ) , incisions for renal surgeries , and nonclassical incisions such as previous drain sites , colostomy sites , bizarre vertical incisions extending from epigastrium up to pubic symphysis in prior surgery for intestinal obstruction , septicemia , and other complex pathologies . two patients had suffered burst abdomen in previous surgery and very thick multiple scars over the abdomen due to delayed , prolonged healing . for instance , if patient had undergone laparotomy with right paramedian incision and a cesarean section with pfannenstiel incision , only right paramedian incision was considered , as probability of having paraumbilical adhesions are more with this type of surgery and incision . complications were defined as events that significantly prolonged or altered the planned procedure , delayed discharge , or lead to a prolongation of convalescence . the stomach was emptied of secretions and air by the use of orogastric tube by anesthetist , after endotracheal intubation . first and foremost , the whole abdomen is inspected , and all incision sites are carefully noted . jain point is marked at the level of umbilicus ( paraumbilical ) on the left side where usually the operating surgeon stands . jain point is placed in a straight line drawn vertically upward from a lower point which is 2.5 cm medial to asis [ figure 2 ] . palmer 's point ( p ) , jain point ( j ) , and anterior superior iliac spine . jain point lies in the left paraumbilical region , in a straight line drawn vertically upward from a point 2.5 cm medial and 1 cm above anterior superior iliac spine veress needle entry at jain point . place the left hand over the left middle quadrant around the jain point , make the skin taut between the index and middle finger , and enter the veress needle in a straight direction . it is important to hold the veress needle in such a manner to control the length of the needle going inside the abdominal wall in the first go . after confirming safe entry by above all tests of veress needle , the intra - peritoneal pressure is transiently raised to 25 mmhg and 5 mm trocar is inserted through the jain point when the total volume of co2 insufflation is around 34 l [ figure 4 ] . the basic aim of making entry from jain point is to avoid abdominal and umbilical adhesions . this entry can be anywhere between the umbilicus and xiphisternum according to the pelvic pathology , size of masses , and adhesion sites . primary port at jain point after introducing the 10 mm port , safe abdominal entry of the 5 mm trocar through jain point is also confirmed . then lower right and left ports are inserted 2.5 cm medial and 1 cm above the asis under vision . this distance of 1012 cm gives a good ergonomic working , and these become the main operating ipsilateral ports . the stomach was emptied of secretions and air by the use of orogastric tube by anesthetist , after endotracheal intubation . first and foremost , the whole abdomen is inspected , and all incision sites are carefully noted . jain point is marked at the level of umbilicus ( paraumbilical ) on the left side where usually the operating surgeon stands . jain point is placed in a straight line drawn vertically upward from a lower point which is 2.5 cm medial to asis [ figure 2 ] . palmer 's point ( p ) , jain point ( j ) , and anterior superior iliac spine . jain point lies in the left paraumbilical region , in a straight line drawn vertically upward from a point 2.5 cm medial and 1 cm above anterior superior iliac spine veress needle entry at jain point . place the left hand over the left middle quadrant around the jain point , make the skin taut between the index and middle finger , and enter the veress needle in a straight direction . it is important to hold the veress needle in such a manner to control the length of the needle going inside the abdominal wall in the first go . after confirming safe entry by above all tests of veress needle , the intra - peritoneal pressure is transiently raised to 25 mmhg and 5 mm trocar is inserted through the jain point when the total volume of co2 insufflation is around 34 l [ figure 4 ] . the basic aim of making entry from jain point is to avoid abdominal and umbilical adhesions . after confirming safe entry , the entry for the 10 mm 30 telescope is optimized according to the mandate of the case . this entry can be anywhere between the umbilicus and xiphisternum according to the pelvic pathology , size of masses , and adhesion sites . it is safe to introduce the 10 mm port under the direct visual guidance of 5 mm telescope introduced through jain point . then lower right and left ports are inserted 2.5 cm medial and 1 cm above the asis under vision . the stomach was emptied of secretions and air by the use of orogastric tube by anesthetist , after endotracheal intubation . first and foremost , the whole abdomen is inspected , and all incision sites are carefully noted . jain point is marked at the level of umbilicus ( paraumbilical ) on the left side where usually the operating surgeon stands . jain point is placed in a straight line drawn vertically upward from a lower point which is 2.5 cm medial to asis [ figure 2 ] . palmer 's point ( p ) , jain point ( j ) , and anterior superior iliac spine . jain point lies in the left paraumbilical region , in a straight line drawn vertically upward from a point 2.5 cm medial and 1 cm above anterior superior iliac spine veress needle entry at jain point . place the left hand over the left middle quadrant around the jain point , make the skin taut between the index and middle finger , and enter the veress needle in a straight direction . it is important to hold the veress needle in such a manner to control the length of the needle going inside the abdominal wall in the first go . the most important test is the low initial veress needle intra - peritoneal pressure , which should not be more than 8 mmhg for initial 10 seconds , as also supported by vilos . after confirming safe entry by above all tests of veress needle , the intra - peritoneal pressure is transiently raised to 25 mmhg and 5 mm trocar is inserted through the jain point when the total volume of co2 insufflation is around 34 l [ figure 4 ] . the basic aim of making entry from jain point is to avoid abdominal and umbilical adhesions . this entry can be anywhere between the umbilicus and xiphisternum according to the pelvic pathology , size of masses , and adhesion sites . then lower right and left ports are inserted 2.5 cm medial and 1 cm above the asis under vision . this distance of 1012 cm gives a good ergonomic working , and these become the main operating ipsilateral ports . of the total 2834 laparoscopic cases done between january 2011 and december 2014 , 624 cases had previous one or multiple surgeries . the previous surgery cases , which we have dealt , include wide variety of cases with 86.37% cases ( 539 out of 624 ) of gynae indication and 13.63% cases ( 85 out of 624 ) of general surgery . in the gynae group , 54.4% ( 293 out of 539 ) had previous laparotomy , and 45.6% ( 246 out of 539 ) had previous laparoscopic procedures . previous cesarean cases showed an alarming rate of adhesions , abdominal wall adhesions were present in 89% cases , and total adherence of the uterus itself to the anterior abdominal wall was noted in 11% cases . in this indication , endometriosis , as is shown in the table , with very extensive obliteration of pouch of douglas and endometriomas has been a very frequent surgery with the previous open approach as well as laparoscopy bizarre adhesions have been noted , in repeat laparoscopy . cases with previous surgery for infectious pathologies such as genital koch 's and septic abortion were also encountered with lots of intra - abdominal and pelvic adhesions . the other indications where we did laparoscopy were in cases of previous pelvic floor repairs and microsurgical tubo tubal reanastomosis . in nongynae cases , 67.1% cases ( 57 out of 85 ) cases had previous surgery by open route and 37.3% ( 28 out of 75 ) cases by laparoscopic route . many of the cases had bowel perforations , and multiple drain sites noted over the abdomen . hence , in nongynae group , we dealt extensive pathologies with high potential of intra - abdominal adhesions [ table 1 and pie diagrams 14 ] . patients profile depending on type and indication of previous abdominal surgery patient profile based on type and indications of previous open gynaecological surgery patient profile based on type and indications of previous laproscopic gynaecological surgery patient profile based on type and indications of previous open non gynaecological surgery patient profile based on type and indications of previous laproscopic non gynaecological surgery a total of 209 ( 33.5% ) patients had midline or paramedian incisions , 120 ( 19.2% ) had pfannenstiel incisions , 242 ( 38.8% ) had laparoscopic ports , and 53 ( 8.5% ) had other incisions table 2 . as we earlier stated that these cases were counted only once for the surgery , which had more chance of adhesions . patients profile depending on type of previous surgical incision patients profile depending on number of previous abdominal surgeries intra - abdominal adhesions were found in a significant majority , i.e. , 487 ( 78.0% ) of previous surgery cases . adhesions in the umbilical region were found in 404 ( 64.7% ) patients with previous abdominal surgeries [ figure 5 ] . no significant entry - related complications were reported in the present study with the use of jain point as primary port . the most important and potentially dangerous first step in laparoscopy is the safe and successful insertion of a primary port since half the complications with laparoscopy occur even before the start of the intended procedure . injuries may occur by advancing the instruments toward the posterior abdominal wall and encountering anatomically normal but distended bowel ( type 1 injuries ) or due to visceral adhesions to the anterior abdominal wall ( type 2 injuries ) . the reported incidence of intra - abdominal adhesions after laparotomy ranges between 30% and 90% . even patients with one previous laparoscopic procedure have been found to have both omentum and bowel adhesions beneath the umbilicus and port sites due to valsalva or accidental suturing . royal college of obstetricians and gynecologists guideline on preventing entry - related gynecological laparoscopic injuries states : the umbilicus may not , therefore , be the most appropriate site for primary trocar insertion following previous abdominal surgery . for this reason , alternative primary approaches to laparoscopic surgery have been introduced for use in patients with a history of laparotomy . to avoid injuries , we can either use alternate entry site avoiding umbilicus or can use modified techniques such as open technique , direct trocar entry , disposable shielded trocar , and optical trocar access through umbilicus . probably the safest initial entry site in high - risk patients is the left upper quadrant ( luq ) known as palmer 's point , first described in 1931 by palmer , which lies 3 cm below the left costal border in midclavicular line . this technique is recommended for obese as well as very thin patients and in patients where intra - peritoneal adhesions are being suspected it is essential at this site , to decompress the stomach using nasogastric suction and then introduction of the veress needle perpendicularly to the skin without lifting the abdominal wall . , they have cautioned regarding the angle of entry of long laparoscopic instruments during the course of surgery due to proximity of retroperitoneal structure like left kidney , which we found commonly located below palmer 's point . comparing the vertebral position of abdominal organs also , we are in a safe zone at jain point at around l4 vertebral level , where stomach ( t10 to l1 ) , left kidney ( t12 to l3 ) , and spleen ( t10 to l1 ) lie out of the way . palmer 's point should be avoided in patients known to have hepatospleenomegaly , portal hypertension , history of previous gastric or spleen surgery , or probable gastropancreatic mass . the other limitation of palmer 's point is that we can not use it as an operative port during further course of surgery due to its higher location while jain point is used as a working port during the whole surgery . moreover , at palmer 's point , it can be more difficult to penetrate the peritoneum with the veress needle than it is at the umbilicus . luq is invariably free of adhesions irrespective of previous incisions , but there is a risk of creating an iatrogenic pneumothorax and also injuring neurovascular bundle and stomach . out of 504 cases , one case had bowel injury by veress insertion , and no other significant entry - related injury was encountered . in another study , the middle upper abdomen was used for insufflation and primary cannula insertion in patients with a history of gynecologic cancer or at least , one laparotomy . an open umbilical method hasson technique first described in 1991 by hasson to avoid bowel and vascular injuries was supposed to be beneficial in obese patients , very thin patients , and in cases who had previous one or more laparotomy with midline vertical incision where chances of adhesion are much more . if bowel is so densely adherent to the umbilicus then it is exposed to the risk of inadvertent injury while making the primary 10 mm umbilical entry ; hence , jain point technique moves the primary entry site away from major vessel or bowel , as it is in the left paraumbilical area . trocar were introduced to the market , the manufacturer claimed that this trocar system works in a way that when it enters the abdominal cavity , the sharp edge retracts and the shield springs so that it avoids contact of the tip of the trocar with intra - abdominal content . a retropective study documented 30% of serious injuries caused by laparoscopic entry , and two deaths out of seven deaths caused by laparoscopic entry injury . optical veress needle is a 2.1 mm visual veress needle , which has a 1.2 mm micro laparoscope , is also available to make the initial veress needle guided entry in previous surgery cases . however , in this visual access system , it is essential for the operator to clearly recognize tissue plane transition on the monitor during entry and appreciate the importance of not applying perpendicular force otherwise , you have to face unintended injury . it is true that we can visualize , the bowel loops through peritoneum but to avoid injury of very densely adherent bowel loop is still a challenge , and we have repeatedly found that jain point is free of bowel loops . it is not only the instrument but also it is a point of the abdomen where we have not seen adherent bowel loops . the present study explores the possibility of an ergonomically lower point which is safe in previous surgery cases and further on usable as a working port for pelvic surgeries . furthermore , it is a normal routine reusable trocar that does not require any sophisticated optical trocar or veress needle or an endotip cannula or a micro laparoscope . no significant entry - related injury was encountered during the use of jain point in the present study . jain point , due to its more lateral location , it avoids intra - abdominal adhesions of the midline vertical and paramedian incision sites . being on the left side , it avoids all other adhesions which could be due to appendiceal , ileocecal , gall bladder , liver pathologies , etc . it is important for surgeons to be well versed in more than one safe laparoscopic access method and be knowledgeable in different entry techniques , as our patients have different medical or surgical needs . with our continued entry through jain point in the various type of cases as we have described , it has been found that despite previous infectious pathologies such as koch 's , septicemia , previous multiple bowel , and pelvic surgery , jain point has been found to be free of bowel adhesions . due to the abdominal wall blood supply , bleeding at the site by veress needle or trocar entry the jain point derives its safety from the fact that it is more lateral ; hence , it avoids intra - abdominal adhesions at previous incision sites . being lower down at paraumbilical position , it is ergonomic to be used as operating port later on in the surgery . since it is much lower than the subcostal margin , it can very well be used in cases of hepatosplenomegaly , portal hypertension , and upper abdomen vertical or kocher 's incisions . jain point promises to be a safe portal for laparoscopic entry in cases with a history of previous surgery .

with a rapid rise in motor vehicle use around the world , large numbers of injuries and deaths due to road traffic accidents ( rtas ) occur annually , as detailed in a report of world health organization ( who ) . in some countries children account for a large proportion of traffic injuries . for example , although children aged 15 years and younger account for about 18% of the population in some developed countries , children injured in crashes account for about one - third of the pedestrian accident dataset . brain injury is still a leading cause of death and disability for children and adolescents involved in rtas . a report by langlois et al . showed that approximately 475 000 tbis occurred each year among children aged 0 to 1 years . it was suggested that trauma to the brainstem is a hallmark of severe head injury because of its important role in physiological function . the mechanism of brain injury has been discovered to substantially involve impact to or rotation of the head , causing deformation of the brain , in which brain injury is associated with strain and strain rate . owing to the geometry of the central nervous system ( cns ) , the brainstem is vulnerable to the high strain induced by the combination of compression , tension , and shear , especially in rotational acceleration loading on the head . head computational models are powerful tools for use in studying adult traumatic brain injury ( tbi ) mechanisms and determining adult brain injury tolerance . to discover age - pertinent mechanisms and estimate the injury threshold of children and young adults , models are needed for use in studying pediatric brain injuries . such tools depend heavily on knowledge of pediatric brain tissue material properties and anatomical structures . owing to the paucity in basic data on the material properties of pediatric brain tissue and skull , the child head finite element model was developed , mostly by scaling the adult head models , in which the child model was regarded as a miniature adult model . however , recent studies show age - related differences in the brain , meaning that it is not reasonable to regard the child s head as a miniature adult head . for example , compressive and tensile tests of brain tissue at quasi - static strain rates were done to study the tissue mechanical properties under surgical conditions owing to considerably lower strain rate . other studies were performed to address the mechanical properties of brain tissue during impact events with compression tests at strain rates of 1 , 10 , and 50/s and tension tests at strain rates of 0.9 , 4.3 , and 25/s . in the experiments involving brain tissue compression and tension by rashid et al . , the 30% strain rates were 30 , 60 , and 90/s . numerous studies have reported the adult brain tissue mechanical properties associated with brain injury in experiments at over 20% strain at strain rats of between 1 and 100/s [ 1318 ] . a few studies have reported on the material properties of immature brain tissue : prange , prange and margulies , duhaime et al . consequently , the brain material properties of children are not well understood , especially the tensile behavior . due to ethics constraints , there is great difficulty in obtaining fresh brain tissue from children ; therefore , in the present study we used immature porcine brain tissue to serve as a substitute to study pediatric brain tensile behavior . the aim of this study was to conduct tensile tests in immature brainstem specimens at high stretch velocity ( i.e. , dynamic strain rates ) and then determine the mechanical behavior of the immature brainstem during traumatic events . the research ethics committee of the third military medical university granted ethics approval prior to the tests . all the tests were performed strictly following the rules listed in the guide for the care and use of laboratory animals , as confirmed by the u.s . a total of 23 immature pigs from the same farm , aged 4 weeks ( at a developmental stage similar to human toddlers ) , with an average weight of 160.5 kg , were killed at a local slaughter house ( chongqing born biological technology co. , ltd . ) . the brain was harvested immediately from the pigs as byproduct , and then stored in a thermally insulated stainless steel vessel filled with a saline solution with 0.9% nacl at 450c to maintain ionic balance during transportation to our lab . the cerebrum , cerebellum , and brainstem were separated in the lab , and then the midbrain of the brainstem was cut into a plate with a thickness of 5 mm along the axis of the upper brainstem . a steel circular trephine of a diameter of 9 mm with sharp edges was used to cut the brainstem plate to produce a specimen with a length of 5 mm and a diameter of 9 mm . in this section the main fibers of the brainstem were considered to orient along the long axis of the cylinder , as illustrated in figure 1 . during the preparation , the samples were sprayed with the physiological solution to maintain the in vitro physical environment . to perform the tensile experiment , we used a computer - controlled , high test precision bose electroforce 3100 machine ( bose corporation , gillingham , uk ) , adapted for testing biological specimens . the instrument contains an electromagnetic motor with a stroke resolution of 0.0015 mm and a maximum stroke length of 5 mm , and a minimum load resolution of 6 mn with a 22n load cell . the equipment was designed to have a high response speed so that the configured loading speed can be achieved instantly . the history of stretch and force experienced by the tissue were recorded when performing tensile tests . owing to the great challenge in gripping the very soft biological tissue in the tensile test , great care was taken during preparation and testing of the specimens . we used tensile grips and clips of the apparatus to rigidly fix 2 glass slides in which the one fixed to the stroke was considered as the top , and the one fixed to the load cell was regarded as the bottom . a thin layer of surgical glue ( cyanoacrylate , low - viscosity z105880 - 1ea , sigma - aldrich ) was used to firmly cover the surface of both slides . the prepared tissue specimen was carefully placed on the bottom slide , and then the stroke was slowly lowered to slightly touch the superior surface of the specimen . the stroke was stopped when the load reached 5 mn . to ensure proper adhesion of the specimen to the top and bottom platen after super- and sub - surfaces of the tissue were fixed to both slides using the procedure detailed above , the tests were carried out at stroke velocities of 10 , 100 , and 500 mm / s , in which the strain rate of the specimen was 2 , 20 , and 100/s , respectively . for all the test specimens , no preconditioning was done , and each sample only experienced 1 loading cycle . the final status of the specimens was checked when finishing the test , and the data were not used in subsequent analysis if the glue between the slides and faces of the specimen failed . the nominal stress , s11 , was calculated using formula ( 1 ) , in which f represents the tension force in newtons , and a was the cross - section area of tested tissue in m. from the conclusion drawn by miller , the stretch ratio for the specimen in the tensile test with confined conditions is related to the height change of the tissue if a value of hh is within 1.0~1.3 , so the constitutive models of immature brainstem tissue may be developed based on quasilinear viscoelastic ( qlv ) theory , in which the relationship between stress and strain tensors was derived from a strain energy potential function . the function , w , is defined using the invariants ( i1 , i2 , i3 ) of the strain tensor . the third strain ( i3 ) is unity , as the tissue is incompressible . as a result the invariants , i1 and i2 , are defined as : in the formulas , the squares of the principal stretch ratios , 21,32 , and 32 , had the relationship of 213232=1 , due to incompressibility . as assumed homogeneous deformation conditions of the brain tissue under tension , the eulerian and lagrangian principal axes of strain and stress are aligned with the tensile directions , with any 2 orthogonal axes x2 , x3 . hence , the stretch ratios are indicated by the formula : in the extensional test , the value of is not less than 1 , so i1 and i2 are expressed by the formula : then , the stresses derived from the experiments may be compared to the predicted using the hyperelastic models from the formula : the material parameters of some common hyperelastic models ( e.g. , fung , gent , and ogden ) may be obtained from fitting the curves . widely used to model soft biological tissues in tension , the fung isotropic energy function depends on the first strain invariant only : yielding the following nominal stress component s11 along the x1-axis : where infinitesimal shear modulus (>0 ) and stiffening parameter b(>0 ) are the 2o constant material parameters . gent strain energy function , widely used to describe viscoelastic material , also depends on the first strain invariant only : in which infinitesimal shear modulus >0 and jm>0 ) are the 2 constant material parameters . ogden strain energy function is given by : producing the stress form : in which >0 is the infinitesimal shear modulus , and is a stiffening parameter . in addition , an exponential model was used to fit the lagrange stress - versus - stretch ratio shown by the following formula : all operations for fitting were done using the software originpro 8.0 . the t test was performed for infinitesimal shear modulus , , in which a value of p<0.05 was considered to be significant . a total of 23 immature pigs from the same farm , aged 4 weeks ( at a developmental stage similar to human toddlers ) , with an average weight of 160.5 kg , were killed at a local slaughter house ( chongqing born biological technology co. , ltd . ) . the brain was harvested immediately from the pigs as byproduct , and then stored in a thermally insulated stainless steel vessel filled with a saline solution with 0.9% nacl at 450c to maintain ionic balance during transportation to our lab . the cerebrum , cerebellum , and brainstem were separated in the lab , and then the midbrain of the brainstem was cut into a plate with a thickness of 5 mm along the axis of the upper brainstem . a steel circular trephine of a diameter of 9 mm with sharp edges was used to cut the brainstem plate to produce a specimen with a length of 5 mm and a diameter of 9 mm . in this section the main fibers of the brainstem were considered to orient along the long axis of the cylinder , as illustrated in figure 1 . during the preparation , the samples were sprayed with the physiological solution to maintain the in vitro physical environment . to perform the tensile experiment , we used a computer - controlled , high test precision bose electroforce 3100 machine ( bose corporation , gillingham , uk ) , adapted for testing biological specimens . the instrument contains an electromagnetic motor with a stroke resolution of 0.0015 mm and a maximum stroke length of 5 mm , and a minimum load resolution of 6 mn with a 22n load cell . the equipment was designed to have a high response speed so that the configured loading speed can be achieved instantly . the history of stretch and force experienced by the tissue were recorded when performing tensile tests . owing to the great challenge in gripping the very soft biological tissue in the tensile test , great care was taken during preparation and testing of the specimens . we used tensile grips and clips of the apparatus to rigidly fix 2 glass slides in which the one fixed to the stroke was considered as the top , and the one fixed to the load cell was regarded as the bottom . a thin layer of surgical glue ( cyanoacrylate , low - viscosity z105880 - 1ea , sigma - aldrich ) was used to firmly cover the surface of both slides . the prepared tissue specimen was carefully placed on the bottom slide , and then the stroke was slowly lowered to slightly touch the superior surface of the specimen . to ensure proper adhesion of the specimen to the top and bottom platen , the stroke would stay for 1 min prior to the tensile test . after super- and sub - surfaces of the tissue were fixed to both slides using the procedure detailed above , the tests were carried out at stroke velocities of 10 , 100 , and 500 mm / s , in which the strain rate of the specimen was 2 , 20 , and 100/s , respectively . for all the test specimens , no preconditioning was done , and each sample only experienced 1 loading cycle . the final status of the specimens was checked when finishing the test , and the data were not used in subsequent analysis if the glue between the slides and faces of the specimen failed . the nominal stress , s11 , was calculated using formula ( 1 ) , in which f represents the tension force in newtons , and a was the cross - section area of tested tissue in m. from the conclusion drawn by miller , the stretch ratio for the specimen in the tensile test with confined conditions is related to the height change of the tissue if a value of hh is within 1.0~1.3 , so the constitutive models of immature brainstem tissue may be developed based on quasilinear viscoelastic ( qlv ) theory , in which the relationship between stress and strain tensors was derived from a strain energy potential function . the function , w , is defined using the invariants ( i1 , i2 , i3 ) of the strain tensor . the third strain ( i3 ) is unity , as the tissue is incompressible . as a result the invariants , i1 and i2 , are defined as : in the formulas , the squares of the principal stretch ratios , 21,32 , and 32 , had the relationship of 213232=1 , due to incompressibility . as assumed homogeneous deformation conditions of the brain tissue under tension , the eulerian and lagrangian principal axes of strain and stress are aligned with the tensile directions , with any 2 orthogonal axes x2 , x3 . hence , the stretch ratios are indicated by the formula : in the extensional test , the value of is not less than 1 , so i1 and i2 are expressed by the formula : then , the stresses derived from the experiments may be compared to the predicted using the hyperelastic models from the formula : the material parameters of some common hyperelastic models ( e.g. , fung , gent , and ogden ) may be obtained from fitting the curves . widely used to model soft biological tissues in tension , the fung isotropic energy function depends on the first strain invariant only : yielding the following nominal stress component s11 along the x1-axis : where infinitesimal shear modulus (>0 ) and stiffening parameter b(>0 ) are the 2o constant material parameters . gent strain energy function , widely used to describe viscoelastic material , also depends on the first strain invariant only : in which infinitesimal shear modulus >0 and jm>0 ) are the 2 constant material parameters . ogden strain energy function is given by : producing the stress form : in which >0 is the infinitesimal shear modulus , and is a stiffening parameter . in addition , an exponential model was used to fit the lagrange stress - versus - stretch ratio shown by the following formula : all operations for fitting were done using the software originpro 8.0 . the t test was performed for infinitesimal shear modulus , , in which a value of p<0.05 was considered to be significant . the history curve of lagrange stresses for the tested brainstem specimens determined according to the formula ( 1 ) are illustrated in figure 2 . the brainstem tissue stretch ratio was determined using equation ( 2 ) from the distance that the tissue was stretched . the curves of the stress - versus - stretch ratio shown in figure 3 were convex in shape . the average lagrange stress for the tested specimen at the 30% strain at the strain rates of 2 , 20 , and 100/s were 273.3114 , 515107 , and 1121197 pa , respectively . from the curves of lagrange stress - versus - stretch ratio illustrated in figure 3 , it was found that the material became stiffer with an increase in strain rate . an interesting phenomenon was that all curves indicated points of inflection at the tensile strain of about 2.5% in each test group . the lagrange stress - against - stretch ratio , , can be fitted with the strain energy function of fung ( 0.820r0.933 ) , ogden ( 0.774r0.940 ) , and gent ( 0.650r0.922 ) , and the exponential model ( 0.852r0.981 ) , as shown in formulas ( 1517 ) . figure 4 exhibits curve fitting of fung , ogden , and gent strain energy function , and the exponential model for the average curve experimentally acquired . we noted that the change trend of stress increase at the 2.5% tensile strain did not agree well with the fitted models : table 1 lists the infinitessimal shear modulus , , derived from the fitted strain energy function , fung , ogden and gent , in which the infinitessimal shear modulus , , was found to have a strong rate dependence . for ogden model in the table , for instance , the infinitessimal shear modulus at the strain rate of 100/s seemed to be 4.2 times higher than that at the strain rate of 2/s . road traffic injuries will increase to become the fifth leading cause of death by 2030 and child safety recently has become an important research field around the world according to the who . head injuries are common in children involved in rtas . to protect children from injury and death , therefore , it is necessary to thoroughly understand the mechanisms of tbi prior to developing the measurements to reduce and eliminate the injury . the finite element model , as a powerful tool , may play a significant role in discovering injury mechanisms in the human body , in which injury response and tolerance could be determined . the characteristics of brain material mechanical properties are of importance in developing accurate computational models , contributing to an increasing research focus on brain tissue mechanical characterization , and many adult brain tissue property studies have been carried out . however , few studies have been performed to investigate pediatric brain tissue properties , especially for the tensile test , because of challenges in the experimental techniques . it should be noted that mechanical characterization of brain tissue at high loading velocities is crucial for modeling tbi . brain tissue properties in tension are not as well defined as in other loading modes , such as compression and shear . therefore , we carried out a series of tests to study the tensile material property of the brainstem . methodological issues could contribute to the apparently disparate material properties reported in some published studies and the experimental technology was regarded as the greatest challenge for tensile tests in very soft biological tissue . much of the large disparity in previously reported data may be explained by the more rigorous approaches to control of sample preparation , test conditions , and the test procedures . in the present study , the brain samples were uniform and came from the same source , the experimental protocol remained consistent , and great care was taken when performing the tensile tests , while there was a huge disparity in each test group , as shown in figures 2 and 3 . because of the paucity of data on pediatric brainstem tissue , the present results are difficult to directly compare with previous reports . the present results show that the average lagrange stress for the tested brainstem specimen at the 30% tensile strain at the strain rate of 2 , 20 , and 100/s was 273114 , 515107 , and 1121197 pa , respectively . in the results of miller and chinzei , at the strain rate of 0.64/s and 0.0064/s , the stress sustained by the tested brain specimen was lower than 300 pa , while rashid et al . reported that under tensile tests at the dynamic strain rates of 30/s , 60/s , and 90/s , the stresses were over 3000 pa . similar to the material behavior of adult brain specimens found in some previous reports [ 13,3236 ] , the immature brainstem tissue showed a significant rate dependence in dynamic tensile tests in the present study . with an increase of the strain rate in the tensile tests , using porcine brain tissue , thibault and margulies performed the first study of the immature brain , reporting a significant increase in the complex shear modulus of cortical gray matter obtained from 23-day - old piglets . some published data on brain tissue material properties at low strain rate show the brain tissue becomes softer with strain increase , but another study reported that at dynamic strain rate the brain tissue became stiffer with an increase of the strain . in the present study , the immature porcine brainstem tissue became softer with strain increase , in which all the curves of stress - versus - stretch ratio were convex in shape . the exponential model seems to best match the immature brainstem tissue material property data in the tensile tests compared to the strain energy function . in addition , we noted that there were significant inflection points at the strain of 2.5% for all the test groups . we presumed that the linear viscoelastic regime of immature porcine brainstem in dynamic tensile tests was within the tensile strain of 2.5% . however , all the models developed for the immature porcine brainstem tissue did not fit the multi - stage experimental data combining the linear regime and non - linear regime , and another model needs to be developed to fit the immature brain material property of multi - stage characterization . our results show that the immature porcine brainstem had strong rate dependence , while the estimated shear modulus increased with an increase of the strain rate . in addition , we found that the shear modulus of immature brainstem tissue in the present study was higher than that of immature cerebellum tissue previously determined in the lab following the same procedures . by conducting oscillatory shear tests , arbogast and margulies reported that the complex modulus and its 2 components , the storage and loss modulus , varied with testing frequency , indicating a viscoelastic response . in their results , the experimentally determined shear modulus of the brainstem was 2 orders of magnitude lower than 168 kpa . compared with the brain test data in the same lab , the brainstem has more complex modulus , particularly for the storage modulus component . computer models of the pediatric head commonly use ratios of adult - to - juvenile porcine brain material properties to extrapolate pediatric material properties from human adult material properties because of the paucity of data on the properties pediatric human brain tissue . it should be noted that age - dependent material properties of brain tissue are not linear , but rather rapidly change during the first few years of life , and then change more gradually in early childhood . axons , rather than the surrounding matrix of astrocytes and oligodendrocytes , contribute more to the stiffness of brain tissue according to some previously reported biomechanical models . axons in the pediatric brain undergo rapid myelination during the first year of life and the pediatric brain reaches adult levels of myelination at approximately 18 months old ; therefore , the composition of the 5-year - old brain is more similar to that of an adult brain . in the present study , only 4-week - old pigs were chosen to test the brainstem material properties , in which the material property of the immature brainstem tissue was carried out in 1 direction , and only the tensile experiment , without the shear and compressive tests , was performed . further research is needed to improve our understanding of the details of brainstem mechanical behavior . we attempted to describe the material properties of immature porcine brainstem tissue at dynamic strain rates to build a finite element model of the pediatric brain . first , the number of samples and valid data for studying the material properties of brainstem tissue from immature pigs was limited . furthermore , pigs aged 4 weeks are similar to human infants , which only account for a small proportion of all children . in high - rate tests , there is a period in which the crosshead is ramping up to the specified speed , and it is not exactly clear how long it took to achieve this speed in our study . the brainstem is anisotropic and therefore may violate the assumption of miller that the materials are isotropic and incompressible . in this study , only tensile tests were done , and it would be better if the compression and shear tests could also be performed in discover the material properties of the brain . finally , immature porcine brain tissue served as a substitute to study the pediatric material tensile behavior in the current study , but a significant difference may exist in material properties between pigs and humans . we attempted to describe the material properties of immature porcine brainstem tissue at dynamic strain rates to build a finite element model of the pediatric brain . first , the number of samples and valid data for studying the material properties of brainstem tissue from immature pigs was limited . furthermore , pigs aged 4 weeks are similar to human infants , which only account for a small proportion of all children . in high - rate tests , there is a period in which the crosshead is ramping up to the specified speed , and it is not exactly clear how long it took to achieve this speed in our study . the brainstem is anisotropic and therefore may violate the assumption of miller that the materials are isotropic and incompressible . in this study , only tensile tests were done , and it would be better if the compression and shear tests could also be performed in discover the material properties of the brain . finally , immature porcine brain tissue served as a substitute to study the pediatric material tensile behavior in the current study , but a significant difference may exist in material properties between pigs and humans . to address the material properties of immature brainstem in tension , dynamic tensile tests at 3 stretch speeds were performed for the brainstem tissue specimens obtained from 4-week - old pigs in the present study . although some limitations exist in our study , the results show that the immature brainstem is a rate - dependent material in dynamic tensile tests , and the tissue becomes stiffer with an increase of strain rate , with inflection points in all the test groups at the strain of about 2.5% . the function of fung , ogden , gent , and exponential model may match well with the material behavior of the brainstem for immature pigs in dynamic tensile testing . the reported results may be useful in understanding brain injuries in children who have been in road traffic accidents .

backgroundmany brain injury cases involve pediatric road traffic accidents , and among these , brainstem injury causes disastrous outcomes . a thorough understanding of the tensile characterization of immature brainstem tissue is crucial in modeling traumatic brain injury sustained by children , but limited experimental data in tension is available for the immature brain tissue at dynamic strain rates.material/methodswe harvested brainstem tissue from immature pigs ( about 4 weeks old , and at a developmental stage similar to that of human toddlers ) as a byproduct from a local slaughter house and very carefully prepared the samples . tensile tests were performed on specimens at dynamic strain rates of 2/s , 20/s , and 100/s using a biological material instrument . the constitutive models , fung , ogden , gent , and exponential function , for immature brainstem tissue material property were developed for the recorded experimental data using originpro 8.0 software . the t test was performed for infinitesimal shear modules.resultsthe curves of stress - versus - stretch ratio were convex in shape , and inflection points were found in all the test groups at the strain of about 2.5% . the average lagrange stress of the immature brainstem specimen at the 30% strain at the strain rates of 2 , 20 , and 100/s was 273114 , 515107 , and 1121197 pa , respectively . the adjusted r - square ( r2 ) of fung , ogden , gent , and exponential model was 0.820r20.933 , 0.774r20.940 , 0.650r20.922 , and 0.852r20.981 , respectively . the infinitesimal shear modulus of the strain energy functions showed a significant association with the strain rate ( p<0.01).conclusionsthe immature brainstem is a rate - dependent material in dynamic tensile tests , and the tissue becomes stiffer with increased strain rate . the reported results may be useful in the study of brain injuries in children who sustain injuries in road traffic accidents . further research in more detail should be performed in the future .

Background Material and Methods Specimen preparation Experimental study Experimental protocol Data processing and analysis Results Discussion Limitations Conclusions

it was suggested that trauma to the brainstem is a hallmark of severe head injury because of its important role in physiological function . the mechanism of brain injury has been discovered to substantially involve impact to or rotation of the head , causing deformation of the brain , in which brain injury is associated with strain and strain rate . owing to the geometry of the central nervous system ( cns ) , the brainstem is vulnerable to the high strain induced by the combination of compression , tension , and shear , especially in rotational acceleration loading on the head . such tools depend heavily on knowledge of pediatric brain tissue material properties and anatomical structures . owing to the paucity in basic data on the material properties of pediatric brain tissue and skull , the child head finite element model was developed , mostly by scaling the adult head models , in which the child model was regarded as a miniature adult model . for example , compressive and tensile tests of brain tissue at quasi - static strain rates were done to study the tissue mechanical properties under surgical conditions owing to considerably lower strain rate . other studies were performed to address the mechanical properties of brain tissue during impact events with compression tests at strain rates of 1 , 10 , and 50/s and tension tests at strain rates of 0.9 , 4.3 , and 25/s . , the 30% strain rates were 30 , 60 , and 90/s . numerous studies have reported the adult brain tissue mechanical properties associated with brain injury in experiments at over 20% strain at strain rats of between 1 and 100/s [ 1318 ] . a few studies have reported on the material properties of immature brain tissue : prange , prange and margulies , duhaime et al . due to ethics constraints , there is great difficulty in obtaining fresh brain tissue from children ; therefore , in the present study we used immature porcine brain tissue to serve as a substitute to study pediatric brain tensile behavior . the aim of this study was to conduct tensile tests in immature brainstem specimens at high stretch velocity ( i.e. , dynamic strain rates ) and then determine the mechanical behavior of the immature brainstem during traumatic events . all the tests were performed strictly following the rules listed in the guide for the care and use of laboratory animals , as confirmed by the u.s . a total of 23 immature pigs from the same farm , aged 4 weeks ( at a developmental stage similar to human toddlers ) , with an average weight of 160.5 kg , were killed at a local slaughter house ( chongqing born biological technology co. , ltd . ) the cerebrum , cerebellum , and brainstem were separated in the lab , and then the midbrain of the brainstem was cut into a plate with a thickness of 5 mm along the axis of the upper brainstem . owing to the great challenge in gripping the very soft biological tissue in the tensile test , great care was taken during preparation and testing of the specimens . we used tensile grips and clips of the apparatus to rigidly fix 2 glass slides in which the one fixed to the stroke was considered as the top , and the one fixed to the load cell was regarded as the bottom . the prepared tissue specimen was carefully placed on the bottom slide , and then the stroke was slowly lowered to slightly touch the superior surface of the specimen . to ensure proper adhesion of the specimen to the top and bottom platen after super- and sub - surfaces of the tissue were fixed to both slides using the procedure detailed above , the tests were carried out at stroke velocities of 10 , 100 , and 500 mm / s , in which the strain rate of the specimen was 2 , 20 , and 100/s , respectively . for all the test specimens , no preconditioning was done , and each sample only experienced 1 loading cycle . the final status of the specimens was checked when finishing the test , and the data were not used in subsequent analysis if the glue between the slides and faces of the specimen failed . the nominal stress , s11 , was calculated using formula ( 1 ) , in which f represents the tension force in newtons , and a was the cross - section area of tested tissue in m. from the conclusion drawn by miller , the stretch ratio for the specimen in the tensile test with confined conditions is related to the height change of the tissue if a value of hh is within 1.0~1.3 , so the constitutive models of immature brainstem tissue may be developed based on quasilinear viscoelastic ( qlv ) theory , in which the relationship between stress and strain tensors was derived from a strain energy potential function . the function , w , is defined using the invariants ( i1 , i2 , i3 ) of the strain tensor . as a result the invariants , i1 and i2 , are defined as : in the formulas , the squares of the principal stretch ratios , 21,32 , and 32 , had the relationship of 213232=1 , due to incompressibility . as assumed homogeneous deformation conditions of the brain tissue under tension , the eulerian and lagrangian principal axes of strain and stress are aligned with the tensile directions , with any 2 orthogonal axes x2 , x3 . , fung , gent , and ogden ) may be obtained from fitting the curves . widely used to model soft biological tissues in tension , the fung isotropic energy function depends on the first strain invariant only : yielding the following nominal stress component s11 along the x1-axis : where infinitesimal shear modulus (>0 ) and stiffening parameter b(>0 ) are the 2o constant material parameters . gent strain energy function , widely used to describe viscoelastic material , also depends on the first strain invariant only : in which infinitesimal shear modulus >0 and jm>0 ) are the 2 constant material parameters . ogden strain energy function is given by : producing the stress form : in which >0 is the infinitesimal shear modulus , and is a stiffening parameter . in addition , an exponential model was used to fit the lagrange stress - versus - stretch ratio shown by the following formula : all operations for fitting were done using the software originpro 8.0 . the t test was performed for infinitesimal shear modulus , , in which a value of p<0.05 was considered to be significant . a total of 23 immature pigs from the same farm , aged 4 weeks ( at a developmental stage similar to human toddlers ) , with an average weight of 160.5 kg , were killed at a local slaughter house ( chongqing born biological technology co. , ltd . ) the cerebrum , cerebellum , and brainstem were separated in the lab , and then the midbrain of the brainstem was cut into a plate with a thickness of 5 mm along the axis of the upper brainstem . owing to the great challenge in gripping the very soft biological tissue in the tensile test , great care was taken during preparation and testing of the specimens . we used tensile grips and clips of the apparatus to rigidly fix 2 glass slides in which the one fixed to the stroke was considered as the top , and the one fixed to the load cell was regarded as the bottom . to ensure proper adhesion of the specimen to the top and bottom platen , the stroke would stay for 1 min prior to the tensile test . after super- and sub - surfaces of the tissue were fixed to both slides using the procedure detailed above , the tests were carried out at stroke velocities of 10 , 100 , and 500 mm / s , in which the strain rate of the specimen was 2 , 20 , and 100/s , respectively . for all the test specimens , no preconditioning was done , and each sample only experienced 1 loading cycle . the final status of the specimens was checked when finishing the test , and the data were not used in subsequent analysis if the glue between the slides and faces of the specimen failed . the nominal stress , s11 , was calculated using formula ( 1 ) , in which f represents the tension force in newtons , and a was the cross - section area of tested tissue in m. from the conclusion drawn by miller , the stretch ratio for the specimen in the tensile test with confined conditions is related to the height change of the tissue if a value of hh is within 1.0~1.3 , so the constitutive models of immature brainstem tissue may be developed based on quasilinear viscoelastic ( qlv ) theory , in which the relationship between stress and strain tensors was derived from a strain energy potential function . the function , w , is defined using the invariants ( i1 , i2 , i3 ) of the strain tensor . as a result the invariants , i1 and i2 , are defined as : in the formulas , the squares of the principal stretch ratios , 21,32 , and 32 , had the relationship of 213232=1 , due to incompressibility . as assumed homogeneous deformation conditions of the brain tissue under tension , the eulerian and lagrangian principal axes of strain and stress are aligned with the tensile directions , with any 2 orthogonal axes x2 , x3 . , fung , gent , and ogden ) may be obtained from fitting the curves . widely used to model soft biological tissues in tension , the fung isotropic energy function depends on the first strain invariant only : yielding the following nominal stress component s11 along the x1-axis : where infinitesimal shear modulus (>0 ) and stiffening parameter b(>0 ) are the 2o constant material parameters . gent strain energy function , widely used to describe viscoelastic material , also depends on the first strain invariant only : in which infinitesimal shear modulus >0 and jm>0 ) are the 2 constant material parameters . ogden strain energy function is given by : producing the stress form : in which >0 is the infinitesimal shear modulus , and is a stiffening parameter . in addition , an exponential model was used to fit the lagrange stress - versus - stretch ratio shown by the following formula : all operations for fitting were done using the software originpro 8.0 . the t test was performed for infinitesimal shear modulus , , in which a value of p<0.05 was considered to be significant . the brainstem tissue stretch ratio was determined using equation ( 2 ) from the distance that the tissue was stretched . the curves of the stress - versus - stretch ratio shown in figure 3 were convex in shape . the average lagrange stress for the tested specimen at the 30% strain at the strain rates of 2 , 20 , and 100/s were 273.3114 , 515107 , and 1121197 pa , respectively . from the curves of lagrange stress - versus - stretch ratio illustrated in figure 3 , it was found that the material became stiffer with an increase in strain rate . an interesting phenomenon was that all curves indicated points of inflection at the tensile strain of about 2.5% in each test group . the lagrange stress - against - stretch ratio , , can be fitted with the strain energy function of fung ( 0.820r0.933 ) , ogden ( 0.774r0.940 ) , and gent ( 0.650r0.922 ) , and the exponential model ( 0.852r0.981 ) , as shown in formulas ( 1517 ) . figure 4 exhibits curve fitting of fung , ogden , and gent strain energy function , and the exponential model for the average curve experimentally acquired . we noted that the change trend of stress increase at the 2.5% tensile strain did not agree well with the fitted models : table 1 lists the infinitessimal shear modulus , , derived from the fitted strain energy function , fung , ogden and gent , in which the infinitessimal shear modulus , , was found to have a strong rate dependence . for ogden model in the table , for instance , the infinitessimal shear modulus at the strain rate of 100/s seemed to be 4.2 times higher than that at the strain rate of 2/s . the finite element model , as a powerful tool , may play a significant role in discovering injury mechanisms in the human body , in which injury response and tolerance could be determined . the characteristics of brain material mechanical properties are of importance in developing accurate computational models , contributing to an increasing research focus on brain tissue mechanical characterization , and many adult brain tissue property studies have been carried out . however , few studies have been performed to investigate pediatric brain tissue properties , especially for the tensile test , because of challenges in the experimental techniques . it should be noted that mechanical characterization of brain tissue at high loading velocities is crucial for modeling tbi . therefore , we carried out a series of tests to study the tensile material property of the brainstem . much of the large disparity in previously reported data may be explained by the more rigorous approaches to control of sample preparation , test conditions , and the test procedures . in the present study , the brain samples were uniform and came from the same source , the experimental protocol remained consistent , and great care was taken when performing the tensile tests , while there was a huge disparity in each test group , as shown in figures 2 and 3 . the present results show that the average lagrange stress for the tested brainstem specimen at the 30% tensile strain at the strain rate of 2 , 20 , and 100/s was 273114 , 515107 , and 1121197 pa , respectively . in the results of miller and chinzei , at the strain rate of 0.64/s and 0.0064/s , the stress sustained by the tested brain specimen was lower than 300 pa , while rashid et al . reported that under tensile tests at the dynamic strain rates of 30/s , 60/s , and 90/s , the stresses were over 3000 pa . similar to the material behavior of adult brain specimens found in some previous reports [ 13,3236 ] , the immature brainstem tissue showed a significant rate dependence in dynamic tensile tests in the present study . with an increase of the strain rate in the tensile tests , using porcine brain tissue , thibault and margulies performed the first study of the immature brain , reporting a significant increase in the complex shear modulus of cortical gray matter obtained from 23-day - old piglets . some published data on brain tissue material properties at low strain rate show the brain tissue becomes softer with strain increase , but another study reported that at dynamic strain rate the brain tissue became stiffer with an increase of the strain . in the present study , the immature porcine brainstem tissue became softer with strain increase , in which all the curves of stress - versus - stretch ratio were convex in shape . the exponential model seems to best match the immature brainstem tissue material property data in the tensile tests compared to the strain energy function . in addition , we noted that there were significant inflection points at the strain of 2.5% for all the test groups . we presumed that the linear viscoelastic regime of immature porcine brainstem in dynamic tensile tests was within the tensile strain of 2.5% . however , all the models developed for the immature porcine brainstem tissue did not fit the multi - stage experimental data combining the linear regime and non - linear regime , and another model needs to be developed to fit the immature brain material property of multi - stage characterization . our results show that the immature porcine brainstem had strong rate dependence , while the estimated shear modulus increased with an increase of the strain rate . in addition , we found that the shear modulus of immature brainstem tissue in the present study was higher than that of immature cerebellum tissue previously determined in the lab following the same procedures . in their results , the experimentally determined shear modulus of the brainstem was 2 orders of magnitude lower than 168 kpa . compared with the brain test data in the same lab , the brainstem has more complex modulus , particularly for the storage modulus component . it should be noted that age - dependent material properties of brain tissue are not linear , but rather rapidly change during the first few years of life , and then change more gradually in early childhood . axons in the pediatric brain undergo rapid myelination during the first year of life and the pediatric brain reaches adult levels of myelination at approximately 18 months old ; therefore , the composition of the 5-year - old brain is more similar to that of an adult brain . in the present study , only 4-week - old pigs were chosen to test the brainstem material properties , in which the material property of the immature brainstem tissue was carried out in 1 direction , and only the tensile experiment , without the shear and compressive tests , was performed . further research is needed to improve our understanding of the details of brainstem mechanical behavior . we attempted to describe the material properties of immature porcine brainstem tissue at dynamic strain rates to build a finite element model of the pediatric brain . first , the number of samples and valid data for studying the material properties of brainstem tissue from immature pigs was limited . furthermore , pigs aged 4 weeks are similar to human infants , which only account for a small proportion of all children . in high - rate tests , there is a period in which the crosshead is ramping up to the specified speed , and it is not exactly clear how long it took to achieve this speed in our study . in this study , only tensile tests were done , and it would be better if the compression and shear tests could also be performed in discover the material properties of the brain . finally , immature porcine brain tissue served as a substitute to study the pediatric material tensile behavior in the current study , but a significant difference may exist in material properties between pigs and humans . we attempted to describe the material properties of immature porcine brainstem tissue at dynamic strain rates to build a finite element model of the pediatric brain . first , the number of samples and valid data for studying the material properties of brainstem tissue from immature pigs was limited . in high - rate tests , there is a period in which the crosshead is ramping up to the specified speed , and it is not exactly clear how long it took to achieve this speed in our study . in this study , only tensile tests were done , and it would be better if the compression and shear tests could also be performed in discover the material properties of the brain . finally , immature porcine brain tissue served as a substitute to study the pediatric material tensile behavior in the current study , but a significant difference may exist in material properties between pigs and humans . to address the material properties of immature brainstem in tension , dynamic tensile tests at 3 stretch speeds were performed for the brainstem tissue specimens obtained from 4-week - old pigs in the present study . although some limitations exist in our study , the results show that the immature brainstem is a rate - dependent material in dynamic tensile tests , and the tissue becomes stiffer with an increase of strain rate , with inflection points in all the test groups at the strain of about 2.5% . the function of fung , ogden , gent , and exponential model may match well with the material behavior of the brainstem for immature pigs in dynamic tensile testing . the reported results may be useful in understanding brain injuries in children who have been in road traffic accidents .

maintaining mobility outside the home is an essential part of independent and active living for the elderly . driving is often the most convenient form of transportation , especially in places with limited or no public transportation services . it is the primary means of transportation among the elderly in the us1,2 and is becoming increasingly important in europe.3,4 visual cues provide the most crucial sensory information for safe driving performance.5,6 although many factors contribute to safe driving , crash rates and driving - related injuries have been associated with deteriorating vision.5,7,8 consequently , licensing authorities have instituted minimum vision requirements for maintaining driving privileges . the number of patients with visual impairments due to progressive eye diseases , such as glaucoma , is expected to rise in coming years as the population ages worldwide.810 because glaucomatous damage progresses to visual impairment , many will fail to meet the vision requirements for legal fitness to drive and will be forced to relinquish their driver s license . for example , former drivers lose their mobility and independence , making it difficult to obtain essential services and daily goods , eg , doctors appointments and groceries.11 they also reduce the frequency of contact with relatives and friends as social opportunities become restricted.12 depressive symptoms are higher in former drivers , possibly as a result of these consequences.1315 the economic consequences of losing driving privileges are not merely limited to the former driver . drivers who lose their license must also rely on others for transportation , which may be costly in terms of a caregiver s time , and monetarily costly if the caregiver needs to make alternative work arrangements . as eye diseases progress to low vision and blindness , the former driver may no longer be able to participate fully in life and/or work activities , and disability pensions may be sought . furthermore , people who lose the ability to drive are also more likely to require long - term care facilities,16 posing a personal and/or societal financial burden . fortunately , effective treatment can slow the rate of disease progression17,18 and potentially increase the length of time that a person is deemed fit to drive . for glaucoma , , researchers and clinicians have become increasingly concerned with slowing the progression of visual field loss,19 which may be as important as achieving target intraocular pressures . as a complement to visual field measurements , treatment effectiveness may also be considered in terms of postponing milestones , such as driver s license forfeiture and receipt of disability benefits . measuring the rate of progression to defined milestones such as these would provide an important link between visual field measurements and patients quality of life . however , the degree of consensus between countries regarding driver s license vision requirements and entitlement to disability pensions has not been clearly defined . the purpose of the present study was to review the requirements for holding a driver s license and rules for obtaining a disability pension due to low vision . results underscore the possibility of using these outcomes as milestones to describe progressive eye disease by relating disease severity to disability . this review was conducted for five european union countries and selected states within the us . research was conducted to identify driver s license rules and requirements for passenger cars and disability pensions due to low vision for six countries ( germany , spain , italy , france , the uk , and the us ) . for each of the six countries , the driver s license regulations were investigated with regard to visual standards . in the us , where the regulations are state - dependent , we focused on regulations from three arbitrarily chosen states ( california , iowa , and new york ) . in europe , the european union member states have their individual driver s license requirements and guidelines , but the european union regulations can potentially overrule these . sources included government reports , research reports , government and nonprofit websites , and journal articles . linguistic experts assisted with the review to include relevant nonenglish documents . national findings and intercountry differences were reviewed , discussed , and verified with ophthalmology specialists from each country . the most important aspects of the driver s license requirements for passenger cars are summarized in table 1 for the relevant european union countries . the medical requirements for driver s licensing in european countries are affected by the minimum standard established by the european union . drivers of cars and motorcycles are required to have a binocular visual acuity of at least 0.5 ( 20/40 feet , 6/12 or 5/10 meters ) with or without correction , and binocular visual field standards are limited to no less than 120.20 for monocular vision , applicants must have a corrected or uncorrected visual acuity of at least 0.6 ( approximately 20/30 feet , 6/9 meters ) . also , medical authorities must certify that the condition has existed sufficiently long enough to allow adaptation and that the field of vision in the subject eye is normal . in cases where a progressive eye disease is detected or declared , driver s licenses may be issued or renewed provided that the applicant undergoes regular medical examinations.20 exceptions can be made in cases duly justified by a favorable medical opinion and a positive practical test . at these examinations , attention is paid to the state of visual function , recovery from glare , contrast sensitivity , and twilight vision.21 driver s licenses are not issued if the medical examination shows any other eye condition that would compromise driving safety.20 european countries generally follow the european union driver s license requirements with a few exceptions . in the uk , visual acuity requirements are based on the number plate eye test in which the applicant must be able to read a registration mark fixed to a motor vehicle.2224 the number plate test has been thought to convert to a snellen acuity of between 6/15 and 6/10 ( 20/5020/40 feet or 0.40.6 decimal).25,26 monocular vision is allowed in the uk if there is a normal monocular visual field and no area of defect caused by pathology.22,27 binocular visual acuity requirements for the remaining european union countries are similar . france and spain follow the european union standard , in that both have a minimum binocular vision requirement of 0.5 ( 20/40 feet , 5/10 meters).28,29 however , in italy , the best - corrected visual acuity must be at least 1.0 ( 10/10 , 20/20 feet , 6/6 meters ) for binocular vision , with a minimum of 0.2 ( 2/10 , 20/100 feet , 6/30 meters ) for the worse - seeing eye.30 in germany , applicants must also have a central visual acuity of at least 1.0 ( 0.7/0.7 , 20/20 feet , 6/6 meters ) with or without corrective means.31 stricter standards are enforced for monocular vision in germany and france , which require visual acuity of the better eye to be at least 0.6.31 in spain and italy , driving with monocular vision is normally not allowed . the visual acuity requirements for holding a driver s license in the european union are similar to those in the us states ( table 2 ) . in california , visual acuity of 20/40 ( 6/12 meters , 0.5 decimal ) with or without correction is required.32,33 visual acuity measurements are obtained for both eyes together and for each eye separately while both eyes remain open , as in normal driving.32,33 in new york and iowa , a driver must also have 20/40 ( 6/12 meters , 0.5 decimal ) vision in at least one eye with or without corrective lenses.34,35 the minimum visual field requirement in the uk is at least 120 horizontally ( table 1 ) . furthermore , there can not be significant defects in the binocular field that encroach within 20 of fixation above or below the horizontal meridian.24,27 the same requirement is observed in germany and france , but the central visual field up to 30 must be normal.28,31 for spain and italy , no diameter is given for visual field requirements . however , the field of vision , whether binocular or monocular , also has to be normal ( eg , 120).29,30 in the us , california does not have visual field requirements ( table 2 ) . the standards are slightly more stringent for new york and iowa compared with european union countries . in new york , for individuals with corrected vision of less than 20/40 but no less than 20/70 , the horizontal binocular field of vision must be no less than 140.36 in iowa , the visual field requirement is also 140 or better.34 to drive in the uk , the applicant has to meet the visual standards . if the driver is unable to meet the standards , he or she must not drive and the license must be refused or revoked . however , the european union regulations can override the uk standards , and the driver can apply to be considered as an exceptional case and granted an exemption.37 in the uk , licenses are normally issued to be valid until the driver is aged 70 years , unless restricted to a shorter duration for medical reasons . there is no upper limit , but after the age of 70 years , renewal is necessary every three years.27,38 in germany , if the applicant does not pass the test , an eye medical investigation is required , and minimum requirements of this test must be fulfilled . visual acuity must not fall below 0.5 in the best eye and 0.2 in the worse eye.31 renewal is not determined by age , and there are no medical requirements for renewal.38 for requirements in france , in case of severe vision loss of an eye , the driver will temporarily lose permission to drive . also , night vision is required . restricted licenses for daytime driving are given only if a specialist deems that the visual field is normal.28 there are no age - related renewal procedures or medical requirements for renewal.38 in the case of alterations of mesopic vision or glare , restrictions and limitations in spain must be determined according to ophthalmologic criteria so that secure driving can be guaranteed . driving with diplopia is not allowed . people in spain with monocular vision and a visual acuity of higher than 0.6 on the better - seeing eye can get permission to drive , provided they fulfill all other requirements and travel no faster than 100 km / hour.39 in spain the renewal cycles are 10 years for drivers younger than 45 years of age . for drivers between 45 and 70 years of age , the renewal cycles are five years . for drivers who are 70 years of age or older , renewal has to take place every other year.40 in italy , a medical / legal specialist and a general ophthalmologist examine the medical certifications , ie , ophthalmic check reports and visual field examinations . candidates must possess sufficient chromatic sense , adequate nocturnal vision , and binocular vision.41 the renewal cycle in italy is 10 years until the age of 50 years . between the ages of 50 and 70 years , the renewal cycle is five years , whereas after age 70 years the renewal cycle is accelerated to an interval of three years.42 driving requirements in the us are varied . in california , candidates who do not pass the vision examination may be referred to a vision specialist , who determines the ability to drive safely and may prescribe eye glasses or a stronger prescription ( table 2).32,33 renewal takes place every five years regardless of the age of the driver . however , drivers who are 70 years of age or older will have to undergo a vision examination to get a renewal.43 if the driver fails the vision test , a supplementary driving performance evaluation ( driving test ) can be given to determine whether the driver can compensate for the vision impairment . if this supplementary driving test is passed , the driver s license can be renewed with restrictions added to the license . restrictions can include requirements for special mechanical devices to be placed on the vehicle , limiting when and where a person may drive ( eg , no freeway driving , no nighttime driving ) , or requiring eye glasses or corrective contact lenses . in new york , if the vision test is not passed , further assessment must be made by a physician , ophthalmologist , or optometrist.36 renewal takes place every five years no matter what age the driver is . for a renewal process , the driver must prove that a visual acuity ( eye ) test has been passed within six months , or within one year as determined by a licensed health care professional.36,43 in iowa , if visual acuity is less than 20/40 but at least 20/50 ( 6/126/15 meters , 0.50.4 ) , no driving is allowed when headlights are required . if visual acuity is less than 20/50 but at least 20/70 , driving at speeds over 35 mph is not allowed , in addition to no driving when headlights are required . driving is not permitted for visual acuity less than 20/70 . in respect of visual field requirements , left and right outside rearview mirrors are required if the field of vision is less than 140 but at least 115 for the single best eye ( 70 temporal and 45 nasal ) . for a field of vision less than 115 , driving is not permitted.34 the normal renewal cycle in iowa is five years , and for drivers who are 70 years of age or older it is two years.43 the requirements for obtaining disability pensions due to low or no vision were found to be unique for each country . the most important aspects of disability pension qualifications have been summarized in table 3 for the relevant european union countries and us states . in the uk , people can claim a disability living allowance if they are certified blind ( severely sight - impaired ) or partially sighted ( sight - impaired ) . severely sight - impaired is defined as a visual acuity below 3/60 or 1/18 ( 20/400 feet ) tested with the snellen test , or with visual acuity better than 3/60 but below 6/60 ( 20/200 feet ) with a very restricted visual field . a person can be certified blind ( or severely sight - impaired ) if he or she is unable to perform work for which eyesight is essential . partially sighted ( sight - impaired ) corresponds to visual acuity from 3/60 to 6/60 ( 20/400 to 20/200 feet ) with a full field or visual acuity up to 6/24 ( 20/80 feet ) with moderate contraction of the field , aphakia ( lens removal ) , or opacities blocking vision in the eye itself , or with visual acuity of 6/18 ( 20/60 feet ) or better if they have a gross visual field defect ( of both eyes , such as hemianopia ) or marked contraction of the visual field ( eg , in retinitis pigmentosa or glaucoma ) . the visual acuity and visual field guidelines in the uk for partially sighted ( sight - impaired ) designations suggest that a person can be certified as such if he or she is substantially and permanently disabled by defective vision caused by congenital defect , illness , or injury.44 furthermore , a person is eligible to receive a disability living allowance only if he or she is below the age of 65 years , has severe sight problems for at least three months , and is expected to have severe sight problems for another six months.45,46 other european union countries have different requirements for receiving disability allowances . a blind person is someone whose central best - corrected visual acuity is 1/50 ( 20/1000 feet , 6/300 meters ) in the better eye.47 for france , 50% of disability corresponds to visual acuity below 3/10 ( approximately 20/66 feet , 6/20 meters ) in both eyes or 50% of visual field loss according to the esterman score ( binocular field ) . eighty percent of disability corresponds to visual acuity below 1/10 ( 20/200 feet , 6/60 meters ) in both eyes , or 80% of visual field loss according to the esterman score ( binocular field ) . in france legal blindness is visual acuity 1/20 ( 20/400 feet , 6/120 meters , 0.05 decimal ) of one eye and visual acuity of the other eye below 1/20 , with peripheral visual field deficiency when the visual field does not exceed 20 in the largest area.48,49 in spain , compensation for the blind relies heavily on once ( spanish national organization for the blind ) . membership conditions are a best - corrected visual acuity of 0.1 ( 1/10 on a wecker scale ) or lower , a visual field reduced to 10 or less , and spanish citizenship.50,51 between normal vision and blindness , there is a wide range of disabilities , eg , visual disability , serious visual disability , and low vision . in all three cases , visual acuity and visual field visual disability is defined as a person who has reduced or very reduced visual capacity ( even though the person is using glasses ) and there is no possibility for improvement with surgical treatment.52 people with disabilities can apply for the pensin no contributiva por invalidez and pensin por incapacidad permanente.53,54 in order to obtain any type of disability pension , it is necessary to pass an examination by specialized doctors . the application procedure is relatively complicated and involves a panel of specialized doctors who decide whether the candidate is sufficiently disabled for eligibility . pensions for the blind ( pensione ciechi civili ) in italy apply only to people with complete blindness . the requirements also include italian citizenship and residency , and the citizen must be older than 18 years . requirements for the partially blind pension ( pensione per ciechi parziali ) include a visual acuity of 1/20 ( 20/400 feet , 6/120 meters ) or lower for both eyes . the candidate can be a european union citizen with an italian residence or a noneuropean union citizen with a work permit in italy.55,56 in the us , people who are legally blind can obtain disability benefits . the requirements and definition for legal blindness are established in the social security act , section 216.57 a person is considered legally blind if central visual acuity is 20/200 ( 6/60 meters , 0.1 decimal ) or less in the better eye with the use of a correcting lens . an eye with a field of vision limitation , such that the widest diameter of the visual field subtends an angle of 20 or less , is considered to have a central visual acuity of 20/200 or less.57,58 a person may also receive disability benefits even if he or she is not considered legally blind , if the vision problems alone or combined with other health problems prevent the person from working . for social security disability benefits , a person must have worked long enough to have paid social security taxes . for supplemental security income payments based on disability and blindness , it is not necessary to have been employed , but income and resources must be below state - imposed limits . the most important aspects of the driver s license requirements for passenger cars are summarized in table 1 for the relevant european union countries . the medical requirements for driver s licensing in european countries are affected by the minimum standard established by the european union . drivers of cars and motorcycles are required to have a binocular visual acuity of at least 0.5 ( 20/40 feet , 6/12 or 5/10 meters ) with or without correction , and binocular visual field standards are limited to no less than 120.20 for monocular vision , applicants must have a corrected or uncorrected visual acuity of at least 0.6 ( approximately 20/30 feet , 6/9 meters ) . also , medical authorities must certify that the condition has existed sufficiently long enough to allow adaptation and that the field of vision in the subject eye is normal . in cases where a progressive eye disease is detected or declared , driver s licenses may be issued or renewed provided that the applicant undergoes regular medical examinations.20 exceptions can be made in cases duly justified by a favorable medical opinion and a positive practical test . at these examinations , attention is paid to the state of visual function , recovery from glare , contrast sensitivity , and twilight vision.21 driver s licenses are not issued if the medical examination shows any other eye condition that would compromise driving safety.20 european countries generally follow the european union driver s license requirements with a few exceptions . in the uk , visual acuity requirements are based on the number plate eye test in which the applicant must be able to read a registration mark fixed to a motor vehicle.2224 the number plate test has been thought to convert to a snellen acuity of between 6/15 and 6/10 ( 20/5020/40 feet or 0.40.6 decimal).25,26 monocular vision is allowed in the uk if there is a normal monocular visual field and no area of defect caused by pathology.22,27 binocular visual acuity requirements for the remaining european union countries are similar . france and spain follow the european union standard , in that both have a minimum binocular vision requirement of 0.5 ( 20/40 feet , 5/10 meters).28,29 however , in italy , the best - corrected visual acuity must be at least 1.0 ( 10/10 , 20/20 feet , 6/6 meters ) for binocular vision , with a minimum of 0.2 ( 2/10 , 20/100 feet , 6/30 meters ) for the worse - seeing eye.30 in germany , applicants must also have a central visual acuity of at least 1.0 ( 0.7/0.7 , 20/20 feet , 6/6 meters ) with or without corrective means.31 stricter standards are enforced for monocular vision in germany and france , which require visual acuity of the better eye to be at least 0.6.31 in spain and italy , driving with monocular vision is normally not allowed . the visual acuity requirements for holding a driver s license in the european union are similar to those in the us states ( table 2 ) . in california , visual acuity of 20/40 ( 6/12 meters , 0.5 decimal ) with or without correction is required.32,33 visual acuity measurements are obtained for both eyes together and for each eye separately while both eyes remain open , as in normal driving.32,33 in new york and iowa , a driver must also have 20/40 ( 6/12 meters , 0.5 decimal ) vision in at least one eye with or without corrective lenses.34,35 the minimum visual field requirement in the uk is at least 120 horizontally ( table 1 ) . furthermore , there can not be significant defects in the binocular field that encroach within 20 of fixation above or below the horizontal meridian.24,27 the same requirement is observed in germany and france , but the central visual field up to 30 must be normal.28,31 for spain and italy , no diameter is given for visual field requirements . however , the field of vision , whether binocular or monocular , also has to be normal ( eg , 120).29,30 in the us , california does not have visual field requirements ( table 2 ) . the standards are slightly more stringent for new york and iowa compared with european union countries . in new york , for individuals with corrected vision of less than 20/40 but no less than 20/70 , the horizontal binocular field of vision must be no less than 140.36 in iowa , the visual field requirement is also 140 or better.34 to drive in the uk , the applicant has to meet the visual standards . if the driver is unable to meet the standards , he or she must not drive and the license must be refused or revoked . however , the european union regulations can override the uk standards , and the driver can apply to be considered as an exceptional case and granted an exemption.37 in the uk , licenses are normally issued to be valid until the driver is aged 70 years , unless restricted to a shorter duration for medical reasons . there is no upper limit , but after the age of 70 years , renewal is necessary every three years.27,38 in germany , if the applicant does not pass the test , an eye medical investigation is required , and minimum requirements of this test must be fulfilled . visual acuity must not fall below 0.5 in the best eye and 0.2 in the worse eye.31 renewal is not determined by age , and there are no medical requirements for renewal.38 for requirements in france , in case of severe vision loss of an eye , the driver will temporarily lose permission to drive . also , night vision is required . restricted licenses for daytime driving are given only if a specialist deems that the visual field is normal.28 there are no age - related renewal procedures or medical requirements for renewal.38 in the case of alterations of mesopic vision or glare , restrictions and limitations in spain must be determined according to ophthalmologic criteria so that secure driving can be guaranteed . driving with diplopia is not allowed . people in spain with monocular vision and a visual acuity of higher than 0.6 on the better - seeing eye can get permission to drive , provided they fulfill all other requirements and travel no faster than 100 km / hour.39 in spain the renewal cycles are 10 years for drivers younger than 45 years of age . for drivers between 45 and 70 years of age , the renewal cycles are five years . for drivers who are 70 years of age or older , renewal has to take place every other year.40 in italy , a medical / legal specialist and a general ophthalmologist examine the medical certifications , ie , ophthalmic check reports and visual field examinations . candidates must possess sufficient chromatic sense , adequate nocturnal vision , and binocular vision.41 the renewal cycle in italy is 10 years until the age of 50 years . between the ages of 50 and 70 years , the renewal cycle is five years , whereas after age 70 years the renewal cycle is accelerated to an interval of three years.42 driving requirements in the us are varied . in california , candidates who do not pass the vision examination may be referred to a vision specialist , who determines the ability to drive safely and may prescribe eye glasses or a stronger prescription ( table 2).32,33 renewal takes place every five years regardless of the age of the driver . however , drivers who are 70 years of age or older will have to undergo a vision examination to get a renewal.43 if the driver fails the vision test , a supplementary driving performance evaluation ( driving test ) can be given to determine whether the driver can compensate for the vision impairment . if this supplementary driving test is passed , the driver s license can be renewed with restrictions added to the license . restrictions can include requirements for special mechanical devices to be placed on the vehicle , limiting when and where a person may drive ( eg , no freeway driving , no nighttime driving ) , or requiring eye glasses or corrective contact lenses . in new york , if the vision test is not passed , further assessment must be made by a physician , ophthalmologist , or optometrist.36 renewal takes place every five years no matter what age the driver is . for a renewal process , the driver must prove that a visual acuity ( eye ) test has been passed within six months , or within one year as determined by a licensed health care professional.36,43 in iowa , if visual acuity is less than 20/40 but at least 20/50 ( 6/126/15 meters , 0.50.4 ) , no driving is allowed when headlights are required . if visual acuity is less than 20/50 but at least 20/70 , driving at speeds over 35 mph is not allowed , in addition to no driving when headlights are required . driving is not permitted for visual acuity less than 20/70 . in respect of visual field requirements , left and right outside rearview mirrors are required if the field of vision is less than 140 but at least 115 for the single best eye ( 70 temporal and 45 nasal ) . for a field of vision less than 115 , driving is not permitted.34 the normal renewal cycle in iowa is five years , and for drivers who are 70 years of age or older it is two years.43 the most important aspects of the driver s license requirements for passenger cars are summarized in table 1 for the relevant european union countries . the medical requirements for driver s licensing in european countries are affected by the minimum standard established by the european union . drivers of cars and motorcycles are required to have a binocular visual acuity of at least 0.5 ( 20/40 feet , 6/12 or 5/10 meters ) with or without correction , and binocular visual field standards are limited to no less than 120.20 for monocular vision , applicants must have a corrected or uncorrected visual acuity of at least 0.6 ( approximately 20/30 feet , 6/9 meters ) . also , medical authorities must certify that the condition has existed sufficiently long enough to allow adaptation and that the field of vision in the subject eye is normal . in cases where a progressive eye disease is detected or declared , driver s licenses may be issued or renewed provided that the applicant undergoes regular medical examinations.20 exceptions can be made in cases duly justified by a favorable medical opinion and a positive practical test . at these examinations , attention is paid to the state of visual function , recovery from glare , contrast sensitivity , and twilight vision.21 driver s licenses are not issued if the medical examination shows any other eye condition that would compromise driving safety.20 european countries generally follow the european union driver s license requirements with a few exceptions . in the uk , visual acuity requirements are based on the number plate eye test in which the applicant must be able to read a registration mark fixed to a motor vehicle.2224 the number plate test has been thought to convert to a snellen acuity of between 6/15 and 6/10 ( 20/5020/40 feet or 0.40.6 decimal).25,26 monocular vision is allowed in the uk if there is a normal monocular visual field and no area of defect caused by pathology.22,27 binocular visual acuity requirements for the remaining european union countries are similar . france and spain follow the european union standard , in that both have a minimum binocular vision requirement of 0.5 ( 20/40 feet , 5/10 meters).28,29 however , in italy , the best - corrected visual acuity must be at least 1.0 ( 10/10 , 20/20 feet , 6/6 meters ) for binocular vision , with a minimum of 0.2 ( 2/10 , 20/100 feet , 6/30 meters ) for the worse - seeing eye.30 in germany , applicants must also have a central visual acuity of at least 1.0 ( 0.7/0.7 , 20/20 feet , 6/6 meters ) with or without corrective means.31 stricter standards are enforced for monocular vision in germany and france , which require visual acuity of the better eye to be at least 0.6.31 in spain and italy , driving with monocular vision is normally not allowed . the visual acuity requirements for holding a driver s license in the european union are similar to those in the us states ( table 2 ) . in california , visual acuity of 20/40 ( 6/12 meters , 0.5 decimal ) with or without correction is required.32,33 visual acuity measurements are obtained for both eyes together and for each eye separately while both eyes remain open , as in normal driving.32,33 in new york and iowa , a driver must also have 20/40 ( 6/12 meters , 0.5 decimal ) vision in at least one eye with or without corrective lenses.34,35 the minimum visual field requirement in the uk is at least 120 horizontally ( table 1 ) . furthermore , there can not be significant defects in the binocular field that encroach within 20 of fixation above or below the horizontal meridian.24,27 the same requirement is observed in germany and france , but the central visual field up to 30 must be normal.28,31 for spain and italy , no diameter is given for visual field requirements . however , the field of vision , whether binocular or monocular , also has to be normal ( eg , 120).29,30 in the us , california does not have visual field requirements ( table 2 ) . the standards are slightly more stringent for new york and iowa compared with european union countries . in new york , for individuals with corrected vision of less than 20/40 but no less than 20/70 , the horizontal binocular field of vision must be no less than 140.36 in iowa , the visual field requirement is also 140 or better.34 if the driver is unable to meet the standards , he or she must not drive and the license must be refused or revoked . however , the european union regulations can override the uk standards , and the driver can apply to be considered as an exceptional case and granted an exemption.37 in the uk , licenses are normally issued to be valid until the driver is aged 70 years , unless restricted to a shorter duration for medical reasons . there is no upper limit , but after the age of 70 years , renewal is necessary every three years.27,38 in germany , if the applicant does not pass the test , an eye medical investigation is required , and minimum requirements of this test must be fulfilled . visual acuity must not fall below 0.5 in the best eye and 0.2 in the worse eye.31 renewal is not determined by age , and there are no medical requirements for renewal.38 for requirements in france , in case of severe vision loss of an eye , the driver will temporarily lose permission to drive . also , night vision is required . restricted licenses for daytime driving are given only if a specialist deems that the visual field is normal.28 there are no age - related renewal procedures or medical requirements for renewal.38 in the case of alterations of mesopic vision or glare , restrictions and limitations in spain must be determined according to ophthalmologic criteria so that secure driving can be guaranteed . driving with diplopia is not allowed . people in spain with monocular vision and a visual acuity of higher than 0.6 on the better - seeing eye can get permission to drive , provided they fulfill all other requirements and travel no faster than 100 km / hour.39 in spain the renewal cycles are 10 years for drivers younger than 45 years of age . for drivers between 45 and 70 years of age , the renewal cycles are five years . for drivers who are 70 years of age or older , renewal has to take place every other year.40 in italy , a medical / legal specialist and a general ophthalmologist examine the medical certifications , ie , ophthalmic check reports and visual field examinations . candidates must possess sufficient chromatic sense , adequate nocturnal vision , and binocular vision.41 the renewal cycle in italy is 10 years until the age of 50 years . between the ages of 50 and 70 years , the renewal cycle is five years , whereas after age 70 years the renewal cycle is accelerated to an interval of three years.42 driving requirements in the us are varied . in california , candidates who do not pass the vision examination may be referred to a vision specialist , who determines the ability to drive safely and may prescribe eye glasses or a stronger prescription ( table 2).32,33 renewal takes place every five years regardless of the age of the driver . however , drivers who are 70 years of age or older will have to undergo a vision examination to get a renewal.43 if the driver fails the vision test , a supplementary driving performance evaluation ( driving test ) can be given to determine whether the driver can compensate for the vision impairment . if this supplementary driving test is passed , the driver s license can be renewed with restrictions added to the license . restrictions can include requirements for special mechanical devices to be placed on the vehicle , limiting when and where a person may drive ( eg , no freeway driving , no nighttime driving ) , or requiring eye glasses or corrective contact lenses . in new york , if the vision test is not passed , further assessment must be made by a physician , ophthalmologist , or optometrist.36 renewal takes place every five years no matter what age the driver is . for a renewal process , the driver must prove that a visual acuity ( eye ) test has been passed within six months , or within one year as determined by a licensed health care professional.36,43 in iowa , if visual acuity is less than 20/40 but at least 20/50 ( 6/126/15 meters , 0.50.4 ) , no driving is allowed when headlights are required . if visual acuity is less than 20/50 but at least 20/70 , driving at speeds over 35 mph is not allowed , in addition to no driving when headlights are required . driving is not permitted for visual acuity less than 20/70 . in respect of visual field requirements , left and right outside rearview mirrors are required if the field of vision is less than 140 but at least 115 for the single best eye ( 70 temporal and 45 nasal ) . for a field of vision less than 115 , driving is not permitted.34 the normal renewal cycle in iowa is five years , and for drivers who are 70 years of age or older it is two years.43 the requirements for obtaining disability pensions due to low or no vision were found to be unique for each country . the most important aspects of disability pension qualifications have been summarized in table 3 for the relevant european union countries and us states . in the uk , people can claim a disability living allowance if they are certified blind ( severely sight - impaired ) or partially sighted ( sight - impaired ) . severely sight - impaired is defined as a visual acuity below 3/60 or 1/18 ( 20/400 feet ) tested with the snellen test , or with visual acuity better than 3/60 but below 6/60 ( 20/200 feet ) with a very restricted visual field . a person can be certified blind ( or severely sight - impaired ) if he or she is unable to perform work for which eyesight is essential . partially sighted ( sight - impaired ) corresponds to visual acuity from 3/60 to 6/60 ( 20/400 to 20/200 feet ) with a full field or visual acuity up to 6/24 ( 20/80 feet ) with moderate contraction of the field , aphakia ( lens removal ) , or opacities blocking vision in the eye itself , or with visual acuity of 6/18 ( 20/60 feet ) or better if they have a gross visual field defect ( of both eyes , such as hemianopia ) or marked contraction of the visual field ( eg , in retinitis pigmentosa or glaucoma ) . the visual acuity and visual field guidelines in the uk for partially sighted ( sight - impaired ) designations suggest that a person can be certified as such if he or she is substantially and permanently disabled by defective vision caused by congenital defect , illness , or injury.44 furthermore , a person is eligible to receive a disability living allowance only if he or she is below the age of 65 years , has severe sight problems for at least three months , and is expected to have severe sight problems for another six months.45,46 other european union countries have different requirements for receiving disability allowances . a blind person is someone whose central best - corrected visual acuity is 1/50 ( 20/1000 feet , 6/300 meters ) in the better eye.47 for france , 50% of disability corresponds to visual acuity below 3/10 ( approximately 20/66 feet , 6/20 meters ) in both eyes or 50% of visual field loss according to the esterman score ( binocular field ) . eighty percent of disability corresponds to visual acuity below 1/10 ( 20/200 feet , 6/60 meters ) in both eyes , or 80% of visual field loss according to the esterman score ( binocular field ) . in france legal blindness is visual acuity 1/20 ( 20/400 feet , 6/120 meters , 0.05 decimal ) of one eye and visual acuity of the other eye below 1/20 , with peripheral visual field deficiency when the visual field does not exceed 20 in the largest area.48,49 in spain , compensation for the blind relies heavily on once ( spanish national organization for the blind ) . membership conditions are a best - corrected visual acuity of 0.1 ( 1/10 on a wecker scale ) or lower , a visual field reduced to 10 or less , and spanish citizenship.50,51 between normal vision and blindness , there is a wide range of disabilities , eg , visual disability , serious visual disability , and low vision . in all three cases , visual acuity and visual field are used to obtain the degree of disability . visual disability is defined as a person who has reduced or very reduced visual capacity ( even though the person is using glasses ) and there is no possibility for improvement with surgical treatment.52 people with disabilities can apply for the pensin no contributiva por invalidez and pensin por incapacidad permanente.53,54 in order to obtain any type of disability pension , it is necessary to pass an examination by specialized doctors . the application procedure is relatively complicated and involves a panel of specialized doctors who decide whether the candidate is sufficiently disabled for eligibility . pensions for the blind ( pensione ciechi civili ) in italy apply only to people with complete blindness . the requirements also include italian citizenship and residency , and the citizen must be older than 18 years . requirements for the partially blind pension ( pensione per ciechi parziali ) include a visual acuity of 1/20 ( 20/400 feet , 6/120 meters ) or lower for both eyes . the candidate can be a european union citizen with an italian residence or a noneuropean union citizen with a work permit in italy.55,56 in the us , people who are legally blind can obtain disability benefits . the requirements and definition for legal blindness are established in the social security act , section 216.57 a person is considered legally blind if central visual acuity is 20/200 ( 6/60 meters , 0.1 decimal ) or less in the better eye with the use of a correcting lens . an eye with a field of vision limitation , such that the widest diameter of the visual field subtends an angle of 20 or less , is considered to have a central visual acuity of 20/200 or less.57,58 a person may also receive disability benefits even if he or she is not considered legally blind , if the vision problems alone or combined with other health problems prevent the person from working . for social security disability benefits , a person must have worked long enough to have paid social security taxes . for supplemental security income payments based on disability and blindness , it is not necessary to have been employed , but income and resources must be below state - imposed limits . progressive eye diseases such as glaucoma can negatively impact vision and driving . maintaining mobility outside the home is a main priority of glaucoma patients and a major contributor to quality of life.5961 evaluating driver s license status and disability entitlements extends beyond typical quality of life assessments by highlighting the practical consequences of progressive eye disease.59 these outcomes may be useful in assessing disease progression and efficacy of treatments . licensing authorities hold the primary responsibility for establishing the fitness of license holders.62 the most common visual requirements for driver s licensing are visual acuity and visual field . in most countries and jurisdictions , monocular vision raises the visual acuity requirements in the better - seeing eye to 0.6 in european countries , whereas in the us , it is maintained at 0.5 in some states and is increased in others . visual field limits are present in some of the driver s license regulations and typically follow the european union standard in european countries . the visual field limit is usually characterized by a radius or diameter , such as 60 or 120. variation in the us is somewhat greater . however , in states with visual field requirements , it is typically between 110 and 140. besides visual acuity and visual field , other characterizations of visual functions are sometimes present but are less well defined . surprisingly , there is little focus on functional vision , such as actual driving performance . in some countries or jurisdictions , measurement levels , defined by mean defect values , are not mentioned explicitly , despite the relevance to patients with eye disease . although we had originally intended to use both driver s license forfeiture and entitlements to disability pension as milestones , our data show that disability pension requirements vary greatly from country to country , partly due to the multitude of rules and requirements that define legal blindness . for example , some countries distinguish between being partially sighted and blind , whereas in other countries , no such separation exists . as a result , disability pension entitlements can not be standardized in a way that would be practical for use as a milestone or as a secondary endpoint in a clinical trial . in the absence of standardized national rules , one option would be to use the world health organization criteria for low vision and blindness to allow better comparability between countries in international trials . the world health organization defines low vision as visual acuity of less than 6/18 but equal to or better than 3/60 , or a corresponding visual field loss to less than 20 , in the better eye with the best possible correction . blindness is defined as visual acuity of less than 3/60 , or a corresponding visual field loss to less than 10 , in the better eye with the best possible correction.63 reductions in intraocular pressure and the rate of visual field progression are commonly used to assess the value of glaucoma treatments,64 but these are not as complete in evaluating the impact of glaucoma on a patient s quality of life . maintaining a driver s license is a performance - based measure and is not subject to the bias of self - reported questionnaires.61,65 using health outcome measures complements clinical measures and may help establish the effectiveness of competing treatments ( figures 1 and 2 ) . for example , if a superior treatment can stop or delay the progression of an eye disease , the milestones can be postponed . treatment goals would include maintaining visual function above the legal driving standard so that glaucoma patients continue to drive for as long as it is safe to do so . visual standards should be set to a binocular visual acuity of 0.5 or better and a visual field of 120 or better in the horizontal meridian . there are a few limitations to the milestone approach . for clinical studies using driver s licensing as a secondary endpoint , it would be important to adjust for education , race , cognitive ability , and disease comorbidities,66,67 which influence driving ability independent of glaucoma status . also , there is considerable heterogeneity for age - related driver s license renewal between countries , and older drivers often voluntarily reduce their driving frequency.68,69 therefore , it may be more accurate to inquire about driving status , not just licensing . it is worth considering whether driving a car would be a better milestone rather than the administrative rules for maintaining a driver s license . hence , the question would become at what stage of the disease do patients stop driving cars , and for what reason ? . a cross - sectional or a longitudinal study approach could be used . with cross - sectional data , the tendency to drive could be estimated as a function of relevant variables , such as disease progression , visual acuity , visual field , and other visual function parameters , sociodemographic and socioeconomic variables , and urbanization . the function could be estimated as a probability regression model , such as a logistic regression . if a longitudinal data set is available , either collected prospectively or retrospectively , the statistical method of survival analysis can be carried out , whereby survival is defined as maintained ability to drive , and the fatal event is defined as giving up driving a car . proportional hazard modeling could be used to estimate the effect of disease progression , visual acuity , visual field , other visual function parameters , sociodemographic and socioeconomic variables , and urbanization . such a study would provide crucial data that would associate the endpoints used in clinical trials with the ability to drive a car . information about driving behavior for eye patients can often be obtained from visual functioning questionnaires such as the national eye institute visual function questionnaire . to study driving behavior in glaucoma patients we focused on the comparison of driver s license regulations between only two states within the us and only five countries ( those that account for more than half the population of europe ) out of 27 countries in the european union . moreover , we did not consider countries with more populations , such as india or china , where the relevant information is more difficult to gather . reductions in intraocular pressure and the rate of visual field progression are commonly used to assess the value of glaucoma treatments,64 but these are not as complete in evaluating the impact of glaucoma on a patient s quality of life . maintaining a driver s license is a performance - based measure and is not subject to the bias of self - reported questionnaires.61,65 using health outcome measures complements clinical measures and may help establish the effectiveness of competing treatments ( figures 1 and 2 ) . for example , if a superior treatment can stop or delay the progression of an eye disease , the milestones can be postponed . treatment goals would include maintaining visual function above the legal driving standard so that glaucoma patients continue to drive for as long as it is safe to do so . visual standards should be set to a binocular visual acuity of 0.5 or better and a visual field of 120 or better in the horizontal meridian . there are a few limitations to the milestone approach . for clinical studies using driver s licensing as a secondary endpoint , it would be important to adjust for education , race , cognitive ability , and disease comorbidities,66,67 which influence driving ability independent of glaucoma status . also , there is considerable heterogeneity for age - related driver s license renewal between countries , and older drivers often voluntarily reduce their driving frequency.68,69 therefore , it may be more accurate to inquire about driving status , not just licensing . it is worth considering whether driving a car would be a better milestone rather than the administrative rules for maintaining a driver s license . hence , the question would become at what stage of the disease do patients stop driving cars , and for what reason ? . a cross - sectional or a longitudinal study approach could be used . with cross - sectional data , the tendency to drive could be estimated as a function of relevant variables , such as disease progression , visual acuity , visual field , and other visual function parameters , sociodemographic and socioeconomic variables , and urbanization . the function could be estimated as a probability regression model , such as a logistic regression . if a longitudinal data set is available , either collected prospectively or retrospectively , the statistical method of survival analysis can be carried out , whereby survival is defined as maintained ability to drive , and the fatal event is defined as giving up driving a car . proportional hazard modeling could be used to estimate the effect of disease progression , visual acuity , visual field , other visual function parameters , sociodemographic and socioeconomic variables , and urbanization . such a study would provide crucial data that would associate the endpoints used in clinical trials with the ability to drive a car . information about driving behavior for eye patients can often be obtained from visual functioning questionnaires such as the national eye institute visual function questionnaire . to study driving behavior in glaucoma patients we focused on the comparison of driver s license regulations between only two states within the us and only five countries ( those that account for more than half the population of europe ) out of 27 countries in the european union . moreover , we did not consider countries with more populations , such as india or china , where the relevant information is more difficult to gather . the driver s license endpoint can be standardized as a best - corrected visual acuity of at least 0.5 and a visual field of at least 120. this is consistent with the driver s license requirements in most countries and jurisdictions . driver s license forfeiture , rather than entitlement to disability pensions , could be considered a milestone or secondary endpoint in the characterization of a progressive eye disease , such as open - angle glaucoma . delaying these milestones is a useful goal of therapeutic interventions , because it may decrease personal and societal burden and cost .

objectivelow vision that causes forfeiture of driver s licenses and collection of disability pension benefits can lead to negative psychosocial and economic consequences . the purpose of this study was to review the requirements for holding a driver s license and rules for obtaining a disability pension due to low vision . results highlight the possibility of using a milestone approach to describe progressive eye disease.methodsgovernment and research reports , websites , and journal articles were evaluated to review rules and requirements in germany , spain , italy , france , the uk , and the us.resultsvisual acuity limits are present in all driver s license regulations . in most countries , the visual acuity limit is 0.5 . visual field limits are included in some driver s license regulations . in europe , binocular visual field requirements typically follow the european union standard of 120. in the us , the visual field requirements are typically between 110 and 140. some countries distinguish between being partially sighted and blind in the definition of legal blindness , and in others there is only one limit.conclusionsloss of driving privileges could be used as a milestone to monitor progressive eye disease . forfeiture could be standardized as a best - corrected visual acuity of < 0.5 or visual field of < 120 , which is consistent in most countries . however , requirements to receive disability pensions were too variable to standardize as milestones in progressive eye disease . implementation of the world health organization criteria for low vision and blindness would help to establish better comparability between countries .

Introduction Methods Results Drivers license requirements Visual acuity Visual field Exemptions and restrictions Entitlement to disability pensions Discussion Milestone approach Conclusion

as eye diseases progress to low vision and blindness , the former driver may no longer be able to participate fully in life and/or work activities , and disability pensions may be sought . the purpose of the present study was to review the requirements for holding a driver s license and rules for obtaining a disability pension due to low vision . results underscore the possibility of using these outcomes as milestones to describe progressive eye disease by relating disease severity to disability . research was conducted to identify driver s license rules and requirements for passenger cars and disability pensions due to low vision for six countries ( germany , spain , italy , france , the uk , and the us ) . at these examinations , attention is paid to the state of visual function , recovery from glare , contrast sensitivity , and twilight vision.21 driver s licenses are not issued if the medical examination shows any other eye condition that would compromise driving safety.20 european countries generally follow the european union driver s license requirements with a few exceptions . in the uk , visual acuity requirements are based on the number plate eye test in which the applicant must be able to read a registration mark fixed to a motor vehicle.2224 the number plate test has been thought to convert to a snellen acuity of between 6/15 and 6/10 ( 20/5020/40 feet or 0.40.6 decimal).25,26 monocular vision is allowed in the uk if there is a normal monocular visual field and no area of defect caused by pathology.22,27 binocular visual acuity requirements for the remaining european union countries are similar . france and spain follow the european union standard , in that both have a minimum binocular vision requirement of 0.5 ( 20/40 feet , 5/10 meters).28,29 however , in italy , the best - corrected visual acuity must be at least 1.0 ( 10/10 , 20/20 feet , 6/6 meters ) for binocular vision , with a minimum of 0.2 ( 2/10 , 20/100 feet , 6/30 meters ) for the worse - seeing eye.30 in germany , applicants must also have a central visual acuity of at least 1.0 ( 0.7/0.7 , 20/20 feet , 6/6 meters ) with or without corrective means.31 stricter standards are enforced for monocular vision in germany and france , which require visual acuity of the better eye to be at least 0.6.31 in spain and italy , driving with monocular vision is normally not allowed . the visual acuity requirements for holding a driver s license in the european union are similar to those in the us states ( table 2 ) . however , the field of vision , whether binocular or monocular , also has to be normal ( eg , 120).29,30 in the us , california does not have visual field requirements ( table 2 ) . in new york , for individuals with corrected vision of less than 20/40 but no less than 20/70 , the horizontal binocular field of vision must be no less than 140.36 in iowa , the visual field requirement is also 140 or better.34 to drive in the uk , the applicant has to meet the visual standards . however , the european union regulations can override the uk standards , and the driver can apply to be considered as an exceptional case and granted an exemption.37 in the uk , licenses are normally issued to be valid until the driver is aged 70 years , unless restricted to a shorter duration for medical reasons . visual acuity must not fall below 0.5 in the best eye and 0.2 in the worse eye.31 renewal is not determined by age , and there are no medical requirements for renewal.38 for requirements in france , in case of severe vision loss of an eye , the driver will temporarily lose permission to drive . for a field of vision less than 115 , driving is not permitted.34 the normal renewal cycle in iowa is five years , and for drivers who are 70 years of age or older it is two years.43 the requirements for obtaining disability pensions due to low or no vision were found to be unique for each country . partially sighted ( sight - impaired ) corresponds to visual acuity from 3/60 to 6/60 ( 20/400 to 20/200 feet ) with a full field or visual acuity up to 6/24 ( 20/80 feet ) with moderate contraction of the field , aphakia ( lens removal ) , or opacities blocking vision in the eye itself , or with visual acuity of 6/18 ( 20/60 feet ) or better if they have a gross visual field defect ( of both eyes , such as hemianopia ) or marked contraction of the visual field ( eg , in retinitis pigmentosa or glaucoma ) . the visual acuity and visual field guidelines in the uk for partially sighted ( sight - impaired ) designations suggest that a person can be certified as such if he or she is substantially and permanently disabled by defective vision caused by congenital defect , illness , or injury.44 furthermore , a person is eligible to receive a disability living allowance only if he or she is below the age of 65 years , has severe sight problems for at least three months , and is expected to have severe sight problems for another six months.45,46 other european union countries have different requirements for receiving disability allowances . a blind person is someone whose central best - corrected visual acuity is 1/50 ( 20/1000 feet , 6/300 meters ) in the better eye.47 for france , 50% of disability corresponds to visual acuity below 3/10 ( approximately 20/66 feet , 6/20 meters ) in both eyes or 50% of visual field loss according to the esterman score ( binocular field ) . in france legal blindness is visual acuity 1/20 ( 20/400 feet , 6/120 meters , 0.05 decimal ) of one eye and visual acuity of the other eye below 1/20 , with peripheral visual field deficiency when the visual field does not exceed 20 in the largest area.48,49 in spain , compensation for the blind relies heavily on once ( spanish national organization for the blind ) . membership conditions are a best - corrected visual acuity of 0.1 ( 1/10 on a wecker scale ) or lower , a visual field reduced to 10 or less , and spanish citizenship.50,51 between normal vision and blindness , there is a wide range of disabilities , eg , visual disability , serious visual disability , and low vision . in all three cases , visual acuity and visual field visual disability is defined as a person who has reduced or very reduced visual capacity ( even though the person is using glasses ) and there is no possibility for improvement with surgical treatment.52 people with disabilities can apply for the pensin no contributiva por invalidez and pensin por incapacidad permanente.53,54 in order to obtain any type of disability pension , it is necessary to pass an examination by specialized doctors . an eye with a field of vision limitation , such that the widest diameter of the visual field subtends an angle of 20 or less , is considered to have a central visual acuity of 20/200 or less.57,58 a person may also receive disability benefits even if he or she is not considered legally blind , if the vision problems alone or combined with other health problems prevent the person from working . at these examinations , attention is paid to the state of visual function , recovery from glare , contrast sensitivity , and twilight vision.21 driver s licenses are not issued if the medical examination shows any other eye condition that would compromise driving safety.20 european countries generally follow the european union driver s license requirements with a few exceptions . in the uk , visual acuity requirements are based on the number plate eye test in which the applicant must be able to read a registration mark fixed to a motor vehicle.2224 the number plate test has been thought to convert to a snellen acuity of between 6/15 and 6/10 ( 20/5020/40 feet or 0.40.6 decimal).25,26 monocular vision is allowed in the uk if there is a normal monocular visual field and no area of defect caused by pathology.22,27 binocular visual acuity requirements for the remaining european union countries are similar . france and spain follow the european union standard , in that both have a minimum binocular vision requirement of 0.5 ( 20/40 feet , 5/10 meters).28,29 however , in italy , the best - corrected visual acuity must be at least 1.0 ( 10/10 , 20/20 feet , 6/6 meters ) for binocular vision , with a minimum of 0.2 ( 2/10 , 20/100 feet , 6/30 meters ) for the worse - seeing eye.30 in germany , applicants must also have a central visual acuity of at least 1.0 ( 0.7/0.7 , 20/20 feet , 6/6 meters ) with or without corrective means.31 stricter standards are enforced for monocular vision in germany and france , which require visual acuity of the better eye to be at least 0.6.31 in spain and italy , driving with monocular vision is normally not allowed . the visual acuity requirements for holding a driver s license in the european union are similar to those in the us states ( table 2 ) . in california , visual acuity of 20/40 ( 6/12 meters , 0.5 decimal ) with or without correction is required.32,33 visual acuity measurements are obtained for both eyes together and for each eye separately while both eyes remain open , as in normal driving.32,33 in new york and iowa , a driver must also have 20/40 ( 6/12 meters , 0.5 decimal ) vision in at least one eye with or without corrective lenses.34,35 the minimum visual field requirement in the uk is at least 120 horizontally ( table 1 ) . however , the field of vision , whether binocular or monocular , also has to be normal ( eg , 120).29,30 in the us , california does not have visual field requirements ( table 2 ) . in new york , for individuals with corrected vision of less than 20/40 but no less than 20/70 , the horizontal binocular field of vision must be no less than 140.36 in iowa , the visual field requirement is also 140 or better.34 to drive in the uk , the applicant has to meet the visual standards . however , the european union regulations can override the uk standards , and the driver can apply to be considered as an exceptional case and granted an exemption.37 in the uk , licenses are normally issued to be valid until the driver is aged 70 years , unless restricted to a shorter duration for medical reasons . visual acuity must not fall below 0.5 in the best eye and 0.2 in the worse eye.31 renewal is not determined by age , and there are no medical requirements for renewal.38 for requirements in france , in case of severe vision loss of an eye , the driver will temporarily lose permission to drive . in the uk , visual acuity requirements are based on the number plate eye test in which the applicant must be able to read a registration mark fixed to a motor vehicle.2224 the number plate test has been thought to convert to a snellen acuity of between 6/15 and 6/10 ( 20/5020/40 feet or 0.40.6 decimal).25,26 monocular vision is allowed in the uk if there is a normal monocular visual field and no area of defect caused by pathology.22,27 binocular visual acuity requirements for the remaining european union countries are similar . france and spain follow the european union standard , in that both have a minimum binocular vision requirement of 0.5 ( 20/40 feet , 5/10 meters).28,29 however , in italy , the best - corrected visual acuity must be at least 1.0 ( 10/10 , 20/20 feet , 6/6 meters ) for binocular vision , with a minimum of 0.2 ( 2/10 , 20/100 feet , 6/30 meters ) for the worse - seeing eye.30 in germany , applicants must also have a central visual acuity of at least 1.0 ( 0.7/0.7 , 20/20 feet , 6/6 meters ) with or without corrective means.31 stricter standards are enforced for monocular vision in germany and france , which require visual acuity of the better eye to be at least 0.6.31 in spain and italy , driving with monocular vision is normally not allowed . the visual acuity requirements for holding a driver s license in the european union are similar to those in the us states ( table 2 ) . however , the field of vision , whether binocular or monocular , also has to be normal ( eg , 120).29,30 in the us , california does not have visual field requirements ( table 2 ) . however , the european union regulations can override the uk standards , and the driver can apply to be considered as an exceptional case and granted an exemption.37 in the uk , licenses are normally issued to be valid until the driver is aged 70 years , unless restricted to a shorter duration for medical reasons . visual acuity must not fall below 0.5 in the best eye and 0.2 in the worse eye.31 renewal is not determined by age , and there are no medical requirements for renewal.38 for requirements in france , in case of severe vision loss of an eye , the driver will temporarily lose permission to drive . for a field of vision less than 115 , driving is not permitted.34 the normal renewal cycle in iowa is five years , and for drivers who are 70 years of age or older it is two years.43 the requirements for obtaining disability pensions due to low or no vision were found to be unique for each country . partially sighted ( sight - impaired ) corresponds to visual acuity from 3/60 to 6/60 ( 20/400 to 20/200 feet ) with a full field or visual acuity up to 6/24 ( 20/80 feet ) with moderate contraction of the field , aphakia ( lens removal ) , or opacities blocking vision in the eye itself , or with visual acuity of 6/18 ( 20/60 feet ) or better if they have a gross visual field defect ( of both eyes , such as hemianopia ) or marked contraction of the visual field ( eg , in retinitis pigmentosa or glaucoma ) . the visual acuity and visual field guidelines in the uk for partially sighted ( sight - impaired ) designations suggest that a person can be certified as such if he or she is substantially and permanently disabled by defective vision caused by congenital defect , illness , or injury.44 furthermore , a person is eligible to receive a disability living allowance only if he or she is below the age of 65 years , has severe sight problems for at least three months , and is expected to have severe sight problems for another six months.45,46 other european union countries have different requirements for receiving disability allowances . a blind person is someone whose central best - corrected visual acuity is 1/50 ( 20/1000 feet , 6/300 meters ) in the better eye.47 for france , 50% of disability corresponds to visual acuity below 3/10 ( approximately 20/66 feet , 6/20 meters ) in both eyes or 50% of visual field loss according to the esterman score ( binocular field ) . in france legal blindness is visual acuity 1/20 ( 20/400 feet , 6/120 meters , 0.05 decimal ) of one eye and visual acuity of the other eye below 1/20 , with peripheral visual field deficiency when the visual field does not exceed 20 in the largest area.48,49 in spain , compensation for the blind relies heavily on once ( spanish national organization for the blind ) . membership conditions are a best - corrected visual acuity of 0.1 ( 1/10 on a wecker scale ) or lower , a visual field reduced to 10 or less , and spanish citizenship.50,51 between normal vision and blindness , there is a wide range of disabilities , eg , visual disability , serious visual disability , and low vision . an eye with a field of vision limitation , such that the widest diameter of the visual field subtends an angle of 20 or less , is considered to have a central visual acuity of 20/200 or less.57,58 a person may also receive disability benefits even if he or she is not considered legally blind , if the vision problems alone or combined with other health problems prevent the person from working . in most countries and jurisdictions , monocular vision raises the visual acuity requirements in the better - seeing eye to 0.6 in european countries , whereas in the us , it is maintained at 0.5 in some states and is increased in others . visual field limits are present in some of the driver s license regulations and typically follow the european union standard in european countries . however , in states with visual field requirements , it is typically between 110 and 140. besides visual acuity and visual field , other characterizations of visual functions are sometimes present but are less well defined . although we had originally intended to use both driver s license forfeiture and entitlements to disability pension as milestones , our data show that disability pension requirements vary greatly from country to country , partly due to the multitude of rules and requirements that define legal blindness . for example , some countries distinguish between being partially sighted and blind , whereas in other countries , no such separation exists . in the absence of standardized national rules , one option would be to use the world health organization criteria for low vision and blindness to allow better comparability between countries in international trials . the world health organization defines low vision as visual acuity of less than 6/18 but equal to or better than 3/60 , or a corresponding visual field loss to less than 20 , in the better eye with the best possible correction . visual standards should be set to a binocular visual acuity of 0.5 or better and a visual field of 120 or better in the horizontal meridian . to study driving behavior in glaucoma patients we focused on the comparison of driver s license regulations between only two states within the us and only five countries ( those that account for more than half the population of europe ) out of 27 countries in the european union . visual standards should be set to a binocular visual acuity of 0.5 or better and a visual field of 120 or better in the horizontal meridian . to study driving behavior in glaucoma patients we focused on the comparison of driver s license regulations between only two states within the us and only five countries ( those that account for more than half the population of europe ) out of 27 countries in the european union . the driver s license endpoint can be standardized as a best - corrected visual acuity of at least 0.5 and a visual field of at least 120. this is consistent with the driver s license requirements in most countries and jurisdictions . driver s license forfeiture , rather than entitlement to disability pensions , could be considered a milestone or secondary endpoint in the characterization of a progressive eye disease , such as open - angle glaucoma .

biological membranes generally contain a mixture of various types of amphiphilic molecules , such as phospholipids , glycolipids , fatty acids , glycerides and amines . although present at levels of only a few percent , fatty acids and amines are important constituents of biomembranes ( kumar et al . they can affect any physiological function , such as enzyme activity ( gennis 2010 ) , particularly the activation of lipid - metabolizing enzymes , and calcium transport ( felix 2006 ) . since fatty acids belong to the lipids that form biological membranes and at the same time also possess a quite simple structure , they are frequently used by researchers for modeling . it is clear that there is a certain equilibrium between the lipid - forming bilayer and lipid molecules and substances present in electrolyte solution . the interactions between membrane lipids are studied by several techniques ; however , quantitative descriptions of the systems are lacking . formation of artificial membranes with built - in study components allows us to research in simpler systems than complicated biological membranes . many investigations have been reported on the effect of fatty acids on physical and chemical properties of model membranes , particularly on the phase behavior of hydrated phospholipid bilayers , using various techniques , such as differential thermal analysis ( ulkowski et al . 2005 ) , differential scanning calorimetry ( ohta and hatta 2002 ; matricarde falleiro et al . 2001 ) , fluorescence spectroscopy ( borst et al . 2000 ) , x - ray diffraction ( kumar et al . 2005 ) , magnetic resonance ( heimburg et al . 1990 ; rama krishna and marsh 1990 ) and fourier transform infrared spectroscopy ( inoue et al . 2001 ) . in this work , the interfacial tension of phosphatidylcholine ( pc)decanoic acid ( da ) and pc decylamine ( de ) membranes was determined within the entire composition range where bilayer formation was possible . the aim of these investigations was to study the mixed pc da and pc de bilayer , characterize the molecular interaction between phospholipids and fatty acids and between phospholipids and amine and compare the properties of these systems : stability constants of the formed complexes and surface areas occupied by pure membrane components . in cases where the membrane components do not form chemical compounds , their interaction can be described by the following set of equations ( petelska and figaszewski 1998 ; petelska et al . 2006a):1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned}&\gamma_{1 } m_{1 } a_{1 } + \gamma_{2 } m_{2 } a_{2 } = \gamma\\&\frac{{m_{1 } } } { { m_{1 } + m_{2 } } } = x_{1}\\&x_{1 } + x_{2 } = 1 \hfill \\ \end{aligned } $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{1}^ { - 1 } , \,a_{2}^ { - 1 } $ $ \end{document } ( mol m ) are the surface concentrations of components 1 and 2 ; m1 , m2 ( mol m ) are the quantities of components 1 and 2 per unit area of the membrane ; 1 , 2 ( n m ) are the interfacial tensions of membranes assembled from pure components 1 and 2 ; ( n m ) is the measured interfacial tension of the membrane ; and x1 , x2 are the solution mole fractions of components 1 and 2 . elimination of m1 and m2 yields the following linear equation:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { \gamma - \gamma_{1 } } \right)x_{1 } = \frac{{a_{2 } } } { { a_{1 } } } \left ( { \gamma_{2 } - \gamma } \right)x_{2 } $ $ \end{document } membranes may also be assembled from two components capable of forming a complex . the stoichiometry of the complex may vary , but because the first stability constant in these complexes is usually the largest ( inczedy 1976 ; beck and nagypal 1990 ) , we assumed that the complexes are primarily of 1:1 stoichiometry . in cases where the membrane components form a 1:1 complex , interactions in the membrane the equilibrium between the individual components and the complex is represented by\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{a}}\ , ( { \text{component}}\ , 1 ) + { \text{b}}\ , ( { \text{component}}\ , 2 ) \leftrightarrow { \text{ab}}\ , ( { \text{complex ) } } $ $ \end{document}and the basic equation describing the interaction between components 1 and 2 can be written as follows ( petelska et al . 2006a , b):3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left [ { \left ( { \gamma - \gamma_{1 } } \right)b_{2 } x_{1 } + \left ( { \gamma - \gamma_{2 } } \right)b_{1 } x_{2 } } \right]\left [ { \left ( { \gamma_{3 } - \gamma_{1 } } \right)b_{2 } x_{1 } + \left ( { \gamma_{3 } - \gamma_{2 } } \right)b_{1 } x_{2 } + \left ( { \gamma_{1 } - \gamma_{2 } } \right)\left ( { x_{1 } - x_{2 } } \right ) } \right ] = ka_{3}^ { - 1 } b_{1 } b_{2 } \left [ { \left ( { \gamma - \gamma_{1 } } \right)\left ( { x_{2 } - x_{1 } } \right ) + \left ( { \gamma_{3 } - \gamma } \right)b_{1 } x_{2 } } \right]\left [ { \left ( { \gamma - \gamma_{2 } } \right)\left ( { x_{1 } - x_{2 } } \right ) + \left ( { \gamma_{3 } - \gamma } \right)b_{2 } x_{1 } } \right ] $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{1 } = { { a_{3 } } \mathord{\left/ { \vphantom { { a_{3 } } { a_{1 } } } } \right . \kern-\nulldelimiterspace } { a_{1 } } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{2 } = { { a_{3 } } \mathord{\left/ { \vphantom { { a_{3 } } { a_{2 } } } } \right . $ $ \end{document } equation 3 may be simplified by taking into account the high stability constant of the complex . applying this simplification results in linear behavior for small ( x2 < x1 ) and large ( x2 > x1 ) x2 values . when calculating the stability constant for the complex , eq . the parameters describing the complex may be used to calculate theoretical points using the equation presented below ( agreement between the theoretical and experimental values implies that the system is well described by the above equations):4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ ka_{1}^ { - 1 } a_{2}^ { - 1 } \left ( { a_{1 } + a_{2 } } \right)\left ( { a_{3 } - a_{1 } } \right)\gamma^{2 } + \left [ { ka_{1}^ { - 1 } a_{2}^ { - 1 } \left ( { \gamma_{1 } a_{1 } - \gamma_{3 } a_{3 } } \right)\left ( { a_{1 } + a_{2 } } \right ) - \,ka_{1}^ { - 1 } a_{2}^ { - 1 } \left ( { \gamma_{2 } a_{1 } + \gamma_{3 } a_{2 } } \right)\left ( { a_{3 } - a_{1 } } \right ) + \ , a_{4 } a_{3}^ { - 1 } \left ( { a_{3 } + a_{2 } } \right ) } \right]\gamma + ka_{1}^ { - 1 } a_{2}^ { - 1 } a_{3 } \gamma_{3 } \left ( { \gamma_{3 } a_{2 } + \gamma_{2 } a_{1 } } \right ) - ka_{1}^ { - 1 } a_{2}^ { - 1 } a_{1 } \gamma_{1 } \left ( { a_{1 } \gamma_{2 } + a_{2 } \gamma_{3 } } \right ) - a_{4 } a_{3}^ { - 1 } \left ( { \gamma_{2 } a_{3 } + \gamma_{1 } a_{2 } } \right ) = 0 $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{gathered } a_{1 } = a_{3}^ { - 1 } \left ( { x_{2 } - x_{1 } } \right ) \hfill \\ a_{2 } = a_{2}^ { - 1 } x_{1 } \hfill \\ a_{3 } = a_{1}^ { - 1 } x_{2 } \hfill \\ a_{4 } = \left [ { a_{3}^ { - 1 } \left ( { \gamma_{1 } - \gamma_{2 } } \right)\left ( { x_{2 } - x_{1 } } \right ) + \,\left ( { \gamma_{1 } - \gamma_{3 } } \right)x_{1 } a_{2}^ { - 1 } + \ , \left ( { \gamma_{2 } - \gamma_{3 } } \right)x_{2 } a_{1}^ { - 1 } } \right ] \hfill \\ \end{gathered } $ $ \end{document } for systems containing two lipid components , 1:1 complex formation was assumed to be the explanation for deviation from the additivity rule . model curves were constructed using calculated parameters such as equilibrium constants , molecular areas of the complexes and the interfacial tension of molecules and complexes . the tension in a lipid bilayer sample is determined by measuring the radius of curvature of the convex surface formed when a pressure difference is applied across the bilayer ( adamson 1960 ) . the apparatus and measurement method were described previously ( petelska and figaszewski 1998 ; petelska et al . the measurement system consists of two glass chambers separated by a mount holding a 1.5-mm - diameter circular teflon element axially pierced by a small orifice . rudin method ( tien and ottova - leitmannova 2003 ) on the flat end of the teflon element . the membrane - forming solution was introduced to the flat wall of the teflon element using a micropipette , and pressure was applied to the left chamber using a manometer . the radius of curvature was determined using this value and the diameter of the teflon element , corresponding to diameter of the lipid cap . the following reagents were used for preparation of the membrane - forming solution:3-sn - phosphatidylcholine ( 99% ; fluka , buchs , switzerland ) isolated from hen egg yolkdecanoic acid ( 98% , fluka)decylamine ( 99.5% , fluka ) 3-sn - phosphatidylcholine ( 99% ; fluka , buchs , switzerland ) isolated from hen egg yolk decanoic acid ( 98% , fluka ) decylamine ( 99.5% , fluka ) the as - received pc and da were purified by dissolving in chloroform and evaporating the solvent under argon . the stock membrane - forming solutions consisted of 20 mg cm of the desired lipids ( pc , da or de ) in 20:1 n - decane : butanol . the solution containing the membrane components was not saturated and could therefore contain the components in any proportion . during membrane formation , the solvent was removed , leaving a membrane composed of lipids in the same ratio as the stock solution . the electrolyte solution contained 0.1 m potassium chloride and was prepared using triple - distilled water and kcl , produced by poch ( gliwice , poland ) . the n - decane was purchased from merck ( darmstadt , germany ) , and the chloroform and butanol were obtained from aldrich ( milwaukee , wi ) . the tension in a lipid bilayer sample is determined by measuring the radius of curvature of the convex surface formed when a pressure difference is applied across the bilayer ( adamson 1960 ) . the apparatus and measurement method were described previously ( petelska and figaszewski 1998 ; petelska et al . the measurement system consists of two glass chambers separated by a mount holding a 1.5-mm - diameter circular teflon element axially pierced by a small orifice . rudin method ( tien and ottova - leitmannova 2003 ) on the flat end of the teflon element . the membrane - forming solution was introduced to the flat wall of the teflon element using a micropipette , and pressure was applied to the left chamber using a manometer . the radius of curvature was determined using this value and the diameter of the teflon element , corresponding to diameter of the lipid cap . the following reagents were used for preparation of the membrane - forming solution:3-sn - phosphatidylcholine ( 99% ; fluka , buchs , switzerland ) isolated from hen egg yolkdecanoic acid ( 98% , fluka)decylamine ( 99.5% , fluka ) 3-sn - phosphatidylcholine ( 99% ; fluka , buchs , switzerland ) isolated from hen egg yolk decanoic acid ( 98% , fluka ) decylamine ( 99.5% , fluka ) the as - received pc and da were purified by dissolving in chloroform and evaporating the solvent under argon . the stock membrane - forming solutions consisted of 20 mg cm of the desired lipids ( pc , da or de ) in 20:1 n - decane : butanol . the solution containing the membrane components was not saturated and could therefore contain the components in any proportion . during membrane formation , the solvent was removed , leaving a membrane composed of lipids in the same ratio as the stock solution . the electrolyte solution contained 0.1 m potassium chloride and was prepared using triple - distilled water and kcl , produced by poch ( gliwice , poland ) . the n - decane was purchased from merck ( darmstadt , germany ) , and the chloroform and butanol were obtained from aldrich ( milwaukee , wi ) . the effect of the presence of da or de on interfacial tension of the membranes formed from pc was studied . the dependence of interfacial tension on the lipid membrane as a function of composition was studied at room temperature ( 293 2 k ) in the entire feasible concentration range . the interfacial tension values reported here refer to the two sides of the bilayer membrane surface area unit . figure 1 contains a graph of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { \gamma - \gamma_{1 } } \right)x_{1 } $ $ \end{document } vs. \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { \gamma_{2 } - \gamma } \right)x_{2 } $ $ \end{document } for the four systems pc da ( fig . according to eq . 2 , when the membrane components do not interact , these functions should yield straight lines . this is clearly not the case , which suggests that a complex or other structure exists in pc da and pc de bilayers . because the use of eq . 3 presupposes the existence of 1:1 complexes , our initial assumption was that the complexes formed were 1:1 . the interfacial tension of the lipid membrane was studied over a wide range of lipid compositions.fig . . 2 for pc da ( a ) and pc de ( b ) , where x2 is the mole fraction of component 2 ( da and de , respectively ) graph of eq . 2 for pc da ( a ) and pc de ( b ) , where x2 is the mole fraction of component 2 ( da and de , respectively ) the dependence of interfacial tension of lipid membrane as a function of membrane composition for pc da and pc de systems was studied over a possible concentration range . the dependence of interfacial tension on the lipid membranes formed from the pc da and pc de systems was executed in the function of the composition to 41% of the da contents ( to 40% of the de ) because only to such contents of component 2 ( da , de ) with lecithin was a bilayer membrane formed.fig . 2the interfacial tension , , of the pc da ( a ) and pc de ( b ) membranes as a molar fraction of da or de , x2 ( experimental values are marked by points and theoretical ones by curves ) the interfacial tension , , of the pc da ( a ) and pc de ( b ) membranes as a molar fraction of da or de , x2 ( experimental values are marked by points and theoretical ones by curves ) the interfacial tension value of pure lecithin membrane ( component 1 ) , 1 , was measured directly and presented earlier ( petelska and figaszewski 1998 ) , which is equal to 1.62 10 n m. there are no accurate data on interfacial tension values for the pure component 2 ( da or de ) because these components do not create a bilayer membrane . however , in order to characterize the course of the experimental curves , the 2 value for the pure components is necessary , which will be used for calculation . in this case , the interfacial tension hypothetical values for membranes built from da and de were determined by adjusting the experimental curve with the polynomial of the other mark extrapolating the x2 = 1 value , which is presented in fig . the interfacial tension values obtained in this way for pure da and de are equal to 2.38 10 and 3.88 10 n m. negative values of interfacial tension for membrane built from pure da and de point to the fact that it is not possible to create bilayer membranes from pure da or de . the thermodynamic potential for this bilayer would have a negative value ; i.e. , the bilayer is not forming.fig . 3plot illustrating the hypothetical interfacial tension values for da ( a ) and de ( b ) membrane calculation plot illustrating the hypothetical interfacial tension values for da ( a ) and de ( b ) membrane calculation the other constants b1 , b2 , 3were determined by assuming that the values of the stability constant of the pc da and pc de complexes were sufficient to be simplified , i.e. , eq . 1 to eqs . 2 and 3 . knowing the b1 , b2 constants , which were determined from eqs . 2 and 3 , it was possible to calculate the interfacial tension values of the pc da and pc de complexes , 3 . the mean values are equal to 7.25 10 n m for pc da and 1.52 10 n m for pc de . determining the interfacial tension value as a function of the composition made it possible to determine the surface concentrations of the membranes composed of pure components . at least one of them is necessary for determination of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{3}^ { - 1 } $ $ \end{document } value . the surface area occupied by a lecithin molecule equal to 85 was determined in previous work ( petelska and figaszewski 2000 ) . the surface areas occupied by da and de are equal to 19 and 22 , respectively ( petelska and figaszewski 2011 ) . as mentioned earlier , the fatty acid forms a dimer ( brzozowska and figaszewski 2003 ; zhao and olesik 2001 ) ; therefore , the surface area occupied by da is equal to 38 . knowing the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{1}^ { - 1 } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{2}^ { - 1 } $ $ \end{document } as well as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{1 } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{2 } $ $ \end{document } values , the surface concentration of the membrane composed of the lecithin da and lecithin de complexes could be determined . the resulting surface concentration values , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{3}^ { - 1 } $ $ \end{document } , for the pc da and pc de complexes were equal to 1.15 10 and 2.03 10 mol m , respectively . this made it possible to determine the area occupied by one lecithin da and one lecithin the value of area occupied by one lecithin da complex molecule is larger than the amount of the surface area occupied by each component of the complex . it is probably connected with the arrangement of lecithin molecules in such a complex and with the structural construction of such a complex . previously ( petelska and figaszewski 2003 ) , we suggested the arrangement of the lecithin molecules in a bilayer membrane at ph > 5 . in these media , one particle from the lecithin molecule in the bilayer ( orientated in this way ) has two straightened chains ; however , the next molecule of lecithin has one straightened and another chain fastened to the membrane surface . how these ions were characterized was previously reported ( petelska and figaszewski 2003 ) : these ions are strongly solvated , and they produce a separation of lecithin particles in the bilayer , which increases the surface occupied by the single molecule of lecithin . the difference between surface area values occupied by one molecule of pc - da and pc - de complexes to appear from the fact than as the chain length increases , van der waals interactions between the chain of the adjacent molecule increase , bringing these molecules closer to each other . when this happens , the carboxylic acid groups of the fatty acids are also packed closer , shielding the hydrogen atom between the two oxygen atoms ( kanicky et al . the only values to be determined were the stability constants of the pc fatty acid and pc amine complexes . 4 when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{1 } = x_{2 } = 0.5 $ $ \end{document } ; these parameters amount to 4.61 10 and 2.38 10 m mol , respectively . during the course of our investigations , we assumed the formation of pc de and pc de complexes . these complexes arise by producing a connection between the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { -}{\mathop { \text{n}}\limits^{\left ( + \right ) } } \left ( { { \text{ch}}_{3 } } \right)_{3 } $ $ \end{document } group from the molecule of lecithin and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - { \text{coo}}^{\left ( - \right ) } $ $ \end{document } groups from the dimer of da , in the case of the complex pc da , and between the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - { \text{po}}^{\left ( - \right ) } $ $ \end{document } group from lecithin and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { -}{\mathop { \text{n}}\limits^{\left ( + \right ) } } { \text{h}}_{3 } $ $ \end{document } group from de . the dissociation constants of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { -}{\mathop { \text{n}}\limits^{\left ( + \right ) } } \left ( { { \text{ch}}_{3 } } \right)_{3 } $ $ \end{document } group from pc and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - { \text{coo}}^{\left ( - \right ) } $ $ \end{document } groups from the dimer of da are equal 10 ( petelska and figaszewski 2000 ) and about 10 ( chemistry tables 2004 ) , respectively . it should be noted that the dissociation constants of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - { \text{po}}^{\left ( - \right ) } $ $ \end{document } group from pc and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { -}{\mathop { \text{n}}\limits^{\left ( + \right ) } } { \text{h}}_{3 } $ $ \end{document } group from de equal 10 ( petelska and figaszewski 2000 ) and about 10 ( chemistry tables 2004 ) , respectively . therefore , the connection between pc and da will be stronger , and we can expect that the stability constant of pc da will be higher than the stability constant of pc de . . 1 ; i.e. , data ( solid lines ) with the experimental data ( points ) in fig . 1 using equation 5 . the values yielding a better agreement of the experimental points with equations describing complex formation between membrane lipid components were chosen . 1 are marked by points and the theoretical ones obtained from equation 5 , by lines . it can be seen from this figure that there is a good agreement between experimental and theoretical points , which verifies the assumption of formation of 1:1 pc da and pc de complexes in the lipid membrane . good agreement of the experimental and theoretical points verifies the assumption of the correct choice of the 2 values for components of the membrane . table 1 lists several physicochemical parameters for membranes containing pc da and pc de complexes.table 1selected physicochemical parameters for two complexes : phosphatidylcholine decanoic acid ( pc da ) and phosphatidylcholine decylamine ( pc de)examined systemsurface area occupied by one molecule of complex ( molecule)stability constant of examined complex ( m mol)complex formation energy ( gibbs free energy ) ( kj mol)pc de82 0.822.38 1032.45 0.97 selected physicochemical parameters for two complexes : phosphatidylcholine decanoic acid ( pc da ) and phosphatidylcholine decylamine ( pc de ) the following conclusions can be drawn from the parameters describing the complexes studied : the stability constant of the pc da complex is 4.61 10 m mol , whereas the stability constant of the pc de complex is 2.38 10 m mol . the values of the stability constants of the lipid da and lipid de complexes are reported for the first time . it can be observed that the stability constants of the fatty acid containing complex are higher . thus , the pc da complex is more stable than the pc de complex.the experimental area occupied by one pc da complex is 144 , whereas the area occupied by the pc de complex is 82 .good agreement of the experimental and theoretical points verifies the assumption of formation of a 1:1 complex in the lipid membrane . a lack of variance between points indicates that complexes at different stoichiometries are not possible in the pc da or pc de membrane . the stability constant of the pc da complex is 4.61 10 m mol , whereas the stability constant of the pc de complex is 2.38 10 m mol . . the values of the stability constants of the lipid da and lipid de complexes are reported for the first time . it can be observed that the stability constants of the fatty acid containing complex are higher . thus , the pc da complex is more stable than the pc de complex . the experimental area occupied by one pc da complex is 144 , whereas the area occupied by the pc de complex is 82 . good agreement of the experimental and theoretical points verifies the assumption of formation of a 1:1 complex in the lipid membrane . a lack of variance between points indicates that complexes at different stoichiometries are not possible in the pc da or pc de membrane .

interfacial tension has been determined for phosphatidylcholine ( pc)decanoic acid ( da ) and pc decylamine ( de ) membranes . pc ( lecithin ) , da and de were used in the experiments ; the interfacial tension values of the pure components are 1.62 103 , 2.38 102 and 3.88 102 n / m ( hypothetical values for da and de ) , respectively . the 1:1 complexes were formed during formation of pc da and pc de membranes . the following parameters describing the complexes were determined : the surface concentrations of the lipid membranes formed from these complexes , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{3}^ { - 1 } $ $ \end{document } ; the interfacial tensions of such membranes , 3 ; and the stability constants of these complexes , k.

Introduction Theory Experimental Procedure Measuring Apparatus and Measuring Procedures Reagents Results and Discussion Conclusion

2006a):1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned}&\gamma_{1 } m_{1 } a_{1 } + \gamma_{2 } m_{2 } a_{2 } = \gamma\\&\frac{{m_{1 } } } { { m_{1 } + m_{2 } } } = x_{1}\\&x_{1 } + x_{2 } = 1 \hfill \\ \end{aligned } $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{1}^ { - 1 } , \,a_{2}^ { - 1 } $ $ \end{document } ( mol m ) are the surface concentrations of components 1 and 2 ; m1 , m2 ( mol m ) are the quantities of components 1 and 2 per unit area of the membrane ; 1 , 2 ( n m ) are the interfacial tensions of membranes assembled from pure components 1 and 2 ; ( n m ) is the measured interfacial tension of the membrane ; and x1 , x2 are the solution mole fractions of components 1 and 2 . the parameters describing the complex may be used to calculate theoretical points using the equation presented below ( agreement between the theoretical and experimental values implies that the system is well described by the above equations):4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ ka_{1}^ { - 1 } a_{2}^ { - 1 } \left ( { a_{1 } + a_{2 } } \right)\left ( { a_{3 } - a_{1 } } \right)\gamma^{2 } + \left [ { ka_{1}^ { - 1 } a_{2}^ { - 1 } \left ( { \gamma_{1 } a_{1 } - \gamma_{3 } a_{3 } } \right)\left ( { a_{1 } + a_{2 } } \right ) - \,ka_{1}^ { - 1 } a_{2}^ { - 1 } \left ( { \gamma_{2 } a_{1 } + \gamma_{3 } a_{2 } } \right)\left ( { a_{3 } - a_{1 } } \right ) + \ , a_{4 } a_{3}^ { - 1 } \left ( { a_{3 } + a_{2 } } \right ) } \right]\gamma + ka_{1}^ { - 1 } a_{2}^ { - 1 } a_{3 } \gamma_{3 } \left ( { \gamma_{3 } a_{2 } + \gamma_{2 } a_{1 } } \right ) - ka_{1}^ { - 1 } a_{2}^ { - 1 } a_{1 } \gamma_{1 } \left ( { a_{1 } \gamma_{2 } + a_{2 } \gamma_{3 } } \right ) - a_{4 } a_{3}^ { - 1 } \left ( { \gamma_{2 } a_{3 } + \gamma_{1 } a_{2 } } \right ) = 0 $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{gathered } a_{1 } = a_{3}^ { - 1 } \left ( { x_{2 } - x_{1 } } \right ) \hfill \\ a_{2 } = a_{2}^ { - 1 } x_{1 } \hfill \\ a_{3 } = a_{1}^ { - 1 } x_{2 } \hfill \\ a_{4 } = \left [ { a_{3}^ { - 1 } \left ( { \gamma_{1 } - \gamma_{2 } } \right)\left ( { x_{2 } - x_{1 } } \right ) + \,\left ( { \gamma_{1 } - \gamma_{3 } } \right)x_{1 } a_{2}^ { - 1 } + \ , \left ( { \gamma_{2 } - \gamma_{3 } } \right)x_{2 } a_{1}^ { - 1 } } \right ] \hfill \\ \end{gathered } $ $ \end{document } for systems containing two lipid components , 1:1 complex formation was assumed to be the explanation for deviation from the additivity rule . at least one of them is necessary for determination of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{3}^ { - 1 } $ $ \end{document } value . knowing the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{1}^ { - 1 } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{2}^ { - 1 } $ $ \end{document } as well as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{1 } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ b_{2 } $ $ \end{document } values , the surface concentration of the membrane composed of the lecithin da and lecithin de complexes could be determined . the resulting surface concentration values , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a_{3}^ { - 1 } $ $ \end{document } , for the pc da and pc de complexes were equal to 1.15 10 and 2.03 10 mol m , respectively . 4 when \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ x_{1 } = x_{2 } = 0.5 $ $ \end{document } ; these parameters amount to 4.61 10 and 2.38 10 m mol , respectively . these complexes arise by producing a connection between the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { -}{\mathop { \text{n}}\limits^{\left ( + \right ) } } \left ( { { \text{ch}}_{3 } } \right)_{3 } $ $ \end{document } group from the molecule of lecithin and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - { \text{coo}}^{\left ( - \right ) } $ $ \end{document } groups from the dimer of da , in the case of the complex pc da , and between the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - { \text{po}}^{\left ( - \right ) } $ $ \end{document } group from lecithin and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { -}{\mathop { \text{n}}\limits^{\left ( + \right ) } } { \text{h}}_{3 } $ $ \end{document } group from de . the dissociation constants of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { -}{\mathop { \text{n}}\limits^{\left ( + \right ) } } \left ( { { \text{ch}}_{3 } } \right)_{3 } $ $ \end{document } group from pc and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - { \text{coo}}^{\left ( - \right ) } $ $ \end{document } groups from the dimer of da are equal 10 ( petelska and figaszewski 2000 ) and about 10 ( chemistry tables 2004 ) , respectively . it should be noted that the dissociation constants of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ - { \text{po}}^{\left ( - \right ) } $ $ \end{document } group from pc and the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { -}{\mathop { \text{n}}\limits^{\left ( + \right ) } } { \text{h}}_{3 } $ $ \end{document } group from de equal 10 ( petelska and figaszewski 2000 ) and about 10 ( chemistry tables 2004 ) , respectively .

, heavy metals from industrial waste contaminate drinking water , soil , fodder , and food . also , the large volume of waste and the intense use of chemicals during past decades have resulted in numerous contaminated sites across europe . contaminated sites could pose significant environmental hazards for terrestrial and aquatic ecosystems as they are important sources of pollution which may result in ecotoxicological effects . management of contaminated sites aims at assessing the adverse effects caused and taking measures to satisfy environmental standards according to current legal requirements . additionally , the impact of soil contamination to health and more specifically the main epidemiological findings relevant to cs are briefly presented below . the implication of soils to human health is direct such as ingestion , inhalation , skin contact , and dermal absorption . some epidemiological examples include geohelminth infection and potentially harmful elements via soil ingestion , cancers caused by the inhalation of fibrous minerals , hookworm disease , and podoconiosis caused by skin contact with soils . ( 2001 ) have found small excess risks of congenital anomalies and low and very low birth weights in populations living near landfill sites . soil contamination is mainly located close to waste landfills , industrial / commercial activities diffusing heavy metals , oil industry , military camps , and nuclear power plants . each year in the eu , 3 billion tonnes of solid wastes are thrown away ( some 90 million tonnes of them are hazardous ) . this amounts to about 6 tonnes of solid waste for every man , woman , and child ( eurostat , environmental data centre on waste ) . the main anthropogenic sources of heavy metals exist in various industrial point sources , for example , present and former mining activities , foundries , smelters , and diffuse sources such as piping , constituents of products , combustion of by products , and traffic related to industrial and human activities . in the us , the army alone has estimated that over 1.2 million tons of soils have been contaminated with explosives , and the impact of explosives contamination in other countries in the world is of similar magnitude . in recent years , growing concerns about the health and ecological threats posed by manmade chemicals have led to studies of the toxicology of explosives , which have identified toxic and mutagenic effects of the common military explosives and their transformation products . papp et al . ( 2002 ) have studied the significant radioactive contamination of soil around a coal - fired thermal power plant . different contaminants have different effects on human health and the environment depending on their properties . the contaminant effect depends on its potential for dispersion , solubility in water or fat , bioavailability , carcinogenicity , and so forth . chlorinated hydrocarbons ( chcs ) are used mainly for the manufacturing of synthetic solvents and insecticides . epidemiological evidence suggests that the increased incidence of a variety of human cancers , such as lymphoma , leukemia , and liver and breast cancers , might be attributed to exposure to these agents . mineral oil large - scale use and various applications lead in many cases to environmental contamination . such contamination may be a consequence of petroleum transport , storage and refining , or accidents . from a quantitative perspective , that humans can tolerate this contaminant without health concerns has not been proven convincingly . the current editorial of the european journal of lipid science and technology concludes that this proof either has to be provided or we have to take measures to reduce our exposure ( from all sources , including cosmetics and pharmaceuticals ) and the environmental contamination . polycyclic aromatic hydrocarbons ( pahs ) are semivolatile , chemically stable , and hydrophobic organic compounds which are ubiquitous in the environment and good markers of urban activities . although several adverse health effects of heavy metals have been known for a long time , exposure to heavy metals continues and is even increasing in some parts of the world , in particular in less developed countries , though emissions have declined in most developed countries over the last 100 years . any metal ( or metalloid ) species may be considered a contaminant if it occurs where it is unwanted , or in a form or concentration that causes a detrimental human or environmental effect . metals / metalloids include lead ( pb ) , cadmium ( cd ) , mercury ( hg ) , arsenic ( as ) , chromium ( cr ) , copper ( cu ) , selenium ( se ) , nickel ( ni ) , silver ( ag ) , and zinc ( zn ) . other less common metallic contaminants include aluminium ( al ) , cesium ( cs ) , cobalt ( co ) , manganese ( mn ) , molybdenum ( mo ) , strontium ( sr ) , and uranium ( u ) . according to who , priority should be given to the pollutants on the basis of toxicity , environmental persistence , mobility , and bioaccumulation . many of the heavy metals such as cadmium , arsenic , chromium , nickel , dioxins , and pahs are considered to be carcinogenic , based on animal studies or studies of people exposed to high levels . in addition to carcinogenicity , many of these substances can produce other toxic effects ( depending on exposure level and duration ) on the central nervous system , liver , kidneys , heart , lungs , skin , reproduction , and so forth . the toxicity and fate of phenolic pollutants in the contaminated soils are associated with the oil - shale industry . phenol has been shown to cause liver and kidney damage , neurotoxic effects , and developmental toxicity in laboratory animals ( environment agency , 2009 ) . however , cyanide contamination is also associated with electroplating factories , road salt storage facilities , and gold mining tailings . cyanide toxicity results from inhibition of cytochrome oxidase thereby limiting the absorption of oxygen at the cellular level . the central nervous system is a major target of acute cyanide toxicity , with a short period of stimulation evidenced by rapid breathing , followed by depression , convulsions , paralysis , and possibly death . benzene , toluene , ethylbenzene , and xylene ( btex ) are classified as hazardous air pollutants ( haps ) . exposure to haps can cause a variety of health problems such as cancerous illnesses , respiratory irritation , and central nervous system damage . the objective of relevant eu policies is to achieve a quality of the environment where the levels of manmade contaminants on sites do not give rise to significant impacts or risks to human health and ecosystems . the most recent developments in soil policy at european level are the introduction of the thematic strategy for the protection of soils and the proposed soil framework directive . soil contamination is recognised as one of the eight soil threats expressed in the thematic strategy and the proposed directive . as there was not a consensus for the establishment of the soil framework directive , legal requirements for the general protection of soil have not been agreed at eu level and only exist individually in most member states . however , the integrated pollution and prevention control directive requires that operations falling under its scope do not create new soil contamination . other eu directives such as the water framework directive and the waste directive , not aimed directly at soil protection , provide indirect controls on soil contamination . notwithstanding these controls , some significant new site contamination still occurs as a result of accidents and illegal actions . while the creation of new contaminated sites is constrained by regulation , a very large number of sites exist with historical contamination that may present unacceptable risks and these sites require management . one example is the environmental disaster following flooding by red sludge in the ajka region in hungary . however , the research and political arena regarding land contamination no longer consider only a few incidents that lead to severe soil contamination , but rather look at it as a wide spread environmental problem . in 2001 , the european environment agency ( eea ) in cooperation with eea affiliated countries started to develop a core set of policy relevant indicators , among which the indicator progress in the management of contaminated sites ( csi015 ) was the only one related to soil . since then , data collections in relation to this indicator were launched four times by eea , the last one in 2006 , with contribution from member countries of the european environment information and observation network ( eionet ) . in the period 2011 - 2012 , the european soil data centre ( esdac ) organized a similar campaign in order to update the csi015 . this indicator quantifies the progress in the management of local contamination , identifies sectors with major contribution to soil contamination , classifies the major contaminants , and finally addresses issues of budgets spent for remediation . the indicator is very important for policy makers as it tracks progress in the management of contaminated sites and the provision of public and private money for remediation . with this indicator the indicator also supports the implementation of existing legislative and regulatory frameworks ( integrated pollution prevention and control directive , landfill directive , water framework directive ) as they should result in less new contamination of soil . the present study presents an overall picture in europe concerning contaminated sites and does not focus on individual countries . instead , there are many other studies , such as the one of ferguson ( 1999 ) , that present the inventories of contaminated sites for individual countries . the overall objective of this paper is to make an overview of the current situation of contaminated sites in europe . specifically , the study intends tofocus on contaminated sites caused by industrial activities;review the type of sources;respond to the main policy questions addressed in the indicator csi015 . focus on contaminated sites caused by industrial activities ; review the type of sources ; respond to the main policy questions addressed in the indicator csi015 . the study makes an assessment of the data collected through eionet and then focuses on the data related to contamination as a consequence of industrial activities . the contaminated sites data ( denoted as eionet - csi from now on ) were collected and managed by the european soil data centre ( esdac ) . the data were collected in 2011 - 2012 through the eionet network which consists of representative organizations from 38 european countries for a number of environmental themes . the appointed organisations for the theme soil are lead institutions in the soil domain at national level , and they provide official country data on specific requests related to soil by esdac . the geographical coverage of eionet includes 27 member states of the european union together with iceland , liechtenstein , norway , switzerland , turkey , and the west balkan cooperating countries : albania , bosnia , herzegovina , croatia , the former yugoslav republic of macedonia , montenegro , and serbia as well as kosovo under the un security council resolution 1244/99 . similar data on contaminated sites have been collected in 2001 , 2002 , 2003 , and 2006 . the data were collected through a standard questionnaire and then compiled in a centralized database . the questionnaire was designed such that received data could feed the compilation of the indicator , the csi015 indicator . there is no legal obligation for the eionet member countries to submit data , and their contribution is on a voluntary basis . in order to minimize the differences in interpretation by individual countries of certain terms used in the questionnaire , esdac provided the following definitions according to eea .contaminated site ( cs ) refers to a well - defined area where the presence of soil contamination has been confirmed and this presents a potential risk to humans , water , ecosystems , or other receptors . risk management measures ( e.g. , remediation ) may be needed depending on the severity of the risk of adverse impacts to receptors under the current or planned use of the site . potentially contaminated site ( pcs ) refers to sites where unacceptable soil contamination is suspected but not verified , and detailed investigations need to be carried out to verify whether there is unacceptable risk of adverse impacts on receptors.management of contaminated sites aims to access and , where necessary , reduce to an acceptable level the risk of adverse impacts on receptors ( remediate ) . the progress in management of cs is traced in 4 management steps starting with preliminary study , continuing with preliminary investigation , followed by site investigation , and concluding with implementation of site remediation ( reduction of risk ) . ( cs ) refers to a well - defined area where the presence of soil contamination has been confirmed and this presents a potential risk to humans , water , ecosystems , or other receptors . risk management measures ( e.g. , remediation ) may be needed depending on the severity of the risk of adverse impacts to receptors under the current or planned use of the site . ( pcs ) refers to sites where unacceptable soil contamination is suspected but not verified , and detailed investigations need to be carried out to verify whether there is unacceptable risk of adverse impacts on receptors . management of contaminated sites aims to access and , where necessary , reduce to an acceptable level the risk of adverse impacts on receptors ( remediate ) . the progress in management of cs is traced in 4 management steps starting with preliminary study , continuing with preliminary investigation , followed by site investigation , and concluding with implementation of site remediation ( reduction of risk ) . there is an important definition in terminology which allows the readers of the article to distinguish between estimated and identified sites . the questionnaire asked the countries to provide estimations on how many css and pcss may be situated in their territory . the questionnaire also asked for identified number of cs and pcs . in this case , the countries report data for which they actually posses available information about local soil properties and hydrology . for a more comprehensive assessment , a number of auxiliary official eurostat datasets were used such as the countries ' populations , the surface area , the gross domestic product ( gdp ) , and the number of enterprises in the industrial / services sectors . those datasets are used for developing statistics with parameters that include the surveyed population , the surveyed area , the density of cs and pcs , the contribution ( % ) of various industrial sectors to contamination , and the proportion of budget spent for management of cs . the study is based on the received data from the countries that participated in the survey , replying to the questionnaire available in the european soil portal . the questionnaire has a user - friendly format as a microsoft excel file and contains 5 main sections : management of contaminated sites , contribution of polluting activities to local soil contamination , environmental impacts , expenditures , remediation targets and technologies . each section includes between 1 and 5 questions requesting the user to submit the data for each of the available options . the questionnaire was requesting numerical values ( not classes or vague responses ) which allowed making aggregations depending on the policy question that was to be addressed . two example questions are the following : percentage ( % ) of sites , where risk reduction measures are completed ; expenditures in million euro per capita per year . as a support , a guidelines document was available with detailed explanation for each of the questions and the possible options plus example responses based on previous data collection exercises . each country represented by its designated eionet national reference centre for soil provided its best assessment based on available data . even if the questionnaire included other data and information , this paper mainly focuses on the local contamination analysis , the type of contamination ( which sectors are contributing the most ) , the distribution of the main contaminants , and the budget spent for remediation . the management of cs will not be analysed in detail as each country follows a different approach concerning the management steps . the analysis is performed in the study area as a whole and not at country level . it should be noted that quite different interpretations of the abovementioned definitions have been applied by individual countries . data on soil contamination per country is a necessary input in order to estimate the scale of soil contamination in europe . the majority of the addressed countries ( 33 out of 38 countries ) , corresponding to 80% of the total population , have responded with data on the identified number of pcs and cs ( figure 1(a ) ) . the missing five countries were bosnia , herzegovina , poland , portugal , slovenia , and turkey . according to figure 1(a ) , around 1,170,000 pcss have been identified in europe till 2011 . more than 10% of them or around 127,000 have been identified or confirmed as css . the ratio of remediated sites ( rss ) to css is around 45% as more than 58,000 css have already been remediated ( figure 1(a ) ) . the data gap for the 5 missing countries can be covered by employing the density of pcs ( 2.4 pcs/1,000 capita ) and cs ( 2.62 cs/10,000 capita ) ( table 1 ) . applying the average of 2.4 identified pcs per 1,000 capita for the 5 missing countries , the identified pcs for the whole europe ( 38 countries ) is estimated to be around 1,470,000 . applying the average of 2.62 identified cs per 10,000 capita ( table 1 , column ( a ) ) for the missing 5 countries , apart from the identified pcs and cs , countries have been asked to provide their estimations for those 2 figures . a subset of 12 countries out of the 33 participating ones has provided estimations about the pcs ( table 1 , column ( b ) ) . as a rule of thumb , the estimations are greater than the identified ones . according to their estimations , 740,000 pcss may exist in their territory with a density of 4.2 pcs/1,000 capita . two types of extrapolations can be performed in order to estimate the total number of pcs . in the first one , the average value of 4.2 pcs/1,000 capita is applied to the total population of the 38 countries , and the total number of estimated pcs is then around 2,553,000 ( figure 1(b ) ) . in the second extrapolation method , identification to estimation for pcs ( 70% ) is applied to the countries which were unable to provide estimations ; then the approximate number of pcs can be estimated to be 2,087,000 . another subset of 11 countries ( not a sub group of the previous 12 ) covering 10% of total population has provided estimations about cs . they estimated more than 32,000 css with a density of 5.7 cs/10,000 capita ( table 1 , column ( c ) ) . those 11 countries have reported 10,036 identified css which result that the ratio identification to estimation for cs is 30.7% . the first method of extrapolation is to apply the average density to the rest of the population ( 90% ) , where data does not exist . according to this estimation , the number of cs in europe is estimated to be around 342,000 which accounts for 14% of the total estimated pcs ( figure 1(b ) ) . in the second extrapolation method , identification to estimation for cs ( 30.7% ) is applied to the whole population , and the estimated number of cs becomes more than 516,000 . when comparing to the last survey of 2006 , the estimated number of pcs was around 3 million , and the estimated number of cs was around 250,000 . the huge differences in the density rates represent the situation of pcs per country and how countries interpret the term of potential contamination . interpreting the metadata that come with the received data , pcss are understood in a different way . for instance , luxemburg , belgium , netherlands , and france include potentially polluting activities in their pcs figures , and this is the reason for high density of pcs in those countries ( figure 2 ) . other countries such as austria , hungary , and norway include in their pcs figures only the sites where there is an evidence of potential contamination . some countries report sites which are important at national level , while others include also small sites such as storage tanks . the countries were asked to allocate a percentage of contribution of each sector to local soil contamination based on the occurrence of incidents . the following seven categories of activities were proposed : waste disposal ( municipal waste disposal and industrial waste disposal).industrial and commercial activities ( mining , oil extraction and production , and power plants).military ( military sites and war affected zones).storages ( oil storage , obsolete chemicals storage , and other storages).transport spills on land ( oil spill sites and other hazardous substance spills sites).nuclear . other sources . industrial and commercial activities ( mining , oil extraction and production , and power plants ) . military ( military sites and war affected zones ) . storages ( oil storage , obsolete chemicals storage , and other storages ) . transport spills on land ( oil spill sites and other hazardous substance spills sites ) . responses related to contributing sectors were received from 22 countries which correspond to circa 53% of the total study population . waste disposal and treatment contribute to more than 37% of soil contamination . inside this category , the industrial and commercial activities contribute to 33.3% share , followed by storage ( 10.5% ) , while of the rest have a contribution of 19.1% . nuclear operations contribute only 0.1% , but contamination from major nuclear players ( e.g. , scores from nuclear power stations ) was not taken into account by some countries . the data can not be compared to 2006 survey as the sample of countries that responded is dissimilar . the countries were asked to assign percentages in each specific industrial sector which contributes to soil contamination . the responses of 17 countries covering 44% of the total study population suggested that the production sector contributes to around 60% of soil contamination , while the service sector has a share of 33% and the mining sector contributes to around 7% ( figure 3 ) . a closer look at the production sector reveals that the textiles , leather , wood , and paper industries are of minor importance for local soil contamination ( circa 5% ) , while metal industries are most frequently reported to be important sources of contamination ( 13% ) followed by chemical industry ( 8% ) , oil industry ( 7% ) , and energy production ( 7% ) that sum up the 35% of the production sector , while all of the rest ( 25% ) are distributed in 6 categories . for the service sector , gasoline stations are the most frequently reported sources of contamination ( 15% ) followed by the car service stations ( around 6% ) . the eurostat data on sectoral breakdown of manufacturing ( nace ) sums up the total number of enterprises in the eu to 2.041 million . the eurostat industrial sectors do not correspond one - to - one with the industrial production sectors considered in the eionet - csi questionnaire ( table 2 , column ( a ) ) . some grouping of the eurostat sectors ( plus sign in column ( c ) ) has taken place to make the correspondence . note that the eurostat data for the mining sector was embedded in the eurostat category from the values in columns d and b , a new value ( column ( e ) ) is computed that expresses how many enterprises of an industrial sector contribute to 1 percent of the local contamination coming from that sector . the resulting figures show for instance that mining sites are individually heavier polluters compared to other sectors . instead , the electronic industry enterprises pollute less compared to the shown sectors ( table 2 ) . the countries were asked to allocate a percentage for the proposed contaminant categories based on the occurrence of soil contamination . distinctions were made between contaminants affecting the solid matrix ( soil , sludge , and sediments ) and the liquid matrix ( groundwater , surface waters , and leachate ) . the following eight categories of contaminants were proposed both for solid and liquid matrices : chlorinated hydrocarbons ( chcs).mineral oil.polycyclic aromatic hydrocarbons ( pahs).heavy metals.phenols.cyanides.aromatic hydrocarbons ( btex : benzene , toluene , ethyl benzene , and xylene).others.the responses were received from 16 countries which correspond to about 40% of the total study population . the analysis based on these responses is of key importance for research and development , the remediation market , and related industries . for instance , if a specific compound is known to be a major soil contaminant , it may be worthwhile to develop new detection methods ( i.e. , in situ detection ) and more efficient remediation techniques . aromatic hydrocarbons ( btex : benzene , toluene , ethyl benzene , and xylene ) . the main contaminant categories are heavy metals and mineral oil contributing jointly to around 60% in soil contamination and 53% of the groundwater contamination ( figure 4 ) . on the contrary , the remaining four categories ( btex , chc , pah , and others ) have similar contributions to soil contamination varying between 8 and 11% and summing up to 40% . in the groundwater contamination , the current distribution is similar to the one proposed after the analysis of the 2006 surveyed results . the cost of managing the cs is an important element taken into account by policy makers . the questionnaire included parts to investigate annual estimation of expenditures , share of private / public money , and share of total expenditure . this is a very important aspect as one of the most criticised issues in the proposed european soil framework directive was the required estimate of annual cost for management of cs . according to the impact assessment of the proposed directive , there was a wide - ranging estimate from 2.4 to 17.3 billion euros . according to the responses of 11 countries covering 23% of the total population ( 139 million out of the 612 million inhabitants for the total area ) , 1,483.2 million euros ( ) were spent annually for the management of cs in these countries . in absolute terms , this is around 10.7 per capita or 0.041% of the gross domestic product ( gdp ) for the 11 countries . the reported data show a small decrease in expenditure for management of cs compared to 2006 ( 12 per capita ) . if this sample of 11 countries is considered representative for the whole europe , then the management of contaminated sites can be estimated to be 6,526 million euros ( ) per year . compared to the impact assessment of the proposed soil framework directive , this amount of money is probably a more precise estimate of the cost of the management of all identified css ( including remediation ) . regarding the share of private / public money , 42% of the total expenditure comes from public budgets while the 58% from private investments . another interesting aspect of the study is the share in the total expenditures for the management of cs for the different management steps . the vast majority ( 80.6% ) is spent for the remediation measures while 15.1% is spent in site investigation and only 4.3% in after care measures and redevelopment of the sites . when considering the budget spent on remediation and the number or remediated sites ( rss ) in the 11 reported countries , it is calculated that the average amount spent per rs annually is around 37.1 thousand euros ( ) in a range varying from 7.5 thousand to 232 thousand annually . as the remediation of sites has a duration of more than 1 year , the majority ( 40% ) of the reported remediation projects fall in the range 50,000 to 500,000 , while a considerable 26.5% of the reported cases fall in the range between 5,000 and 50,000. in terms of estimations , around 1,170,000 pcss have been identified which are circa 45% of the total estimated pcss . also , around 127,000 css have been already identified which are circa 27% of the total estimated 342,000 css . moreover , around 46% of the total identified css have been remediated ( 58,300 rss ) . the identified figures for cs , pcss and rs are based on reported data from 33 countries , while the estimated css and pcss have been extrapolated based on data from a limited sample ( 11 or 12 countries ) . notwithstanding the positive outcomes of the eionet - csi data collection , it could be noted that the data submitted were not homogeneous since there are differences in the way that countries interpret the terms of contaminated sites . as shown in figure 2 this variability is explained by the large uncertainty both in terms of methodology and data . some countries run their own cs management system which may not fit perfectly to the definition of the csi015 indicator , and this contributes to methodology uncertainty . moreover , the reported data are usually based on expert judgement which includes a high degree of uncertainly . the countries may interpret the data specifications in different ways , and this increases the heterogeneity in the data reported . the reported data on csi015 indicator are based on the exceed of limits in concentrations of hazardous chemicals . however , common limits are unlikely to be established at the european level since they may be strongly influenced by local soil and geological properties . an adequate response to the high data variability could be to make a pan - european training event with the participation of competent national eionet authorities , with the objective to apply the same terminology in all countries in subsequent data collections . the heterogeneity of responses can also be decreased if the provided documentation is taken into account . in general , there are difficulties in getting the data on soil contamination , but improvement in data availability and data quality over the years can be observed . at this moment , the resulting dataset is the best picture that can be achieved based on national data . the eionet - csi data collection has taken place 5 years after the previous one of 2006 . this 5-year period between data collections seems to be more appropriate than the 2-year period applied in the past , since the data on cs are not changing considerably in such a relative short time . the direct and indirect costs to a country for dealing with the problem of cs depend on the amount and characteristics of cs in its territory . generally , the presence of cs can affect company profits , business confidence , and attractiveness to investors . the remediation cost of cs , even if only a very little percentage of gdp , seems to be a major issue , and investments to improve the land quality through remediation are not readily made . countries should weigh the costs of dealing with local land contamination against benefits to public health , improvement of the environment ( e.g. , water quality ) , land regeneration , and sustainable use of soil . restrictions set by privacy law in europe are a major obstacle to identification and management of land contamination . status and data on private land are not easily accessible to public authorities as this may have some implications for the land owner . however , the situation of his land is affecting public health , water quality , and ecosystem services . in cases of proven soil contamination the conflicts between public interest and privacy regarding land and in general environmental problems should be resolved at a legal basis . the eionet - csi dataset will be supplemented with heavy metals data at european level . in 2009 , 22,000 soil samples were taken in european union countries during a soil survey named lucas . those soil samples have been analysed for some of the most important soil attributes such as soil organic carbon , and the results assist to estimate better the overall situation in europe . currently , these soil samples are analysed for heavy metals , and the expected output results will facilitate better assessment of soil contamination in european union . the lucas heavy metals dataset will face the issue of privacy which can overcome with the application of digital soil mapping for the development of interpolated maps . the combination of lucas heavy metals with eionet - csi will be an important step in assessing soil contamination in europe . the proposed datasets and the current study can be considered by public health professionals for epidemiological assessments . the study of human exposure pathways is a key issue on contaminated sites , and certainly the integration of eionet - csi datasets with epidemiological data would be a very important step forward in this direction . moreover , as the majority of food is growing in soil , biomonitoring and other research should investigate the pathways and routes from producers to consumers .

under the european union ( eu ) thematic strategy for soil protection , the european commission has identified soil contamination as a priority for the collection of policy - relevant soil data at european scale . in order to support eu soil management policies , soil - related indicators need to be developed which requires appropriate data collection and establishment of harmonized datasets for the eu member states . in 2011 - 12 , the european soil data centre of the european commission conducted a project to collect data on contaminated sites from national institutions in europe using the european environment information and observation network for soil ( eionet - soil ) . this paper presents the results obtained from analysing the soil contaminated sites data submitted by participating countries . according to the received data , the number of estimated potential contaminated sites is more than 2.5 million and the identified contaminated sites around 342 thousand . municipal and industrial wastes contribute most to soil contamination ( 38% ) , followed by the industrial / commercial sector ( 34% ) . mineral oil and heavy metals are the main contaminants contributing around 60% to soil contamination . in terms of budget , the management of contaminated sites is estimated to cost around 6 billion euros ( ) annually .

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

, heavy metals from industrial waste contaminate drinking water , soil , fodder , and food . also , the large volume of waste and the intense use of chemicals during past decades have resulted in numerous contaminated sites across europe . management of contaminated sites aims at assessing the adverse effects caused and taking measures to satisfy environmental standards according to current legal requirements . additionally , the impact of soil contamination to health and more specifically the main epidemiological findings relevant to cs are briefly presented below . soil contamination is mainly located close to waste landfills , industrial / commercial activities diffusing heavy metals , oil industry , military camps , and nuclear power plants . this amounts to about 6 tonnes of solid waste for every man , woman , and child ( eurostat , environmental data centre on waste ) . the main anthropogenic sources of heavy metals exist in various industrial point sources , for example , present and former mining activities , foundries , smelters , and diffuse sources such as piping , constituents of products , combustion of by products , and traffic related to industrial and human activities . in the us , the army alone has estimated that over 1.2 million tons of soils have been contaminated with explosives , and the impact of explosives contamination in other countries in the world is of similar magnitude . the current editorial of the european journal of lipid science and technology concludes that this proof either has to be provided or we have to take measures to reduce our exposure ( from all sources , including cosmetics and pharmaceuticals ) and the environmental contamination . although several adverse health effects of heavy metals have been known for a long time , exposure to heavy metals continues and is even increasing in some parts of the world , in particular in less developed countries , though emissions have declined in most developed countries over the last 100 years . other less common metallic contaminants include aluminium ( al ) , cesium ( cs ) , cobalt ( co ) , manganese ( mn ) , molybdenum ( mo ) , strontium ( sr ) , and uranium ( u ) . according to who , priority should be given to the pollutants on the basis of toxicity , environmental persistence , mobility , and bioaccumulation . many of the heavy metals such as cadmium , arsenic , chromium , nickel , dioxins , and pahs are considered to be carcinogenic , based on animal studies or studies of people exposed to high levels . the central nervous system is a major target of acute cyanide toxicity , with a short period of stimulation evidenced by rapid breathing , followed by depression , convulsions , paralysis , and possibly death . the most recent developments in soil policy at european level are the introduction of the thematic strategy for the protection of soils and the proposed soil framework directive . soil contamination is recognised as one of the eight soil threats expressed in the thematic strategy and the proposed directive . as there was not a consensus for the establishment of the soil framework directive , legal requirements for the general protection of soil have not been agreed at eu level and only exist individually in most member states . however , the integrated pollution and prevention control directive requires that operations falling under its scope do not create new soil contamination . other eu directives such as the water framework directive and the waste directive , not aimed directly at soil protection , provide indirect controls on soil contamination . while the creation of new contaminated sites is constrained by regulation , a very large number of sites exist with historical contamination that may present unacceptable risks and these sites require management . however , the research and political arena regarding land contamination no longer consider only a few incidents that lead to severe soil contamination , but rather look at it as a wide spread environmental problem . in 2001 , the european environment agency ( eea ) in cooperation with eea affiliated countries started to develop a core set of policy relevant indicators , among which the indicator progress in the management of contaminated sites ( csi015 ) was the only one related to soil . since then , data collections in relation to this indicator were launched four times by eea , the last one in 2006 , with contribution from member countries of the european environment information and observation network ( eionet ) . in the period 2011 - 2012 , the european soil data centre ( esdac ) organized a similar campaign in order to update the csi015 . this indicator quantifies the progress in the management of local contamination , identifies sectors with major contribution to soil contamination , classifies the major contaminants , and finally addresses issues of budgets spent for remediation . the indicator is very important for policy makers as it tracks progress in the management of contaminated sites and the provision of public and private money for remediation . the present study presents an overall picture in europe concerning contaminated sites and does not focus on individual countries . instead , there are many other studies , such as the one of ferguson ( 1999 ) , that present the inventories of contaminated sites for individual countries . the overall objective of this paper is to make an overview of the current situation of contaminated sites in europe . specifically , the study intends tofocus on contaminated sites caused by industrial activities;review the type of sources;respond to the main policy questions addressed in the indicator csi015 . focus on contaminated sites caused by industrial activities ; review the type of sources ; respond to the main policy questions addressed in the indicator csi015 . the study makes an assessment of the data collected through eionet and then focuses on the data related to contamination as a consequence of industrial activities . the contaminated sites data ( denoted as eionet - csi from now on ) were collected and managed by the european soil data centre ( esdac ) . the data were collected in 2011 - 2012 through the eionet network which consists of representative organizations from 38 european countries for a number of environmental themes . the appointed organisations for the theme soil are lead institutions in the soil domain at national level , and they provide official country data on specific requests related to soil by esdac . the geographical coverage of eionet includes 27 member states of the european union together with iceland , liechtenstein , norway , switzerland , turkey , and the west balkan cooperating countries : albania , bosnia , herzegovina , croatia , the former yugoslav republic of macedonia , montenegro , and serbia as well as kosovo under the un security council resolution 1244/99 . similar data on contaminated sites have been collected in 2001 , 2002 , 2003 , and 2006 . the questionnaire was designed such that received data could feed the compilation of the indicator , the csi015 indicator . there is no legal obligation for the eionet member countries to submit data , and their contribution is on a voluntary basis . in order to minimize the differences in interpretation by individual countries of certain terms used in the questionnaire , esdac provided the following definitions according to eea .contaminated site ( cs ) refers to a well - defined area where the presence of soil contamination has been confirmed and this presents a potential risk to humans , water , ecosystems , or other receptors . , remediation ) may be needed depending on the severity of the risk of adverse impacts to receptors under the current or planned use of the site . potentially contaminated site ( pcs ) refers to sites where unacceptable soil contamination is suspected but not verified , and detailed investigations need to be carried out to verify whether there is unacceptable risk of adverse impacts on receptors.management of contaminated sites aims to access and , where necessary , reduce to an acceptable level the risk of adverse impacts on receptors ( remediate ) . the progress in management of cs is traced in 4 management steps starting with preliminary study , continuing with preliminary investigation , followed by site investigation , and concluding with implementation of site remediation ( reduction of risk ) . , remediation ) may be needed depending on the severity of the risk of adverse impacts to receptors under the current or planned use of the site . ( pcs ) refers to sites where unacceptable soil contamination is suspected but not verified , and detailed investigations need to be carried out to verify whether there is unacceptable risk of adverse impacts on receptors . management of contaminated sites aims to access and , where necessary , reduce to an acceptable level the risk of adverse impacts on receptors ( remediate ) . the progress in management of cs is traced in 4 management steps starting with preliminary study , continuing with preliminary investigation , followed by site investigation , and concluding with implementation of site remediation ( reduction of risk ) . in this case , the countries report data for which they actually posses available information about local soil properties and hydrology . for a more comprehensive assessment , a number of auxiliary official eurostat datasets were used such as the countries ' populations , the surface area , the gross domestic product ( gdp ) , and the number of enterprises in the industrial / services sectors . those datasets are used for developing statistics with parameters that include the surveyed population , the surveyed area , the density of cs and pcs , the contribution ( % ) of various industrial sectors to contamination , and the proportion of budget spent for management of cs . the study is based on the received data from the countries that participated in the survey , replying to the questionnaire available in the european soil portal . the questionnaire has a user - friendly format as a microsoft excel file and contains 5 main sections : management of contaminated sites , contribution of polluting activities to local soil contamination , environmental impacts , expenditures , remediation targets and technologies . two example questions are the following : percentage ( % ) of sites , where risk reduction measures are completed ; expenditures in million euro per capita per year . as a support , a guidelines document was available with detailed explanation for each of the questions and the possible options plus example responses based on previous data collection exercises . even if the questionnaire included other data and information , this paper mainly focuses on the local contamination analysis , the type of contamination ( which sectors are contributing the most ) , the distribution of the main contaminants , and the budget spent for remediation . the management of cs will not be analysed in detail as each country follows a different approach concerning the management steps . data on soil contamination per country is a necessary input in order to estimate the scale of soil contamination in europe . the majority of the addressed countries ( 33 out of 38 countries ) , corresponding to 80% of the total population , have responded with data on the identified number of pcs and cs ( figure 1(a ) ) . according to figure 1(a ) , around 1,170,000 pcss have been identified in europe till 2011 . applying the average of 2.4 identified pcs per 1,000 capita for the 5 missing countries , the identified pcs for the whole europe ( 38 countries ) is estimated to be around 1,470,000 . applying the average of 2.62 identified cs per 10,000 capita ( table 1 , column ( a ) ) for the missing 5 countries , apart from the identified pcs and cs , countries have been asked to provide their estimations for those 2 figures . as a rule of thumb , the estimations are greater than the identified ones . two types of extrapolations can be performed in order to estimate the total number of pcs . in the first one , the average value of 4.2 pcs/1,000 capita is applied to the total population of the 38 countries , and the total number of estimated pcs is then around 2,553,000 ( figure 1(b ) ) . in the second extrapolation method , identification to estimation for pcs ( 70% ) is applied to the countries which were unable to provide estimations ; then the approximate number of pcs can be estimated to be 2,087,000 . the first method of extrapolation is to apply the average density to the rest of the population ( 90% ) , where data does not exist . according to this estimation , the number of cs in europe is estimated to be around 342,000 which accounts for 14% of the total estimated pcs ( figure 1(b ) ) . in the second extrapolation method , identification to estimation for cs ( 30.7% ) is applied to the whole population , and the estimated number of cs becomes more than 516,000 . when comparing to the last survey of 2006 , the estimated number of pcs was around 3 million , and the estimated number of cs was around 250,000 . interpreting the metadata that come with the received data , pcss are understood in a different way . waste disposal and treatment contribute to more than 37% of soil contamination . inside this category , the industrial and commercial activities contribute to 33.3% share , followed by storage ( 10.5% ) , while of the rest have a contribution of 19.1% . the countries were asked to assign percentages in each specific industrial sector which contributes to soil contamination . the responses of 17 countries covering 44% of the total study population suggested that the production sector contributes to around 60% of soil contamination , while the service sector has a share of 33% and the mining sector contributes to around 7% ( figure 3 ) . a closer look at the production sector reveals that the textiles , leather , wood , and paper industries are of minor importance for local soil contamination ( circa 5% ) , while metal industries are most frequently reported to be important sources of contamination ( 13% ) followed by chemical industry ( 8% ) , oil industry ( 7% ) , and energy production ( 7% ) that sum up the 35% of the production sector , while all of the rest ( 25% ) are distributed in 6 categories . for the service sector , gasoline stations are the most frequently reported sources of contamination ( 15% ) followed by the car service stations ( around 6% ) . the eurostat data on sectoral breakdown of manufacturing ( nace ) sums up the total number of enterprises in the eu to 2.041 million . the eurostat industrial sectors do not correspond one - to - one with the industrial production sectors considered in the eionet - csi questionnaire ( table 2 , column ( a ) ) . note that the eurostat data for the mining sector was embedded in the eurostat category from the values in columns d and b , a new value ( column ( e ) ) is computed that expresses how many enterprises of an industrial sector contribute to 1 percent of the local contamination coming from that sector . instead , the electronic industry enterprises pollute less compared to the shown sectors ( table 2 ) . the countries were asked to allocate a percentage for the proposed contaminant categories based on the occurrence of soil contamination . the following eight categories of contaminants were proposed both for solid and liquid matrices : chlorinated hydrocarbons ( chcs).mineral oil.polycyclic aromatic hydrocarbons ( pahs).heavy metals.phenols.cyanides.aromatic hydrocarbons ( btex : benzene , toluene , ethyl benzene , and xylene).others.the responses were received from 16 countries which correspond to about 40% of the total study population . the main contaminant categories are heavy metals and mineral oil contributing jointly to around 60% in soil contamination and 53% of the groundwater contamination ( figure 4 ) . on the contrary , the remaining four categories ( btex , chc , pah , and others ) have similar contributions to soil contamination varying between 8 and 11% and summing up to 40% . in the groundwater contamination , the current distribution is similar to the one proposed after the analysis of the 2006 surveyed results . this is a very important aspect as one of the most criticised issues in the proposed european soil framework directive was the required estimate of annual cost for management of cs . according to the impact assessment of the proposed directive , there was a wide - ranging estimate from 2.4 to 17.3 billion euros . according to the responses of 11 countries covering 23% of the total population ( 139 million out of the 612 million inhabitants for the total area ) , 1,483.2 million euros ( ) were spent annually for the management of cs in these countries . in absolute terms , this is around 10.7 per capita or 0.041% of the gross domestic product ( gdp ) for the 11 countries . if this sample of 11 countries is considered representative for the whole europe , then the management of contaminated sites can be estimated to be 6,526 million euros ( ) per year . compared to the impact assessment of the proposed soil framework directive , this amount of money is probably a more precise estimate of the cost of the management of all identified css ( including remediation ) . another interesting aspect of the study is the share in the total expenditures for the management of cs for the different management steps . the vast majority ( 80.6% ) is spent for the remediation measures while 15.1% is spent in site investigation and only 4.3% in after care measures and redevelopment of the sites . when considering the budget spent on remediation and the number or remediated sites ( rss ) in the 11 reported countries , it is calculated that the average amount spent per rs annually is around 37.1 thousand euros ( ) in a range varying from 7.5 thousand to 232 thousand annually . as the remediation of sites has a duration of more than 1 year , the majority ( 40% ) of the reported remediation projects fall in the range 50,000 to 500,000 , while a considerable 26.5% of the reported cases fall in the range between 5,000 and 50,000. in terms of estimations , around 1,170,000 pcss have been identified which are circa 45% of the total estimated pcss . notwithstanding the positive outcomes of the eionet - csi data collection , it could be noted that the data submitted were not homogeneous since there are differences in the way that countries interpret the terms of contaminated sites . as shown in figure 2 this variability is explained by the large uncertainty both in terms of methodology and data . some countries run their own cs management system which may not fit perfectly to the definition of the csi015 indicator , and this contributes to methodology uncertainty . however , common limits are unlikely to be established at the european level since they may be strongly influenced by local soil and geological properties . in general , there are difficulties in getting the data on soil contamination , but improvement in data availability and data quality over the years can be observed . the eionet - csi data collection has taken place 5 years after the previous one of 2006 . this 5-year period between data collections seems to be more appropriate than the 2-year period applied in the past , since the data on cs are not changing considerably in such a relative short time . restrictions set by privacy law in europe are a major obstacle to identification and management of land contamination . status and data on private land are not easily accessible to public authorities as this may have some implications for the land owner . the eionet - csi dataset will be supplemented with heavy metals data at european level . those soil samples have been analysed for some of the most important soil attributes such as soil organic carbon , and the results assist to estimate better the overall situation in europe . currently , these soil samples are analysed for heavy metals , and the expected output results will facilitate better assessment of soil contamination in european union . the lucas heavy metals dataset will face the issue of privacy which can overcome with the application of digital soil mapping for the development of interpolated maps . the combination of lucas heavy metals with eionet - csi will be an important step in assessing soil contamination in europe . the study of human exposure pathways is a key issue on contaminated sites , and certainly the integration of eionet - csi datasets with epidemiological data would be a very important step forward in this direction .

most individuals diagnosed with schizophrenia must cope with some form of stigmatization during their lives.1,2 different types of public stigma , self - stigma , and label avoidance may have profoundly negative consequences for individuals with a mental disorder.38 the stigma consists of two basic factors , negative attitudes and discrimination . the stigma then operates at three levels , public ( ie , how the stigma manifests in a given society , culture , media , everyday habits , and attitudes ) , structural ( institutional , ie , at the level of functioning of organizations , agencies , and employers ) , and personal ( ie , how patients perceive themselves).9 significant terminological distinction exists between the public stigma ( when the general population supports the prejudices and discriminates against people with mental illness ) and the personal stigma , which also consists of the following three parts : 1 ) the perceived stigma ; 2 ) the experienced stigma ; and 3 ) the self - stigma , ie , how stigma itself accepted.10 the perceived or anticipated stigma is associated with patients beliefs about the attitudes of the general public to the members of stigmatized groups.11 the experienced stigma is related to the emotional experience of discrimination . the self - stigma is accompanied by a loss of positive beliefs about themselves , a decrease in self - esteem and self - confidence , and poorer insight among the individuals with psychoses.10,1216 internalization of the stigma can start when the individuals notice that others begin to act differently to them because of their mental struggles and realize the prejudices that lead to such action . subsequently , they begin to believe that the views and attitudes toward people with a mental disorder are justified . in the last stage of internalization of the stigma , this process leads to the increase in the intensity of symptoms and worse overall prognosis.17,18 the stigma affects not only the patients but also their families . hand in hand with accepting the reality of mental illness often appears the fear of stigmatization.19 a meta - analysis of 54 studies performed by gerlinger et al20 shows that the neighbors negative perception of stigma affects a significant number of patients with schizophrenia . on average , 64.5% of the patients feel stigmatized , and up to 55.9% actually experience stigmatization . according to the authors , the perceived or experienced stigma is associated with more severe depressive symptoms , greater social anxiety and avoidant behavior , low self - confidence , poorer social functioning , and overall lower quality of life . two other studies have pointed to the relationship between the personal stigma and the social anxiety already during the first psychotic episode.21,22 patients with comorbid social anxiety showed an increased perception of the stigma.21 the aim of this study was to investigate the degree of self - stigma in patients with schizophrenia and its association with clinical and demographic factors . according to our hypotheses , the level of self - stigma is linked with , 1 ) the severity of the disorder , 2 ) the duration or early onset of the disorder , 3 ) the number of hospitalizations , 4 ) the employment status , 5 ) the level of education , and 6 ) the partner status . a total of 197 patients of both sexes , attending outpatient psychiatric facilities in stable condition ( they did not require hospitalization , changes in medication , or other therapeutic intervention ) , participated in the study . the inclusion criteria were as follows : age : 1865 years.diagnosis of schizophrenia spectrum disorders according to international classification of diseases tenth edition.23 diagnosis of schizophrenia spectrum disorders according to international classification of diseases tenth edition.23 patients suffering from severe physical disease , mental retardation , or an organic mental disorder were excluded . after a short education , the patients completed assessment scales and questionnaires . the severity of illness was determined by a thorough interview with the doctor , who evaluated the severity on the objective version of clinical global impression ( cgi ) severity scale ( objcgi - s ) . the following assessment tools were used : the internalized stigma of mental illness ( ismi ) scale : the scale consists of 29 items with a 4-point likert scale and evaluates five areas of self - stigma alienation , stereotype endorsement , perceived discrimination , social withdrawal , and stigma resistance.24,25 the czech version of the ismi scale was standardized by ociskov et al.26 internal consistency of the czech text of the scale was excellent ( cronbach s =0.91).cgi : the cgi is the assessment of the overall severity of the disorder.27 the objcgi - s is an overall evaluation of the patient s mental state by a physician . in the subjective version of cgi severity scale ( subjcgi - s ) , the patient himself / herself evaluates his / her overall condition . the scale ranges from 1 ( normal , with no signs of illness ) to 7 ( extremely severe symptoms of the disease).demographic questionnaire contained the basic information : sex , age , marital status , education , employment , disability , age at onset of the disorder , duration of attendance to the psychiatric services , number of hospitalizations , time since last hospitalization , number of visited psychiatrists , medication , and medication discontinuation in the past ( on the recommendation of a psychiatrist or willingly ) . the internalized stigma of mental illness ( ismi ) scale : the scale consists of 29 items with a 4-point likert scale and evaluates five areas of self - stigma alienation , stereotype endorsement , perceived discrimination , social withdrawal , and stigma resistance.24,25 the czech version of the ismi scale was standardized by ociskov et al.26 internal consistency of the czech text of the scale was excellent ( cronbach s =0.91 ) . cgi : the cgi is the assessment of the overall severity of the disorder.27 the objcgi - s is an overall evaluation of the patient s mental state by a physician . in the subjective version of cgi severity scale ( subjcgi - s ) , the patient himself / herself evaluates his / her overall condition . the scale ranges from 1 ( normal , with no signs of illness ) to 7 ( extremely severe symptoms of the disease ) . demographic questionnaire contained the basic information : sex , age , marital status , education , employment , disability , age at onset of the disorder , duration of attendance to the psychiatric services , number of hospitalizations , time since last hospitalization , number of visited psychiatrists , medication , and medication discontinuation in the past ( on the recommendation of a psychiatrist or willingly ) . the statistical programs prism 3 ( graphpad software , inc . , la jolla , ca , usa ) and spss 24.0 ( spss inc . , chicago , il , usa ) demographic data and the average scores in the scales were evaluated using descriptive statistics ( mean , median , standard deviation , and the characteristics of data distribution ) . fisher s exact test was used to verify the relationship between dichotomous variables ( sex , marital status , dropout of medication ) . the general rule of selection was that backward stepwise regression tested only variables that were statistically significant in the univariate analysis . the research was conducted according to the latest version of the declaration of helsinki and recommendations for good clinical practice.28 all patients signed an informed consent form . all patients were in outpatient care at the psychiatric clinic and were currently in stable condition medication was administered according to the recommended guidelines for the treatment of schizophrenia.29 a total of 195 patients with psychotic spectrum disorders ( 99.0% ) reported that they used recommended medication prescribed by an attending psychiatrist . most of them declared that they were using the medication regularly in prescribed doses ( n=137 ; 69.5% ) , 58 ( 29.4% ) used medication irregularly , and two patients ( 0.01% ) stated that they did not use the medication at all . a total of 197 patients of both sexes , attending outpatient psychiatric facilities in stable condition ( they did not require hospitalization , changes in medication , or other therapeutic intervention ) , participated in the study . the inclusion criteria were as follows : age : 1865 years.diagnosis of schizophrenia spectrum disorders according to international classification of diseases tenth edition.23 diagnosis of schizophrenia spectrum disorders according to international classification of diseases tenth edition.23 patients suffering from severe physical disease , mental retardation , or an organic mental disorder were excluded . after a short education , the patients completed assessment scales and questionnaires . the severity of illness was determined by a thorough interview with the doctor , who evaluated the severity on the objective version of clinical global impression ( cgi ) severity scale ( objcgi - s ) . the following assessment tools were used : the internalized stigma of mental illness ( ismi ) scale : the scale consists of 29 items with a 4-point likert scale and evaluates five areas of self - stigma alienation , stereotype endorsement , perceived discrimination , social withdrawal , and stigma resistance.24,25 the czech version of the ismi scale was standardized by ociskov et al.26 internal consistency of the czech text of the scale was excellent ( cronbach s =0.91).cgi : the cgi is the assessment of the overall severity of the disorder.27 the objcgi - s is an overall evaluation of the patient s mental state by a physician . in the subjective version of cgi severity scale ( subjcgi - s ) , the patient himself / herself evaluates his / her overall condition . the scale ranges from 1 ( normal , with no signs of illness ) to 7 ( extremely severe symptoms of the disease).demographic questionnaire contained the basic information : sex , age , marital status , education , employment , disability , age at onset of the disorder , duration of attendance to the psychiatric services , number of hospitalizations , time since last hospitalization , number of visited psychiatrists , medication , and medication discontinuation in the past ( on the recommendation of a psychiatrist or willingly ) . the internalized stigma of mental illness ( ismi ) scale : the scale consists of 29 items with a 4-point likert scale and evaluates five areas of self - stigma alienation , stereotype endorsement , perceived discrimination , social withdrawal , and stigma resistance.24,25 the czech version of the ismi scale was standardized by ociskov et al.26 internal consistency of the czech text of the scale was excellent ( cronbach s =0.91 ) . cgi : the cgi is the assessment of the overall severity of the disorder.27 the objcgi - s is an overall evaluation of the patient s mental state by a physician . in the subjective version of cgi severity scale ( subjcgi - s ) , the patient himself / herself evaluates his / her overall condition . the scale ranges from 1 ( normal , with no signs of illness ) to 7 ( extremely severe symptoms of the disease ) . demographic questionnaire contained the basic information : sex , age , marital status , education , employment , disability , age at onset of the disorder , duration of attendance to the psychiatric services , number of hospitalizations , time since last hospitalization , number of visited psychiatrists , medication , and medication discontinuation in the past ( on the recommendation of a psychiatrist or willingly ) . the statistical programs prism 3 ( graphpad software , inc . , la jolla , ca , usa ) and spss 24.0 ( spss inc . , chicago , il , usa ) were used for the statistical evaluation of the results . demographic data and the average scores in the scales were evaluated using descriptive statistics ( mean , median , standard deviation , and the characteristics of data distribution ) . fisher s exact test was used to verify the relationship between dichotomous variables ( sex , marital status , dropout of medication ) . the general rule of selection was that backward stepwise regression tested only variables that were statistically significant in the univariate analysis . the research was conducted according to the latest version of the declaration of helsinki and recommendations for good clinical practice.28 all patients signed an informed consent form . all patients were in outpatient care at the psychiatric clinic and were currently in stable condition medication was administered according to the recommended guidelines for the treatment of schizophrenia.29 a total of 195 patients with psychotic spectrum disorders ( 99.0% ) reported that they used recommended medication prescribed by an attending psychiatrist . most of them declared that they were using the medication regularly in prescribed doses ( n=137 ; 69.5% ) , 58 ( 29.4% ) used medication irregularly , and two patients ( 0.01% ) stated that they did not use the medication at all . the data were collected in the period from may 2015 to august 2016 . the battery of scales was offered to 197 patients with schizophrenia and related psychotic disorders who attended the private psychiatric practices or the outpatient psychiatric ward in olomouc ( overall in 30 outpatient psychiatrists ) . there were slightly more women than men in the sample ( 54.3% of women ) . the levels of education were as follows : 23 ( 11.7% ) patients had a primary school education , 52 ( 26.4% ) had vocational training , 84 ( 42.6% ) had a secondary education , and 37 ( 18.8% ) had university level of education . there were 66.0% of unemployed patients and 33.5% worked as employees or were self - employed . overall , 51.8% of the participants had a full disability pension , 16.8% had a partial disability pension , and 5.5% were taking the senior age rent . regarding marital status , more patients were single ( 63.5% ) , 20.3% were married , and 13.2% were divorced . nearly a third of the patients had a partner ( 29.4% ; table 1 ) . the psychiatrist assessed the current severity of the disorders on average between mildly ill to moderately ill and the patients in their subjective scale on average between borderline mentally ill and mildly ill . the subjective assessment of the disease severity by the patients differed ( was lower ) significantly from an objective evaluation ( mann whitney [ mw ] test , u=11,470 ; p<0.001 ) . still , the subjective and objective assessment of the severity of the disorder significantly correlated with each other ( spearman s r=0.45 , p<0.001 ) . the primary diagnosis was a schizophrenia spectrum disorder in all 197 patients ( 139 were diagnosed with schizophrenia , five with delusional disorder , 12 with acute and transient psychotic disorders , and 41 with schizoaffective disorder ) . the total score of the ismi significantly correlated with certain demographic and clinical factors ( table 2 ) . the age of the patient and the age at onset of the disorder did not significantly correlate with the ismi total score and most of the ismi domains , with the exception that the age positively correlated with the stereotype endorsement and negatively with the perceived discrimination ( table 2 ) . the number of hospitalizations significantly correlated with the ismi total score and all domains of the ismi scale except stigma resistance . in addition , the length of the treatment significantly correlated with the ismi total score and all its domains except stigma resistance . the objcgi - s significantly positively correlated with the ismi total score and with domains alienation , perceived discrimination , and social withdrawal , but not with stereotype endorsement or stigma resistance . the subjcgi - s correlated with ismi total score and all domains of ismi scale . the difference between objcgi - s and subjcgi - s negatively correlated with all domains of ismi scale and the ismi total score except perceived discrimination ( table 2 ) . the men and women differed according to their mean age ( the average age of men and women was 37.411.2 years and 42.411.3 years , respectively ; the independent t - test : t=3.106 ; df=195 ; p<0.01 ) . there were no significant differences in the mean level of self - stigma in the ismi total score or any ismi domain according to the sex of the patients ( table 3 ) . there were several statistically significant differences between the patients with and without jobs according to their age ( unemployed : 41.412.2 years versus employed : 37.69.5 years ; the independent t - test : t=2.203 ; df = 194 ; p<0.05 ) , the mean number of hospitalizations ( unemployed : 4.43.8 versus employed : 2.52.0 ; the mw test : u=3,554 ; p<0.001 ) , the mean duration of the treatment ( unemployed : 15.010.4 years versus employed : 9.48.5 years ; the mw test : u=2,850 ; p<0.001 ) , objcgi - s ( unemployed : 3.81.2 versus employed : 3.11.2 ; the mw test : u=2,881 ; p<0.001 ) , and subjcgi - s ( unemployed : 2.91.6 versus employed : 2.21.3 ; the mw test : u=3,208 ; p<0.01 ) . there was also a statistically significant difference according to the occupation in the self - stigma measured by the ismi total score and in the domains , stereotype endorsement and social withdrawal ( table 3 ) . the differences in self - stigma according to the marital status of the patients were calculated without widows because this category was not sufficiently large ( there were only six widowed individuals ) . there was a statistically significant difference between subgroups according to the marital status , in their mean age ( one - way analysis of variance [ anova ] : f=0.85 ; df = 190 ; p<0.001 ) . the bonferroni s multiple comparison test showed that the differences were between the singles and the married patients ( p<0.001 ) and the singles and the divorced patients ( p<0.001 ) , but not between the married and divorced patients . there were also statistically significant differences between the subgroups of the patients divided according to the marital status at the onset of the disorder ( one - way anova : f=26.07 ; df=189 ; p<0.001 ) . the patients did not significantly differ in both the mean scores of the objcgi - s ( the singles scored 3.61.3 , the married 3.21.2 , and the divorced 3.81.3 ; the kruskal wallis [ kw ] test = 3.574 ; non significant [ ns ] ) and the subjcgi - s ( the singles reached 2.61.5 , the married 2.61.4 , and the divorced 3.01.6 ; the kw test : = 1.186 ; ns ) according to the marital status . there was no statistically significant difference between the subgroups according to the marital status in the mean ismi scores ( table 3 ) . the patients , who had a partner , were older ( the individuals without a partner had the mean age of 37.911.4 years versus the patients with a partner had the mean age of 45.59.8 years ; the independent t - test : t=4.462 ; df=195 ; p<0.001 ) , their disorder started later in life ( without a partner , the average age at the beginning of the disorder was 25.08.7 years versus with a partner mean age was 30.210.8 years ; mw test ; u=2,754 ; p<0.001 ) . there were no significant differences according to the partnership in the average number of the hospitalizations , the duration of the treatment , and the objective or subjective measure of the severity of the disorder ( all mw tests : ns ) . the partnership had no effect on the self - stigma measured by the ismi scale ( table 3 ) . there were only 51 patients without any pension or rent in the sample ( table 1 ) . there was a significant difference in the age according to the pension status in the subgroups ( the mean age in the patients without the disability pension was 34.911.2 years , in the full disability pension 40.89.2 years , in the partial disability pension 38.49.0 , and in the senior rent 63.210.0 years ; one - way anova : f=25.77 ; df=196 ; p<0.001 ) . the bonferroni s multiple comparison test showed that the significant difference between the age was in comparison to the subgroups of the senior rent and all another group ( all p<0.001 ) and among the patients without the pension and the patients with the full disability pension ( p<0.01 ) . there were also statistically significant differences between the subgroups divided according to the pension status in the number of the hospitalizations . the patients without any pension were hospitalized 1.81.5 times , the patients with the full disability pension 4.83.0 times , the patients with the partial disability pension 3.32.5 times , and the patients with the senior rent 4.22.5 times ( the kw test : = 35.57 ; p<0.001 ) . the dunn s multiple comparison test showed that the differences were between the group of the patients without the pension and other groups with all types of a pension , or rent ( all : p<0.05 ) . in addition , there were statistically significant differences between the subgroups of patients divided according to their pension status in length of the treatment . the patients without any pension had been treated for 6.48.4 years , the full pensioned patients 14.88.4 years , the partially pensioned patients 12.97.8 years , and the senior rent patients 29.613.4 years ( the kw test : = 53.05 ; p<0.001 ) . the dunn s multiple comparison test showed that the differences were between the group without the pension and all other type pension groups ( all three comparisons : p<0.001 ) and the senior rent group and both pension groups ( both : p<0.05 ) . there were also significant differences in the objectively rated severity of the disorder between the groups divided according to the pension status . the patients without any pension were rated by their psychiatrists with the mean score of 2.81.2 , those with the full disability pension with the average rating of 4.01.2 , the individuals with the partial disability pension with the mean rating of 3.61.1 , and those with the senior rent with the mean rating of 3.71.4 ( the kw test : = 30.4 ; p<0.001 ) . the dunn s multiple comparison test showed that the differences were only in the group of the patients without any pension , the group with the full disability pension ( p<0.05 ) , and the group with the partial disability pension ( p<0.05 ) , but not in other intergroup comparisons . the subjcgi - s showed significant differences among the groups divided according to the pension , too . the patients without any pension rated their severity of the disorders as 1.91.2 , the patients with the full disability pension as 2.91.5 , the patients with the partial disability pension as 3.11.6 , and the patients with the senior rent as 2.41.4 ( the kw test : = 17.97 ; p<0.001 ) . according to the dunn s multiple comparison test , the differences were only in the group without any pension , the group with the full disability pension ( p<0.05 ) , and the group with a partial disability pension ( p<0.05 ) , but not in other intergroup comparisons . the difference between the ismi total score in groups divided according to the pension status reached statistical significance ( table 3 ) . according to the bonferroni s multiple comparison test , the difference was only among patients without any pension and the patients with the full disability pension ( p<0.05 ) , but not in other intergroup comparisons . from the domains of the ismi scale , stereotype endorsement showed the highest difference between the groups divided according to the pension status ( table 3 ) and the bonferroni s multiple comparison test showed that there were significant differences between the group without any pension and all other groups with the pension ( with the full disability pension : p<0.001 ; with the partial disability pension : p<0.001 ; with the senior rent : p<0.05 ) . furthermore , there were significant differences in the domain of the ismi scale , social withdrawal , between the groups divided according to the pension status ( table 3 ) . in the bonferroni s multiple comparison test , the significant difference was only among patients without the pension and the patients with the full disability pension , but not in other subgroup interactions . there were no significant differences between groups divided according to the education in the demographical data ( age , onset of the disorder , length of the treatment ) , or the severity of the disorder . the degree of the self - stigma measured by the ismi total score did not differ according to the level of education , but one domain of the ismi scale , stereotype endorsement , correlated with the degree of education ( table 3 ) . to identify the most significant variables connected to self - stigma , a multiple regression analysis was carried out , namely backward stepwise regression . independent variables were the number of hospitalizations , the length of the treatment , objcgi - s , subjcgi - s , the difference between the objcgi - s and subjcgi - s , occupation , and pension . the objcgi had the tolerance score of 0.708 and variance inflation factor ( vif ) of 1.412 ; and the difference of objcgi - subjcgi had a tolerance score 0.823 and vif of 1.214 . the subjcgi was removed at the very beginning of the first step of regression . in the four steps of the backward stepwise regression , three factors remained , and all of them were statistically significant ( table 4 ) . the data were collected in the period from may 2015 to august 2016 . the battery of scales was offered to 197 patients with schizophrenia and related psychotic disorders who attended the private psychiatric practices or the outpatient psychiatric ward in olomouc ( overall in 30 outpatient psychiatrists ) . there were slightly more women than men in the sample ( 54.3% of women ) . the levels of education were as follows : 23 ( 11.7% ) patients had a primary school education , 52 ( 26.4% ) had vocational training , 84 ( 42.6% ) had a secondary education , and 37 ( 18.8% ) had university level of education . there were 66.0% of unemployed patients and 33.5% worked as employees or were self - employed . overall , 51.8% of the participants had a full disability pension , 16.8% had a partial disability pension , and 5.5% were taking the senior age rent . regarding marital status , more patients were single ( 63.5% ) , 20.3% were married , and 13.2% were divorced . nearly a third of the patients had a partner ( 29.4% ; table 1 ) . the psychiatrist assessed the current severity of the disorders on average between mildly ill to moderately ill and the patients in their subjective scale on average between borderline mentally ill and mildly ill . the subjective assessment of the disease severity by the patients differed ( was lower ) significantly from an objective evaluation ( mann whitney [ mw ] test , u=11,470 ; p<0.001 ) . still , the subjective and objective assessment of the severity of the disorder significantly correlated with each other ( spearman s r=0.45 , p<0.001 ) . the primary diagnosis was a schizophrenia spectrum disorder in all 197 patients ( 139 were diagnosed with schizophrenia , five with delusional disorder , 12 with acute and transient psychotic disorders , and 41 with schizoaffective disorder ) . the total score of the ismi significantly correlated with certain demographic and clinical factors ( table 2 ) . the age of the patient and the age at onset of the disorder did not significantly correlate with the ismi total score and most of the ismi domains , with the exception that the age positively correlated with the stereotype endorsement and negatively with the perceived discrimination ( table 2 ) . the number of hospitalizations significantly correlated with the ismi total score and all domains of the ismi scale except stigma resistance . in addition , the length of the treatment significantly correlated with the ismi total score and all its domains except stigma resistance . the objcgi - s significantly positively correlated with the ismi total score and with domains alienation , perceived discrimination , and social withdrawal , but not with stereotype endorsement or stigma resistance . the subjcgi - s correlated with ismi total score and all domains of ismi scale . the difference between objcgi - s and subjcgi - s negatively correlated with all domains of ismi scale and the ismi total score except perceived discrimination ( table 2 ) . the men and women differed according to their mean age ( the average age of men and women was 37.411.2 years and 42.411.3 years , respectively ; the independent t - test : t=3.106 ; df=195 ; p<0.01 ) . there were no significant differences in the mean level of self - stigma in the ismi total score or any ismi domain according to the sex of the patients ( table 3 ) . there were several statistically significant differences between the patients with and without jobs according to their age ( unemployed : 41.412.2 years versus employed : 37.69.5 years ; the independent t - test : t=2.203 ; df = 194 ; p<0.05 ) , the mean number of hospitalizations ( unemployed : 4.43.8 versus employed : 2.52.0 ; the mw test : u=3,554 ; p<0.001 ) , the mean duration of the treatment ( unemployed : 15.010.4 years versus employed : 9.48.5 years ; the mw test : u=2,850 ; p<0.001 ) , objcgi - s ( unemployed : 3.81.2 versus employed : 3.11.2 ; the mw test : u=2,881 ; p<0.001 ) , and subjcgi - s ( unemployed : 2.91.6 versus employed : 2.21.3 ; the mw test : u=3,208 ; p<0.01 ) . there was also a statistically significant difference according to the occupation in the self - stigma measured by the ismi total score and in the domains , stereotype endorsement and social withdrawal ( table 3 ) . the differences in self - stigma according to the marital status of the patients were calculated without widows because this category was not sufficiently large ( there were only six widowed individuals ) . there was a statistically significant difference between subgroups according to the marital status , in their mean age ( one - way analysis of variance [ anova ] : f=0.85 ; df = 190 ; p<0.001 ) . the bonferroni s multiple comparison test showed that the differences were between the singles and the married patients ( p<0.001 ) and the singles and the divorced patients ( p<0.001 ) , but not between the married and divorced patients . there were also statistically significant differences between the subgroups of the patients divided according to the marital status at the onset of the disorder ( one - way anova : f=26.07 ; df=189 ; p<0.001 ) . the patients did not significantly differ in both the mean scores of the objcgi - s ( the singles scored 3.61.3 , the married 3.21.2 , and the divorced 3.81.3 ; the kruskal wallis [ kw ] test = 3.574 ; non significant [ ns ] ) and the subjcgi - s ( the singles reached 2.61.5 , the married 2.61.4 , and the divorced 3.01.6 ; the kw test : = 1.186 ; ns ) according to the marital status . there was no statistically significant difference between the subgroups according to the marital status in the mean ismi scores ( table 3 ) . the patients , who had a partner , were older ( the individuals without a partner had the mean age of 37.911.4 years versus the patients with a partner had the mean age of 45.59.8 years ; the independent t - test : t=4.462 ; df=195 ; p<0.001 ) , their disorder started later in life ( without a partner , the average age at the beginning of the disorder was 25.08.7 years versus with a partner mean age was 30.210.8 years ; mw test ; u=2,754 ; p<0.001 ) . there were no significant differences according to the partnership in the average number of the hospitalizations , the duration of the treatment , and the objective or subjective measure of the severity of the disorder ( all mw tests : ns ) . the partnership had no effect on the self - stigma measured by the ismi scale ( table 3 ) . there were only 51 patients without any pension or rent in the sample ( table 1 ) . there was a significant difference in the age according to the pension status in the subgroups ( the mean age in the patients without the disability pension was 34.911.2 years , in the full disability pension 40.89.2 years , in the partial disability pension 38.49.0 , and in the senior rent 63.210.0 years ; one - way anova : f=25.77 ; df=196 ; p<0.001 ) . the bonferroni s multiple comparison test showed that the significant difference between the age was in comparison to the subgroups of the senior rent and all another group ( all p<0.001 ) and among the patients without the pension and the patients with the full disability pension ( p<0.01 ) . there were also statistically significant differences between the subgroups divided according to the pension status in the number of the hospitalizations . the patients without any pension were hospitalized 1.81.5 times , the patients with the full disability pension 4.83.0 times , the patients with the partial disability pension 3.32.5 times , and the patients with the senior rent 4.22.5 times ( the kw test : = 35.57 ; p<0.001 ) . the dunn s multiple comparison test showed that the differences were between the group of the patients without the pension and other groups with all types of a pension , or rent ( all : p<0.05 ) . in addition , there were statistically significant differences between the subgroups of patients divided according to their pension status in length of the treatment . the patients without any pension had been treated for 6.48.4 years , the full pensioned patients 14.88.4 years , the partially pensioned patients 12.97.8 years , and the senior rent patients 29.613.4 years ( the kw test : = 53.05 ; p<0.001 ) . the dunn s multiple comparison test showed that the differences were between the group without the pension and all other type pension groups ( all three comparisons : p<0.001 ) and the senior rent group and both pension groups ( both : p<0.05 ) . there were also significant differences in the objectively rated severity of the disorder between the groups divided according to the pension status . the patients without any pension were rated by their psychiatrists with the mean score of 2.81.2 , those with the full disability pension with the average rating of 4.01.2 , the individuals with the partial disability pension with the mean rating of 3.61.1 , and those with the senior rent with the mean rating of 3.71.4 ( the kw test : = 30.4 ; p<0.001 ) . the dunn s multiple comparison test showed that the differences were only in the group of the patients without any pension , the group with the full disability pension ( p<0.05 ) , and the group with the partial disability pension ( p<0.05 ) , but not in other intergroup comparisons . the subjcgi - s showed significant differences among the groups divided according to the pension , too . the patients without any pension rated their severity of the disorders as 1.91.2 , the patients with the full disability pension as 2.91.5 , the patients with the partial disability pension as 3.11.6 , and the patients with the senior rent as 2.41.4 ( the kw test : = 17.97 ; p<0.001 ) . according to the dunn s multiple comparison test , the differences were only in the group without any pension , the group with the full disability pension ( p<0.05 ) , and the group with a partial disability pension ( p<0.05 ) , but not in other intergroup comparisons . the difference between the ismi total score in groups divided according to the pension status reached statistical significance ( table 3 ) . according to the bonferroni s multiple comparison test , the difference was only among patients without any pension and the patients with the full disability pension ( p<0.05 ) , but not in other intergroup comparisons . from the domains of the ismi scale , stereotype endorsement showed the highest difference between the groups divided according to the pension status ( table 3 ) and the bonferroni s multiple comparison test showed that there were significant differences between the group without any pension and all other groups with the pension ( with the full disability pension : p<0.001 ; with the partial disability pension : p<0.001 ; with the senior rent : p<0.05 ) . furthermore , there were significant differences in the domain of the ismi scale , social withdrawal , between the groups divided according to the pension status ( table 3 ) . in the bonferroni s multiple comparison test , the significant difference was only among patients without the pension and the patients with the full disability pension , but not in other subgroup interactions . there were no significant differences between groups divided according to the education in the demographical data ( age , onset of the disorder , length of the treatment ) , or the severity of the disorder . the degree of the self - stigma measured by the ismi total score did not differ according to the level of education , but one domain of the ismi scale , stereotype endorsement , correlated with the degree of education ( table 3 ) . to identify the most significant variables connected to self - stigma , a multiple regression analysis was carried out , namely backward stepwise regression . as the dependent variable independent variables were the number of hospitalizations , the length of the treatment , objcgi - s , subjcgi - s , the difference between the objcgi - s and subjcgi - s , occupation , and pension . the objcgi had the tolerance score of 0.708 and variance inflation factor ( vif ) of 1.412 ; and the difference of objcgi - subjcgi had a tolerance score 0.823 and vif of 1.214 . the subjcgi was removed at the very beginning of the first step of regression . in the four steps of the backward stepwise regression , three factors remained , and all of them were statistically significant ( table 4 ) . the aim of this study was to investigate the factors that may be significantly associated with the internalized stigma in patients with schizophrenia and related disorders . the first hypothesis that the level of self - stigma is associated with the severity of the disorder was confirmed . the severity of the disorder , assessed both objectively and subjectively , significantly positively correlated with the total level of self - stigma and also with most domains of this scale . most research on the topic of self - stigma shows a significant association between the internalized stigma and the overall severity of the disorder in various psychiatric diagnoses including schizophrenia.3036 the results of this study confirm these findings in a group of patients with schizophrenia and related disorders . the second hypothesis that the level of self - stigma is linked to the duration or the early onset of the disorder was not confirmed , if one takes into account only the ismi total score . the rates of the self - stigma evaluated by the ismi total score also did not correlate with the age of the patient or the age at the onset of the disorder . this finding is consistent with the results of holubova et al35 in an outpatient population of patients with schizophrenia spectrum disorders . only the domain , perceived discrimination negatively correlated with the age at the onset of the disorder . this means that the patients with the early onset of the disorder feel that they are discriminated more often than the individuals with later onset . early onset of schizophrenia may impair the development of personality and the patient s social roles before he or she can learn how to manage these situations . earlier onset of schizophrenia is also connected with low self - esteem.14,37 the third hypothesis that the level of self - stigma is associated with the number of hospitalizations was confirmed . the number of hospitalizations positively correlated with the ismi total score and also with most of the domains except stigma resistance . in addition , our previous crossover study in various psychiatric diagnoses showed the same result.34 it is hard to conclude what was first , whether it was the self - stigma or more frequent hospitalizations . it is impossible to answer such a question from the viewpoint of a cross - sectional study . a longitudinal investigation is needed for an adequate answer . the fourth hypothesis that the level of the stigma the patients with a job had a lower mean of the ismi total score than the patients without a job . the ismi total score in connection with the employment status was supported by domains , stereotype endorsement and social withdrawal , which both were significantly higher in the subgroup without a job . the link between self - stigma and unemployment was also described in another study.18,38 nevertheless , in the regression analysis , the employment status was removed from the variables . the fifth hypothesis that the level of self - stigma the mean ismi total score did not differ among the subgroups with varying degrees of education . the only exception was the domain , stereotype endorsement , which was lower in the patients with the university level of education than in the less educated groups . our previous study showed that the degree of education might influence self - stigma.34 the sixth hypothesis that the degree of self - stigma is associated with the partnership status was not confirmed . neither the ismi total score nor any of the domains were affected by the partnership status . the result confirms the findings of our previous study.31 looking at the results of the regression analysis , there were three most significant independent factors that passed through , the number of hospitalizations , the objcgi , and the difference between the objcgi and subjcgi . the number of hospitalizations and the objcgi were linked to increased levels of self - stigma . the number of admissions probably reflects the severity of the long - term course of the disorder with severe psychotic episodes and separation from home environment , often with hopelessness and shame . the higher severity of the disorder is often associated with the disapproving behavior of others , and it might increase the self - stigma . the difference between the assessment of the severity of the disorder by the psychiatrist and by the higher the difference , the lower the self - stigma , and vice versa . this could reflect a controversial issue the patients with the evaluation closer to that of the therapist show a higher self - stigma . other variables , which can explain ismi values , could be personality traits , especially harm avoidance , and the level of hope and dissociation , as seen in patients with anxiety disorders.30 these variables were not involved in the current research but should be included in the studies in future . another variable , which could influence self - stigma , is the quality of life.39 our group explored this topic in the study by holubova et al.35 the study described a negative impact of self - stigma on the quality of life in outpatients suffering from schizophrenia spectrum disorders . many people who develope schizophrenia do not recognize the symptoms and consequences of their illness . poor insight has frequently been detected in patients with schizophrenia and has been long recognized as a powerful obstacle to the treatment adherence and a risk factor for poorer results of the treatment.40 in contrary , if the patients recognize the symptoms and consequences of their illness it can increase the level of depression , decrease self - esteem , and can increase a risk of suicide.41 self - stigma and difficulties in social cognition and metacognition can be the reasons for insight to convert into negative outcomes.42 in lysaker et als42 study of 65 patients suffering from schizophrenia spectrum disorders , the patients with fair insight and moderate depression described a higher level of self - stigma than patients with poor insight and minimal depression . on the other hand , the decreases in self - stigma in the rehabilitation program were correlated with increased self - esteem.43 the influence of stigma resistance on the functioning of patients with schizophrenia was studied by firmin et al.44 their meta - analysis showed a large negative association between stigma resistance and self - stigma , the substantial positive relation between stigma resistance and quality of life , hope , and recovery , and the medium and small relationship between stigma resistance and insight and symptoms of the disorder . nabors et al45 showed that fewer negative symptoms , greater metacognitive capacity , and higher self - esteem might be aspects that increase the stigma resistance . yanos et al46 elaborated a narrative review of articles focused on interventions targeting self - stigma . they identified six approaches that discussed interventions specifically targeting self - stigma in patients with a severe mental illness . they use psychoeducation and information to counteract myths about mental disease , cognitive techniques that offer opportunities to learn and practice skills to identify and combat self - stigmatizing thoughts and beliefs , an emphasis on narration and its potential to help patients make sense and create meaning out of past experiences , and some degree of behavioral decision making , and they offer tools and experiences designed to increase or elicit hope , empowerment , and motivation to act toward one s goals according to one s values . in particular , it was not possible to explain the causality of the described correlations , because of the cross - sectional study design . another limitation was the fact that the data were collected through scales filled out by the patients themselves . the preponderance of the subjective judgment of the assessment methods , which are dependent on the ability of introspection of the patient and his / her willingness to testify , has its limitations , particularly in patients with schizophrenia who may have a considerable degree of cognitive dysfunction . filling out the questionnaires also could have been modified by various levels of fatigue , current status , and motivation of the patients . the current study has several limitations . in particular , it was not possible to explain the causality of the described correlations , because of the cross - sectional study design . another limitation was the fact that the data were collected through scales filled out by the patients themselves . the preponderance of the subjective judgment of the assessment methods , which are dependent on the ability of introspection of the patient and his / her willingness to testify , has its limitations , particularly in patients with schizophrenia who may have a considerable degree of cognitive dysfunction . filling out the questionnaires also could have been modified by various levels of fatigue , current status , and motivation of the patients . stigma is a major social problem that significantly affects the quality of life of the patients with schizophrenia . understanding the issue of self - stigma in practice can play a major role in the treatment of the patients with schizophrenia spectrum disorders , not only for a psychiatrist but also for other mental health professionals . the management of self - stigma in the patient s care should be given due attention .

objectivethe aim of this study was to investigate the degree of self - stigma in schizophrenia and its association with clinical and demographic factors.patients and methodsa total of 197 outpatients ( 54.3% females ) diagnosed with schizophrenia spectrum disorders ( schizophrenia , schizoaffective disorder , delusional disorder ) according to international classification of diseases tenth edition participated in the study . the mean age of the patients was 40.1011.49 years . all individuals completed the internalized stigma of mental illness ( ismi ) scale and a demographic questionnaire . the disorder severity was assessed by both a psychiatrist ( the objective version of clinical global impression severity scale [ objcgi - s ] ) and the patients ( the subjective version of clinical global impression severity scale [ subjcgi - s ] ) . treatment with antipsychotics stabilized the patients.resultsthe overall level of self - stigma measured by the total score of the ismi was 63.3213.59 . the total score of the ismi positively correlated with the severity of the disorder measured by the objcgi - s and subjcgi - s . in addition , self - stigma positively correlated with the treatment duration and the number of psychiatric hospitalizations . the backward stepwise regression was applied to identify the most significant factors connected to self - stigma . the regression analysis identified the following regressors as the most relevant to self - stigma : the number of previous psychiatric hospitalizations , the severity of the disorder rated by a psychiatrist , and the difference between the objective rating and the subjective rating of the severity of the disorder.conclusionoutpatients with schizophrenia spectrum disorders , who have undergone a higher number of psychiatric hospitalizations , who dispose of a higher severity of the disorder and show a higher discrepancy between their rating of the severity and the psychiatric rating , showed a greater degree of self - stigma . the management of self - stigma in patients with schizophrenia should be implemented in the routine care .

Introduction Patients and methods Sample Assessment instruments Statistics and ethics Medication management Results Description of the sample Self-stigma and demographic and clinical factors Regression analysis Discussion Limitations of the study Conclusion

the stigma then operates at three levels , public ( ie , how the stigma manifests in a given society , culture , media , everyday habits , and attitudes ) , structural ( institutional , ie , at the level of functioning of organizations , agencies , and employers ) , and personal ( ie , how patients perceive themselves).9 significant terminological distinction exists between the public stigma ( when the general population supports the prejudices and discriminates against people with mental illness ) and the personal stigma , which also consists of the following three parts : 1 ) the perceived stigma ; 2 ) the experienced stigma ; and 3 ) the self - stigma , ie , how stigma itself accepted.10 the perceived or anticipated stigma is associated with patients beliefs about the attitudes of the general public to the members of stigmatized groups.11 the experienced stigma is related to the emotional experience of discrimination . two other studies have pointed to the relationship between the personal stigma and the social anxiety already during the first psychotic episode.21,22 patients with comorbid social anxiety showed an increased perception of the stigma.21 the aim of this study was to investigate the degree of self - stigma in patients with schizophrenia and its association with clinical and demographic factors . according to our hypotheses , the level of self - stigma is linked with , 1 ) the severity of the disorder , 2 ) the duration or early onset of the disorder , 3 ) the number of hospitalizations , 4 ) the employment status , 5 ) the level of education , and 6 ) the partner status . the inclusion criteria were as follows : age : 1865 years.diagnosis of schizophrenia spectrum disorders according to international classification of diseases tenth edition.23 diagnosis of schizophrenia spectrum disorders according to international classification of diseases tenth edition.23 patients suffering from severe physical disease , mental retardation , or an organic mental disorder were excluded . the severity of illness was determined by a thorough interview with the doctor , who evaluated the severity on the objective version of clinical global impression ( cgi ) severity scale ( objcgi - s ) . the following assessment tools were used : the internalized stigma of mental illness ( ismi ) scale : the scale consists of 29 items with a 4-point likert scale and evaluates five areas of self - stigma alienation , stereotype endorsement , perceived discrimination , social withdrawal , and stigma resistance.24,25 the czech version of the ismi scale was standardized by ociskov et al.26 internal consistency of the czech text of the scale was excellent ( cronbach s =0.91).cgi : the cgi is the assessment of the overall severity of the disorder.27 the objcgi - s is an overall evaluation of the patient s mental state by a physician . the scale ranges from 1 ( normal , with no signs of illness ) to 7 ( extremely severe symptoms of the disease).demographic questionnaire contained the basic information : sex , age , marital status , education , employment , disability , age at onset of the disorder , duration of attendance to the psychiatric services , number of hospitalizations , time since last hospitalization , number of visited psychiatrists , medication , and medication discontinuation in the past ( on the recommendation of a psychiatrist or willingly ) . the internalized stigma of mental illness ( ismi ) scale : the scale consists of 29 items with a 4-point likert scale and evaluates five areas of self - stigma alienation , stereotype endorsement , perceived discrimination , social withdrawal , and stigma resistance.24,25 the czech version of the ismi scale was standardized by ociskov et al.26 internal consistency of the czech text of the scale was excellent ( cronbach s =0.91 ) . demographic questionnaire contained the basic information : sex , age , marital status , education , employment , disability , age at onset of the disorder , duration of attendance to the psychiatric services , number of hospitalizations , time since last hospitalization , number of visited psychiatrists , medication , and medication discontinuation in the past ( on the recommendation of a psychiatrist or willingly ) . the inclusion criteria were as follows : age : 1865 years.diagnosis of schizophrenia spectrum disorders according to international classification of diseases tenth edition.23 diagnosis of schizophrenia spectrum disorders according to international classification of diseases tenth edition.23 patients suffering from severe physical disease , mental retardation , or an organic mental disorder were excluded . the severity of illness was determined by a thorough interview with the doctor , who evaluated the severity on the objective version of clinical global impression ( cgi ) severity scale ( objcgi - s ) . the following assessment tools were used : the internalized stigma of mental illness ( ismi ) scale : the scale consists of 29 items with a 4-point likert scale and evaluates five areas of self - stigma alienation , stereotype endorsement , perceived discrimination , social withdrawal , and stigma resistance.24,25 the czech version of the ismi scale was standardized by ociskov et al.26 internal consistency of the czech text of the scale was excellent ( cronbach s =0.91).cgi : the cgi is the assessment of the overall severity of the disorder.27 the objcgi - s is an overall evaluation of the patient s mental state by a physician . in the subjective version of cgi severity scale ( subjcgi - s ) , the patient himself / herself evaluates his / her overall condition . the scale ranges from 1 ( normal , with no signs of illness ) to 7 ( extremely severe symptoms of the disease).demographic questionnaire contained the basic information : sex , age , marital status , education , employment , disability , age at onset of the disorder , duration of attendance to the psychiatric services , number of hospitalizations , time since last hospitalization , number of visited psychiatrists , medication , and medication discontinuation in the past ( on the recommendation of a psychiatrist or willingly ) . the internalized stigma of mental illness ( ismi ) scale : the scale consists of 29 items with a 4-point likert scale and evaluates five areas of self - stigma alienation , stereotype endorsement , perceived discrimination , social withdrawal , and stigma resistance.24,25 the czech version of the ismi scale was standardized by ociskov et al.26 internal consistency of the czech text of the scale was excellent ( cronbach s =0.91 ) . demographic questionnaire contained the basic information : sex , age , marital status , education , employment , disability , age at onset of the disorder , duration of attendance to the psychiatric services , number of hospitalizations , time since last hospitalization , number of visited psychiatrists , medication , and medication discontinuation in the past ( on the recommendation of a psychiatrist or willingly ) . the primary diagnosis was a schizophrenia spectrum disorder in all 197 patients ( 139 were diagnosed with schizophrenia , five with delusional disorder , 12 with acute and transient psychotic disorders , and 41 with schizoaffective disorder ) . the age of the patient and the age at onset of the disorder did not significantly correlate with the ismi total score and most of the ismi domains , with the exception that the age positively correlated with the stereotype endorsement and negatively with the perceived discrimination ( table 2 ) . in addition , the length of the treatment significantly correlated with the ismi total score and all its domains except stigma resistance . the objcgi - s significantly positively correlated with the ismi total score and with domains alienation , perceived discrimination , and social withdrawal , but not with stereotype endorsement or stigma resistance . the difference between objcgi - s and subjcgi - s negatively correlated with all domains of ismi scale and the ismi total score except perceived discrimination ( table 2 ) . there were no significant differences in the mean level of self - stigma in the ismi total score or any ismi domain according to the sex of the patients ( table 3 ) . there were several statistically significant differences between the patients with and without jobs according to their age ( unemployed : 41.412.2 years versus employed : 37.69.5 years ; the independent t - test : t=2.203 ; df = 194 ; p<0.05 ) , the mean number of hospitalizations ( unemployed : 4.43.8 versus employed : 2.52.0 ; the mw test : u=3,554 ; p<0.001 ) , the mean duration of the treatment ( unemployed : 15.010.4 years versus employed : 9.48.5 years ; the mw test : u=2,850 ; p<0.001 ) , objcgi - s ( unemployed : 3.81.2 versus employed : 3.11.2 ; the mw test : u=2,881 ; p<0.001 ) , and subjcgi - s ( unemployed : 2.91.6 versus employed : 2.21.3 ; the mw test : u=3,208 ; p<0.01 ) . there was also a statistically significant difference according to the occupation in the self - stigma measured by the ismi total score and in the domains , stereotype endorsement and social withdrawal ( table 3 ) . the patients did not significantly differ in both the mean scores of the objcgi - s ( the singles scored 3.61.3 , the married 3.21.2 , and the divorced 3.81.3 ; the kruskal wallis [ kw ] test = 3.574 ; non significant [ ns ] ) and the subjcgi - s ( the singles reached 2.61.5 , the married 2.61.4 , and the divorced 3.01.6 ; the kw test : = 1.186 ; ns ) according to the marital status . the patients , who had a partner , were older ( the individuals without a partner had the mean age of 37.911.4 years versus the patients with a partner had the mean age of 45.59.8 years ; the independent t - test : t=4.462 ; df=195 ; p<0.001 ) , their disorder started later in life ( without a partner , the average age at the beginning of the disorder was 25.08.7 years versus with a partner mean age was 30.210.8 years ; mw test ; u=2,754 ; p<0.001 ) . there were no significant differences according to the partnership in the average number of the hospitalizations , the duration of the treatment , and the objective or subjective measure of the severity of the disorder ( all mw tests : ns ) . the patients without any pension were rated by their psychiatrists with the mean score of 2.81.2 , those with the full disability pension with the average rating of 4.01.2 , the individuals with the partial disability pension with the mean rating of 3.61.1 , and those with the senior rent with the mean rating of 3.71.4 ( the kw test : = 30.4 ; p<0.001 ) . the patients without any pension rated their severity of the disorders as 1.91.2 , the patients with the full disability pension as 2.91.5 , the patients with the partial disability pension as 3.11.6 , and the patients with the senior rent as 2.41.4 ( the kw test : = 17.97 ; p<0.001 ) . the degree of the self - stigma measured by the ismi total score did not differ according to the level of education , but one domain of the ismi scale , stereotype endorsement , correlated with the degree of education ( table 3 ) . to identify the most significant variables connected to self - stigma , a multiple regression analysis was carried out , namely backward stepwise regression . independent variables were the number of hospitalizations , the length of the treatment , objcgi - s , subjcgi - s , the difference between the objcgi - s and subjcgi - s , occupation , and pension . the primary diagnosis was a schizophrenia spectrum disorder in all 197 patients ( 139 were diagnosed with schizophrenia , five with delusional disorder , 12 with acute and transient psychotic disorders , and 41 with schizoaffective disorder ) . the age of the patient and the age at onset of the disorder did not significantly correlate with the ismi total score and most of the ismi domains , with the exception that the age positively correlated with the stereotype endorsement and negatively with the perceived discrimination ( table 2 ) . in addition , the length of the treatment significantly correlated with the ismi total score and all its domains except stigma resistance . the objcgi - s significantly positively correlated with the ismi total score and with domains alienation , perceived discrimination , and social withdrawal , but not with stereotype endorsement or stigma resistance . the difference between objcgi - s and subjcgi - s negatively correlated with all domains of ismi scale and the ismi total score except perceived discrimination ( table 2 ) . there were no significant differences in the mean level of self - stigma in the ismi total score or any ismi domain according to the sex of the patients ( table 3 ) . there were several statistically significant differences between the patients with and without jobs according to their age ( unemployed : 41.412.2 years versus employed : 37.69.5 years ; the independent t - test : t=2.203 ; df = 194 ; p<0.05 ) , the mean number of hospitalizations ( unemployed : 4.43.8 versus employed : 2.52.0 ; the mw test : u=3,554 ; p<0.001 ) , the mean duration of the treatment ( unemployed : 15.010.4 years versus employed : 9.48.5 years ; the mw test : u=2,850 ; p<0.001 ) , objcgi - s ( unemployed : 3.81.2 versus employed : 3.11.2 ; the mw test : u=2,881 ; p<0.001 ) , and subjcgi - s ( unemployed : 2.91.6 versus employed : 2.21.3 ; the mw test : u=3,208 ; p<0.01 ) . there was also a statistically significant difference according to the occupation in the self - stigma measured by the ismi total score and in the domains , stereotype endorsement and social withdrawal ( table 3 ) . the patients did not significantly differ in both the mean scores of the objcgi - s ( the singles scored 3.61.3 , the married 3.21.2 , and the divorced 3.81.3 ; the kruskal wallis [ kw ] test = 3.574 ; non significant [ ns ] ) and the subjcgi - s ( the singles reached 2.61.5 , the married 2.61.4 , and the divorced 3.01.6 ; the kw test : = 1.186 ; ns ) according to the marital status . the patients , who had a partner , were older ( the individuals without a partner had the mean age of 37.911.4 years versus the patients with a partner had the mean age of 45.59.8 years ; the independent t - test : t=4.462 ; df=195 ; p<0.001 ) , their disorder started later in life ( without a partner , the average age at the beginning of the disorder was 25.08.7 years versus with a partner mean age was 30.210.8 years ; mw test ; u=2,754 ; p<0.001 ) . there were no significant differences according to the partnership in the average number of the hospitalizations , the duration of the treatment , and the objective or subjective measure of the severity of the disorder ( all mw tests : ns ) . the bonferroni s multiple comparison test showed that the significant difference between the age was in comparison to the subgroups of the senior rent and all another group ( all p<0.001 ) and among the patients without the pension and the patients with the full disability pension ( p<0.01 ) . the patients without any pension were rated by their psychiatrists with the mean score of 2.81.2 , those with the full disability pension with the average rating of 4.01.2 , the individuals with the partial disability pension with the mean rating of 3.61.1 , and those with the senior rent with the mean rating of 3.71.4 ( the kw test : = 30.4 ; p<0.001 ) . the patients without any pension rated their severity of the disorders as 1.91.2 , the patients with the full disability pension as 2.91.5 , the patients with the partial disability pension as 3.11.6 , and the patients with the senior rent as 2.41.4 ( the kw test : = 17.97 ; p<0.001 ) . the degree of the self - stigma measured by the ismi total score did not differ according to the level of education , but one domain of the ismi scale , stereotype endorsement , correlated with the degree of education ( table 3 ) . to identify the most significant variables connected to self - stigma , a multiple regression analysis was carried out , namely backward stepwise regression . as the dependent variable independent variables were the number of hospitalizations , the length of the treatment , objcgi - s , subjcgi - s , the difference between the objcgi - s and subjcgi - s , occupation , and pension . the aim of this study was to investigate the factors that may be significantly associated with the internalized stigma in patients with schizophrenia and related disorders . the first hypothesis that the level of self - stigma is associated with the severity of the disorder was confirmed . the severity of the disorder , assessed both objectively and subjectively , significantly positively correlated with the total level of self - stigma and also with most domains of this scale . most research on the topic of self - stigma shows a significant association between the internalized stigma and the overall severity of the disorder in various psychiatric diagnoses including schizophrenia.3036 the results of this study confirm these findings in a group of patients with schizophrenia and related disorders . the rates of the self - stigma evaluated by the ismi total score also did not correlate with the age of the patient or the age at the onset of the disorder . the result confirms the findings of our previous study.31 looking at the results of the regression analysis , there were three most significant independent factors that passed through , the number of hospitalizations , the objcgi , and the difference between the objcgi and subjcgi . the difference between the assessment of the severity of the disorder by the psychiatrist and by the higher the difference , the lower the self - stigma , and vice versa . poor insight has frequently been detected in patients with schizophrenia and has been long recognized as a powerful obstacle to the treatment adherence and a risk factor for poorer results of the treatment.40 in contrary , if the patients recognize the symptoms and consequences of their illness it can increase the level of depression , decrease self - esteem , and can increase a risk of suicide.41 self - stigma and difficulties in social cognition and metacognition can be the reasons for insight to convert into negative outcomes.42 in lysaker et als42 study of 65 patients suffering from schizophrenia spectrum disorders , the patients with fair insight and moderate depression described a higher level of self - stigma than patients with poor insight and minimal depression . on the other hand , the decreases in self - stigma in the rehabilitation program were correlated with increased self - esteem.43 the influence of stigma resistance on the functioning of patients with schizophrenia was studied by firmin et al.44 their meta - analysis showed a large negative association between stigma resistance and self - stigma , the substantial positive relation between stigma resistance and quality of life , hope , and recovery , and the medium and small relationship between stigma resistance and insight and symptoms of the disorder . understanding the issue of self - stigma in practice can play a major role in the treatment of the patients with schizophrenia spectrum disorders , not only for a psychiatrist but also for other mental health professionals .

standard techniques employed for the production of drug nanocrystals are high pressure homogenization and wet bead milling . however , they have drawbacks , such as long processing times and the necessity of employing a micronized drug as the starting material . next - generation technologies involve combinative particle size reduction methods to improve the particle size reduction effectiveness of the standard techniques . there are basically two approaches to produce drug nanocrystals , with a third one ( the combinative approach ) being a combination of the first two techniques . this approach is called bottom - up , as the size of the particles is increased . this approach is called top - down , as the size of already existing particles is decreased . the third approach involves combinations of bottom - up and/or top - down steps to improve the particle size reduction effectiveness of the single - unit processes . the first step is usually a bottom - up process employed as a drug pretreatment to obtain a brittle , friable starting material for a subsequent comminution step . combinations of two different top - down steps ( such as bead milling followed by high pressure homogenization ) have also been developed [ 5 , 6 ] . this is a classical precipitation process , also known as via humida paratum ( latin for produced in a wet process ) . the challenges of this technique are to minimize the crystal growth into the nanometer range ( controlled crystallization ) and to control the solid state of the crystals , that is , to produce them in crystalline or amorphous form . technology is the first process involving a bottom - up step to produce drug nanoparticles ; this technology was developed by sucker and nowadays belongs to novartis [ 7 , 8 ] . the drug has to be soluble in at least one solvent and the process involves organic solvents that need to be removed . there are apparently no products on the market that use this technology , perhaps due to the difficulty of avoiding the crystal growth . this technology yields amorphous drug nanoparticles , which have the advantage of higher saturation solubility and a faster dissolution rate compared to the crystalline form . however , drawbacks include undesired compound recrystallization to the crystalline state with a subsequent decrease in bioavailability . additionally , drug releases of up to 80% after 10 min of dissolution testing were reported . the special features of this bottom - up technology are the production of crystalline drug nanoparticles and the ability for large - scale production [ 12 , 13 ] . freeze - drying as well as spray - drying are precipitation processes widely employed in the pharmaceutical industry to obtain dry intermediates or final drug powders . both bottom - up technologies can also be employed to modify drug materials to make them more suitable ( i.e. , friable and brittle ) for a subsequent comminution process . the pearl or bead milling technique was developed by liversidge et al . and is owned by alkermes plc . this technology comprises a milling chamber with an agitator , which is loaded with the milling material , a dispersium medium ( usually water ) , surfactants as a stabilization system , and the drug to be nanosized . the milling material is usually small beads of stainless steel , glass , ceramic ( e.g. , yttrium stabilized zirconium dioxide ) , or highly cross - linked polystyrene resin , the last two being preferred due to reduced contamination to the product . the milling pearls have different sizes ( e.g. , 0.1 , 0.2 , or 0.5 mm ) . the collision frequency during the comminution process is increased with the reduction in size of the milling beads . other factors affecting the comminution effectiveness are the hardness of the drug , the surfactant and its concentration , temperature , the viscosity of the dispersion medium , and so forth . the forces producing the particle size reduction include shear forces , and particle collision produced by the movement of the milling material inside the chamber . the nanocrystal technology is regarded as a successful technology : the first product containing drug nanocrystals ( rapamune by wyeth pharmaceuticals in 2000 ) came to the market only 10 years after the development of the technology [ 16 , 17 ] . the hph technique is a high - energy disintegration process that employs high pressure to reduce the particle size of drug particles in liquid media with surfactants for stabilization purposes . the hph involves the principles of piston - gap homogenization and jet - stream homogenization ( microfluidization ) [ 1 , 18 ] . when the piston - gap hph technique is employed , the particle size reduction is achieved by cavitation , shear forces and particle collision . the suspension is forced through a small gap , which reduces the diameter from 3 cm to approximately 25 m . because of the tremendous diameter change , according to bernoulli 's law , the dynamic pressure raises and the static pressure falls . as a result of the latter , the liquid starts boiling in the homogenization gap , as the static pressure is lower than the vapor pressure of the liquid . this phenomenon results in the formation of gas bubbles that implode after leaving the gap ( cavitation ) . the shear forces and the particle collisions are developed during the process due to the high pressures involved ( usually up to 1500 bar ) . the equipment employed for piston - gap homogenization is produced , for example , by apv , gaulin and avestin [ 18 , 20 ] . employing piston - gap homogenizers , mller and coworkers developed the dissocubes technology ( now belonging to skyepharma plc ) and the nanopure technology ( now belonging to abbott gmbh & co. kg ) [ 18 , 21 ] . the dissocubes technology produces drug nanoparticles in an aqueous dispersion at room temperature . on the contrary , the nanopure process employs nonaqueous media ( e.g. , oils or liquid polyethylene glycols ) or water - reduced media ( e.g. , employing glycerol / water mixtures ) . one interesting feature of the nanopure technology is that the oil dispersions can be employed to subsequently fill capsules as the final dosage form . then , the suspension is put into the homogenizer device for processing . during the homogenization process , two jet streams are forced to circulate at high pressure ( up to 1700 bar ) through two different interaction chambers ( y and z ) . this produces particle collision and shear forces as well as cavitation , which results in the disintegration of the drug particles . employs a microfluidizer homogenizer for its idd - p ( insoluble drug delivery particles ) technology , which produces submicronic nanosuspensions . in summary , the known limitations of the standard processes ( wbm , hph ) for the production of drug nanocrystals are the necessity of a micronized drug as the starting material and the long runtimes for the top - down equipment . the combinative particle size reduction techniques have been developed to overcome these drawbacks and to improve the particle size reduction effectiveness of the standard processes . nowadays , five combinative methods are known : nanoedge ( microprecipitation followed by a high - energy step such as hph ) , h 69 ( microprecipitation immediately followed by hph , also called cavi - precipitation ) , h 42 ( spray - drying followed by hph ) , h 96 ( freeze - drying followed by hph ) , and the ct combinative technology ( media milling followed by hph ) . the applications of the combinative technologies for a variety of drugs are shown in table 1 . the nanoedge technology from baxter is the first combinative particle size reduction method developed for the production of drug nanosuspensions . this production technique combines a microprecipitation step ( a solvent - antisolvent technique ) followed by a high - energy process . the drug is first dissolved in a suitable solvent , usually a water - miscible organic solvent . the drug solution is then mixed with a second aqueous liquid in which the drug is less soluble . the aqueous liquid can contain surfactants for stabilization , and it is added to the drug solution in a controlled manner using , for example , an infuser device . the microprecipitation is a pretreatment and the drug particles can be obtained in amorphous or semicrystalline form . then , the drug particles are reduced in size and transformed to the more stable crystalline state after employing a high - energy annealing step , such as high pressure homogenization [ 24 , 25 ] . the objective of the annealing step is to improve the thermodynamic stability of the nanosuspensions by preventing the crystal growth of the precipitated particles to the micrometer range . the change to the more stable form is produced by the high - energy input of the top - down step , which promotes the formation of low - energy , more stable structures , either by enhancing the crystallinity of the particles ( reordering of the lattice structure ) or by rearrangement of the stabilizing system at the surface of the drug nanocrystals . the fast microprecipitation improves the particle size reduction effectiveness of the top - down step due to induced friable material , drug crystal defects , and dendritic morphology . the top - down process is usually high pressure homogenization but other techniques such as sonication or microfluidization can also be employed [ 25 , 26 ] . the residues of organic solvents in the nanosuspension are a major problem associated with this combinative technology , which becomes more complicated in the case of large - scale production ( i.e. , larger amounts of solvent to be removed from the final drug product ) . another drawback is that this technology achieves particle sizes markedly bigger than with standard technologies . as the baxter development is mainly focused on injectables , the solvent - removal processes as well as the production lines need to be performed under sterilized conditions . this technology has no marketed products to date , as i.v .- injectable products are more complicated to develop as oral products . the nanoedge platform has been employed to formulate poorly soluble anticancer drugs such as paclitaxel to improve the plasma concentration and thereby the pharmacologic efficacy . nanosuspensions formulated with 15% ( w / v ) drug presented particle sizes of around 1000 nm . the drug nanosuspensions produced by this technology could achieve higher drug loads and a more flexible administration , such as oral and injectable routes . the therapy efficacy for an anticancer compound described as the nanosuspension formulation showed better tolerability in rats than the drug formulated with standard techniques , which enable the implementation of higher doses . injection and after oral administration . with another poorly soluble , poorly bioavailable anticancer drug described as b , a linear relationship was found between the decreasing in nanosuspension particle size ( particle sizes of 4001000 nm , administered at 300 mg / kg ) and the increase of oral bioavailability measured from the plasma of rats . in this case , the formulation as a nanosuspension employing the nanoedge process resulted in an up to 30-fold bioavailability increase in the rat models compared to the control formulation . the nanoedge combinative technology was also employed to reformulate a paclitaxel product ( taxol , bristol myers squibb company ) . the objective was to eliminate chremophor el ( polyethoxylated castor oil ) as an excipient to avoid its incompatibilities and toxicity . functionalized polyethylene glycols were employed as surfactants to minimize the opsonization of the drug nanocrystals , which had a mean particle size of 200 nm [ 27 , 28 ] . it was dissolved in n - methyl-2-pyrrolidone ( nmp ) and then precipitated by adding an aqueous diluent with surfactants . sonication for one minute at 10000 hertz ( hz ) and 400 watts ( w ) employed as the annealing step resulted in drug nanoparticles with a mean particle size of 177 nm . in another study , an itraconazol nanosuspension for i.v . its resulting bioavailability was compared to the results of a marketed itraconazol solution ( sporanox iv , janssen pharmaceutica ) , which is formulated with cyclodextrin technology and presents some degree of toxicity due to the high cyclodextrin load . in this case , hph was used as the annealing step , achieving a final mean particle size of 581 nm . the nanosuspension formulation led to better bioavailability and tolerability , enabling the use of higher drug doses . the subject survival was superior with the nanoparticulated itraconazol due to higher drug concentrations in the target organs compared to the standard solution formulation [ 28 , 29 ] . carbamazepin and prednisolone presented a needle - shaped form and a mean particle size of approximately 2 m after precipitation . canada ) was employed for the high - energy step to process the three drug macrosuspensions , which resulted in final mean particle sizes of 400 nm for carbamazepin , 640 nm for prednisolone and 930 nm for nabumetone . nanoedge - like processes involving a microprecipitation step followed by a high - energy process ( hph or sonication ) , although they are not addressed as nanoedge . the antitumor alkaloid 10-hydroxycamptothecin ( 10-hcpt ) was processed by employing a microprecipitation - homogenization process . the drug was dissolved in dimethyl sulfoxide ( dmso ) , and then it was precipitated by adding an aqueous surfactant solution . the drug suspension was then homogenized employing an ats ah110d piston - gap homogenizer ( ats engineer inc . , china ) . the drug particles were obtained in the amorphous state and the best final mean particle size of 131 nm was obtained by homogenizing the drug suspension for 20 cycles at 1000 bar . the drug was dissolved in 2-propanol and then precipitated by adding an aqueous solution containing surfactants for stabilization purposes . this macrosuspension was then processed by hph employing a piston - gap homogenizer ( gea niro soavi inc . this drug was dissolved in dimethylformamide ( dmf ) and then precipitated by adding the drug solution to an aqueous solution containing surfactants . the drug particles were then further processed either by ultrasonication ( 20 min 300 w with a fs-5 sonicator , frontline ltd . , india ) or by hph ( 15 cycles at 500 bar with an ats ah110d homogenizer ) . the results showed a final mean particle size of 259 nm with the sonication method and 212 nm with the hph technique . however , the amount of larger crystals was considerably smaller when hph was used as the reduction step . additionally , low pressure could be maintained ( 500 bar ) , as higher pressures ( or a higher number of homogenization cycles ) did not improve the particle size reduction . in the case of nitrendipine , the drug was first dissolved in a 1 : 1 peg 200 : acetone mixture , and then it was precipitated by adding a polyvinyl alcohol aqueous solution . the drug particles were subsequently processed by employing ultrasonication ( ningbo scientz biotechnology co. ltd . , the best mean particle size result of 209 nm was obtained by employing 20000 hz and 400 w as sonication conditions for 15 min . all - trans retinoic acid is a poorly soluble , heat - sensitive , anticancer drug . a microprecipitation - sonication process under controlled temperature was chosen to produce nanoparticles of the drug to eventually improve its dissolution rate - dependent bioavailability . the drug was first dissolved in acetone , and then it was mixed with demineralized water to produce the precipitation . the drug particles were immediately sonicated employing an eq-250e medical ultrasonicator ( kunshan ultrasonic instrument corporation , china ) for 30 min . a final mean particle size of 155 nm was obtained . however , this result was only slightly improved compared to the precipitation process without sonication ( 176 nm mean particle size ) . this drug was dissolved in ethanol and then precipitated by adding an aqueous surfactant solution . the process factors that were investigated included solvent : antisolvent flow rate and drug concentration . a mean particle size of 80 nm could be achieved under optimized conditions . the precipitation process modified the high crystallinity of the starting material leading to amorphous drug nanoparticles [ 35 , 36 ] . the drug was dissolved in acetone and then added to an aqueous solution containing surfactants . the precipitated drug particles were further sonicated for 60 min employing a sonic dismembrator model 550 ( fisher scientific international inc . , usa ) . different surfactants were screened for the precipitation step and the best stabilization results were achieved with hpmc k3 , which led to a mean particle size of 702 nm after sonication . the h 69 process was developed by mller and mschwitzer , and it belongs to the smartcrystal technology family . it combines a microprecipitation step involving organic solvents , followed by high pressure homogenization for particle size reduction . the difference is that with the h 69 technology , the cavitation takes place at the same time as the particle formation ( cavi - precipitation ) or at most two seconds thereafter . to employ this combinative technique , the drug is dissolved in a suitable solvent ( liquid 1 ) , which is then mixed with an aqueous nonsolvent ( liquid 2 ) . the nonsolvent is added to the solvent in a controlled manner using , for example , an infuser device such as the perfusor from b. braun melsungen , germany . to do this , the liquid flows come in contact , which results in the precipitation of the drug . the particle formation takes place in the high - energy zone of a homogenizer , where the just - formed drug particles are immediately treated with cavitation , particle collision , and shear forces . the microfluidizer or the emulsiflex c5 from avestin are suitable homogenizers to process the liquid flows directly in the high - energy zone of the device . as with all precipitation methods , the challenge is to control the particle crystallization by avoiding crystal growth . technique , where the drug particles formed are immediately treated with a high - energy annealing process . the top - down step not only reduces the particle size but also stabilizes the drug nanocrystals with the energy application . another advantage of the annealing step is that it promotes the more stable crystalline form . a drawback of this combinative process is that the resulting nanosuspensions contain organic solvent residues that need to be removed before further processing , just as with the nanoedge technology . the drug was dissolved in ethanol , mixed with demineralized water as a nonsolvent for precipitation , and then directly homogenized at high pressure . a mean particle size of 113 nm could be achieved after one minute of homogenization . these results improved to 27 nm after 5 min and to 22 nm after 6 min . afterwards , the drug nanocrystals dissolved due to the increased dissolution pressure at these small particle sizes . the drugs hydrocortisone acetate ( hca ) and omeprazol were processed employing the h 69 process , achieving mean particle sizes of 787 nm and 921 nm , respectively , after 20 cycles of homogenization at 1500 bar . ibuprofen and resveratrol are other examples of drugs processed with the h 69 technology . in the case of ibuprofen , best results were achieved when the drug was dissolved in tetrahydrofuran and then precipitated by adding demineralized water with surfactants . the drug crystals were immediately homogenized employing a micron lab 40 device ( apv gaulin , germany ) for 10 cycles at 1500 bar or the emulsiflex c5 homogenizer for 10 cycles at 1200 bar . the latter equipment produced the smallest ibuprofen nanocrystals , which presented a mean particle size of 170 nm . the emulsiflex c5 has the advantage that the precipitated drug particles can be directly homogenized at the high - energy zone of the device . thus , it is possible to immediately stabilize the drug nanocrystals to ensure small particle sizes . in the case of resveratrol , a final mean particle size of 150 nm could be achieved in the case of resveratrol . the reduction of the time between precipitation and the top - down step and the proper selection of the organic solvent to dissolve the drug prior to the precipitation step were identified as critical factors in obtaining the smallest drug nanocrystals . the h 42 process this combinative technology combines spray - drying ( sd ) as a precipitation and pretreatment step , followed by hph for particle size reduction . the organic solvent is eliminated during the bottom - up step , which differentiates this technology from the nanoedge and h 69 processes.in the first unit operation ( sd ) , the poorly soluble compound is dissolved in organic solvents . surfactants such as poloxamer or sugars such as mannitol can be added to the drug solution to improve the results of the drying step . the ideal organic solvent should possess good dissolving properties as well as suitable both boiling point and vapor pressure to ensure an efficient process and spray - dried powders free of solvent residues . the objective of the drug modification by means of sd is to produce suitable , more breakable drug powders for the subsequent comminution process . the obtained spray - dried drug powders are then dispersed in aqueous media containing surfactants for stabilization purposes . the suspensions are further processed to nanosuspensions by employing the hph technique , using homogenization equipment such as the micron lab 40 . the h 42 combinative technology has advantages such as relatively short processing times during sd , solvent - free dry intermediates , and small drug nanocrystals after a reduced number of hph cycles . its drawback is the employment of high temperatures during sd , which could make this technology unsuitable to process thermolabile compounds . in the first experiments the modified powders were then homogenized for 20 cycles at 1500 bar , reaching a mean particle size of 636 nm ( original value without modification : 1172 nm ) . the spray - dried ibuprofen powders showed almost no crystallinity change compared to the unprocessed material , which was confirmed by employing the differential scanning calorimetry ( dsc ) technique . the melting points and the normalized melting enthalpies of unmodified and spray - dried modified ibuprofen were compared and showed almost no difference . in this case , the improved reduction effectiveness was not linked to a change in the solid state behavior of the drug , but to the enhanced friability of the starting material . this model compound was dissolved in a 1 : 19 dmso / methanol mixture and then spray - dried . the drug powders were homogenized at 1500 bar for 20 cycles using peg 300 as a dispersion medium with the purpose of employing the nanosuspension to directly fill capsules . the drug was dissolved in ethanol , with different amounts of poloxamer 188 being added to the drug solution . the spray - dried drug processed with a 9 : 1 drug / surfactant ratio brought the best particle size results , which were 281 nm after 20 homogenization cycles at 1500 bar . the micronized , unmodified hca led to a final mean particle size of 551 nm under the same process conditions . the improved drug structure of the best spray - dried powder was analyzed employing the scanning electron microscopy ( sem ) technique , which showed spherical drug particles . further , the solid state behavior of the spray - dried powders was analyzed by using the powder x - ray diffraction ( pxrd ) technique . these results showed that the spray - dried powders stayed as crystalline as the unmodified drug material . small amounts of the surfactant positively impacted the characteristics of the spray - dried powders , such as flowability and millability . on the contrary , high surfactant amounts ( i.e. , 1 : 1 drug / surfactant ratio ) negatively impacted the powders ' characteristics and the subsequent particle size reduction effectiveness . the processing times could also be drastically reduced . when the best modified material was employed , only one cycle at 1500 bar was necessary to achieve smaller particle sizes than by homogenizing micronized drug material for 20 cycles . the influence of both surfactant and drug concentration during the bottom - up step was tested in this study . the effect of these parameters on the solid state behavior and morphology of the drug , as well as on the particle size reduction effectiveness of the top - down step , was analyzed . the degree of crystallinity ( dc ) of the drug powders was established employing the dsc technique . it was discovered that the spray - dried glibenclamide powders showed , in general , a reduced crystallinity ( dcs of between 20% and 30% ) compared to the unmodified drug ( 100% dc ) . however , the drug solutions processed with medium and high drug concentrations ( both sprayed with a 0.2% docusate sodium salt ethanolic solution ) produced spray - dried powders with very low dcs : 8.1% and 8.3% . both powders led after the homogenization step to nanosuspensions presenting mean particle sizes of about 236 nm , which were the best of all the results . additionally , the sem analysis of these glibenclamide samples revealed the formation of spherical drug particles . both solid state modification leading to an amorphous drug and the morphology change due to the precipitation process positively impacted the particle size reduction effectiveness of the top down - step . with the antioxidant compound resveratrol , experiments were also performed with the h 42 process employing a design of experiments . resveratrol was dissolved in ethanol containing different amounts of the surfactant sodium cholate , and then the drug solutions were spray - dried for further homogenization . the best mean particle size obtained was 200 nm , which was an improvement compared to the 428 nm mean particle size obtained with unmodified resveratrol . additionally , the amount of larger crystals was drastically reduced by employing the spray - dried modified drug instead of micronized material : from 2.2 m ( d90% ) to 0.736 m ( d90% ) . finally , the number of hph cycles at 1500 bar necessary to achieve a proper nanosuspension could be reduced from 20 cycles with the standard method to only one cycle with the modified drug . however , it was difficult to establish a link between the dc and the smallest drug particle sizes with resveratrol as the model drug . the h 96 combinative technology was developed by mschwitzer and lemke and belongs to the smartcrystal technology family ( abbott / soliqs , germany ) . this process involves freeze - drying ( fd ) as a bottom - up and pretreatment step , followed by hph for particle size reduction . the bottom - up step eliminates the organic solvent content , just as with the h 42 technology . the drug solution is then frozen ( e.g. , with instant freezing or snap - freezing ) with liquid nitrogen and further freeze - dried . the aim of the drug pretreatment is to modify the starting material to improve the particle size reduction effectiveness of the hph [ 5 , 41 ] . the solvents need to be carefully selected to optimize the process and the characteristics of the freeze - dried powders . the critical solvent characteristics that determine the process performance are , among others , the freezing point , vapor pressure , and toxicity . for fd purposes , it is important to employ organic solvents presenting relatively high freezing points . in this way the selected solvent should also possess a high vapor pressure to ensure a complete elimination during the primary drying step . the complete removal of residues of organic solvents is necessary to ensure patient safety and product quality [ 42 , 43 ] . mixtures of organic solvents can also be implemented to improve the performance of the lyophilization process . for example , experiments using glibenclamide as a model compound had employed mixtures of dimethyl sulfoxide ( dmso ) and tert - butyl alcohol ( tba ) for fd . dmso dissolves the model compound but has a low vapor pressure , which resulted in low - quality freeze - dried powders ( i.e. , wet and sticky due to incomplete elimination of the solvent ) . tba has both a high freezing point and vapor pressure , which makes it an ideal solvent for lyophilization purposes . dmso contributed to the process with the necessary dissolving force for glibenclamide , and tba was added to the solution to improve the characteristics of the freeze - dried cakes [ 43 , 44 ] . the h 96 technology is especially suitable to process thermolabile or expensive drugs due to the low temperatures and the high yields of the fd . additionally , as the lyophilization step eliminates the organic solvent content , the subsequently produced nanosuspensions are ready to be further processed or used . its drawback is the extension of the lyophilization step . during first experiments employing the h 96 technology , amphotericin b was dissolved in dmso , snap - frozen with liquid nitrogen , and then lyophilized . the freeze - dried drug powder was processed to a nanosuspension employing a micron lab 40 homogenizer for five cycles at 1500 bar producing drug nanocrystals of a 62 nm mean particle size . the snap - freezing or instant freezing with liquid nitrogen was necessary to achieve this very low particle size , as slowly freezing the drug solution resulted in bigger particle sizes after the top - down step ( 186 nm ) . in addition to the ultrasmall particle size , the process became extremely cost - effective by reducing the number of homogenization cycles from 20 with the standard technique to only one cycle at 1500 bar with the combinative technology . in another study , human erythrocytes were loaded with an amphotericin b nanosuspension produced with the h 96 technology . the antifungal treatment could be improved due to the enhanced pharmacological profile of the amphotericin b nanocrystals [ 45 , 46 ] . hca was also processed as described for amphotericin b. after dissolving the drug in dmso , the drug solution was snap - frozen with liquid nitrogen . the drug powder was processed to a nanosuspension employing the hph technique for 10 cycles at 1500 bar . another drug processed with the h 96 process was cyclosporine a. this compound was dissolved in a 1 : 1 ethanol : dmso mixture , freeze - dried employing the snap - freezing technique , and further homogenized for 15 cycles at 1500 bar . a mean particle size of 440 nm was reported in this case . further experiments employing glibenclamide revealed a relationship between the crystallization conditions and the particle size reduction effectiveness of the top - down step . the different ratios of a dmso - tba mixture ( 90 : 10 to 10 : 90 v / v ) and the drug concentration during the bottom - up process modified the solid state behavior of the drug as well as its morphology . the micronized and freeze - dried glibenclamide powders were analyzed with the dsc technique to determine their dc . the micronized glibenclamide possesses a dc of 100% while most of the lyophilized powders showed dcs between 50% and 60% . however , when a design of experiments for the assessment of the critical crystallization factors was employed , it was found that solvent mixtures containing a high tba proportion ( i.e. , dmso : tba : 10 : 90 v / v ) and a low drug concentration favored the formation of highly amorphous glibenclamide . it was found that the h 96 technology is able to produce drug powders in either a crystalline or an amorphous state , depending on the process conditions and additives . additionally , the process conditions modified the morphology of glibenclamide from a hard , rough structure to a fine , subtle , and brittle structure , determined by the sem technique . a mean particle size of 164 nm could be obtained under optimized conditions , which was markedly improved compared to the unmodified glibenclamide ( 772 nm ) . the homogenization length could also be reduced from 20 cycles to only one cycle , which was sufficient to produce a nanosuspension with a smaller particle size than after 20 cycles with the standard method . in another study also employing glibenclamide as a model drug , the comminution effectiveness of the wbm and hph processes when employing lyophilized drug as a starting material was compared . the fd solvents were dmso : tba mixtures prepared with solvent ratios of 90 : 10 to 10 : 90 ( v / v ) . the drug concentration was kept constant at 5% for both bottom - up and top - down steps . both methods were an improvement over the standard process using unmodified material . in the case of the wbm , the process time was reduced from 24 hours to only one hour to achieve a proper nanosuspension . smaller particle sizes can be achieved much faster by modifying the drug structure . in the case of the hph , the number of homogenization cycles was reduced from 20 to only five cycles to achieve a sufficiently small particle size . a mean particle size of 160 nm was reported employing wbm on modified material after 24 hours of processing . in addition , a mean particle size of 335 nm was obtained using hph on a freeze - dried modified drug . for the wbm process , the higher friability and volume of the drug powders , which remained crystalline , were beneficial . for the homogenization process , the change in the drug crystal behavior from crystalline to amorphous to achieve smaller particle sizes was beneficial . this feature was confirmed by assessing the dc of the drug powders employing the dsc technique . the ct process combines a low - energy pearl milling step , followed by high pressure homogenization for particle size reduction . the shear forces and particle collision are combined with the cavitation for an innovative particle size reduction process . this step achieves , in general , drug particle sizes between 600 nm and 1500 nm . the subsequent homogenization process improves the homogeneity of the nanosuspension by reducing the particle size and the amount of larger crystals . the latter feature also enhances the physical stability by avoiding crystal growth ( ostwald ripening ) , which improves the long - term stability of the drug nanosuspensions during storage . interestingly , it was reported that lower homogenization pressures ( 100500 bar ) resulted in smaller drug nanocrystals and more homogeneous nanosuspensions than higher homogenization pressures ( 1500 bar ) after the pearl milling step . the advantages of this technology are the reduction of the homogenization pressure and process length , as well as the improved physical stability of the nanosuspensions . however , the ct process leads to particle sizes that are relatively bigger compared to the other combinative technologies . the flavonoid hesperidin is an example of a poorly soluble drug processed with the ct technology . a mean particle size of 599 nm was reported for hesperidin nanosuspensions , which also showed improved long - term stability . special features of the production of nanoparticulated hesperidin employing the ct process were the reduction of the homogenization cycles ( from 20 to five ) and of the necessary pressure ( from 1500 bar to 1000 bar ) to achieve a nanosuspension . in this manner , it is possible to reduce the energy input and the wearing of the machines . these drugs are flavonoids showing antioxidant properties with potential applications in pharmaceutical and cosmetic products . the first cosmetic product formulated employing nanotechnology contains rutin nanocrystals and was launched by juvena , switzerland . hesperidin nanocrystals can be found in the platinum rare cosmetic product ( la prairie , switzerland ) . the topical route has been reported as full of potential for nanoparticulate applications , as the drug nanocrystals enhance the compound penetration to the skin . the electrolytes could lead to aggregation by reducing the zeta potential ( i.e. , the electrostatic repulsion ) of the drug nanocrystals , thus producing the loss of their fast dissolution properties . employing apigenin , the ct technology led to a final mean particle size of 275 nm after only one homogenization cycle at 300 bar using an avestin c50 homogenizer . the pearl - milled product presented a mean particle size of 412 nm , which was further reduced by the homogenization step . interestingly , in this case lower pressures had an advantage in achieving smaller drug nanocrystals . in the case of rutin , a suspension of the drug was pearl - milled with zirconium oxide beads ( 0.3 mm ) to a mean particle size of about 1000 nm . this premilled suspension was then homogenized employing the avestin c50 for one cycle at different pressures . the best mean particle size of 604 nm was achieved employing low pressure ( 100 bar ) . the nanosuspension production could be scaled from a 20 g batch to a 3 kg batch . the milling process was performed using an agitating pearl mill bhler pml 2 ( bhler ag , switzerland ) with zirconium oxide beads ( bead size : 0.40.6 mm ) . the homogenization part of the ct process was performed employing the avestin c50 for one cycle at 300 bar . the premilling step resulted in drug particles with a mean particle size of 413 nm , which remained constant after the homogenization process . however , the homogenization produced a narrowing of the particle size distribution , manifested through a decreasing polydispersity index . the particle size , crystallinity , and physical stability of the nanosuspension were maintained when up - scaling the process , which is necessary for industrial production . further , the ct technology could drastically reduce the number of homogenization cycles to just one , which is more cost - effective . new formulation technologies are the key to overcoming the increasing problem of poor aqueous solubility among emerging compounds . the combinative particle size reduction processes have been presented as a part of the new enabling technologies . a schematic description of the standard particle size reduction processes ( left side ) and the combinative technologies ( right side ) is shown in figure 1 . employing the combinative methods , combinative processes such as the h 42 , h 96 , h 69 , and the nanoedge technologies enable the direct processing of a drug solution after synthesis without previously performing a crystallization step . however , as the h 69 and nanoedge technologies involve the precipitation of particles in liquid media that usually contain organic solvents , these nanosuspensions are not ready to be used further ( see figure 1 ) . extra drying steps need to be performed to eliminate the organic solvent content , which makes the process longer , more expensive , and more complicated regarding regulatory aspects [ 5 , 43 ] . on the contrary , when employing the h 42 and h 96 technologies , the organic solvent necessary to dissolve the poorly soluble drugs is eliminated during the bottom - up step . in this manner , the nanosuspensions produced with the dried intermediates can be directly used or down - streamed for the production of solid dosage forms [ 5 , 18 , 51 ] . a wide variety of drugs processed with the combinative technologies is shown in table 1 . the nanoedge technology is the only process with a main focus on injectables ( i.v . the other technologies are focused on nanosizing for dissolution rate improvement for oral administration or formulation for topical administration . the formulation for cosmetic and nutraceutical applications , such as those discussed by petersen in the ct technology patent , has also been successful . when comparing the production length of the processes , the nanoedge and the h 69 techniques are relatively fast due to the rapid precipitation step . however , the organic solvent content of the nanosuspensions needs to be removed when employing these technologies , and so they lose the advantage of producing nanosuspensions in a fast process . the h 96 technology is more time - consuming due to the lengthy fd process . however , the lyophilization technique results in yields near to 100% , which is important in the case of expensive compounds . additionally , the h 96 nanosuspensions do not contain amounts of organic solvents , which enable their direct usage after production . finally , the h 42 technology produces nanosuspensions in a fast process . the sd is a rapid production step that can be performed in continuous mode . the h 42 nanosuspensions can also be subsequently directly processed or used , as they do not contain organic solvents . regarding the particle size reduction effectiveness , the h 96 , h 69 , and h 42 technologies are the processes achieving the smallest particle sizes for a variety of drugs ( table 1 ) . also , some microprecipitation - high - energy approaches ( nanoedge and nanoedge - like ) led to small mean particle sizes . by processing the same drug with different combinative techniques , it is possible to compare the reduction effectiveness and performance of the technologies . for example , amphotericin b and glibenclamide were both more effectively processed with the h 96 technology than with the h 42 process . in the case of amphotericin b , final mean particle sizes of 62 nm and 172 nm were achieved employing the h 96 and h 42 technologies , respectively [ 39 , 41 ] . in the case of glibenclamide , final mean particle sizes of 164 nm and 236 nm were achieved employing the h 96 and h 42 technologies , respectively . the factors influencing the particle size reduction effectiveness were the porosity and the crystallinity of the drug powders . both technologies produced drug powders with porous and brittle drug structures as well as with modified crystallinity . the h 96 technology produced , under optimized conditions , glibenclamide powders with 1% dc , which subsequently led to the low mean particle size of 164 nm . in comparison , the h 42 process led to glibenclamide powders with relatively higher dc ( 8.1% ) , which also resulted in relatively bigger particle sizes after the homogenization step ( 236 nm ) . however , both technologies achieved homogeneous dispersed nanosuspensions with a low particle size . additionally , the h 42 technology had the advantage of being a much faster process [ 3 , 44 ] . when comparing the h 42 and h 69 performances , the results are diverse . in the case of ibuprofen , better particle size results these results were 170 nm with the h 69 process and 636 nm with the h 42 technology . however , the homogenizing equipment was different : the avestin c5 for 10 cycles at 1200 bar in the case of the h 69 process and the micron lab 40 for 20 cycles at 1500 bar in the case of the h 42 process [ 38 , 39 ] . ibuprofen was also processed employing a microprecipitation - sonication technique . however , this approach led to bigger mean particle sizes than with the other combinative approaches ( 702 nm ) . with hca as a model drug , the best results were achieved with the h 42 process with a final mean particle size of 281 nm after 20 cycles at 1500 bar . however , the h 96 process achieved a final mean particle size of 414 nm after 10 cycles at 1500 bar . finally , the h 69 process produced with hca a nanosuspension with a mean particle size of 787 nm after 20 cycles at 1500 bar . the latter result was considerably bigger than with the first two techniques [ 2 , 37 ] . in the case of resveratrol as a model compound , the h 69 combinative processes led to a final mean particle size of 150 nm and the h 42 process achieved a final mean particle size of 200 nm . however , the homogenization conditions were different : the h 69 process was performed with an avestin c5 for 10 cycles at 1200 bar and the h 42 technology was performed with a micron lab 40 for 1 cycle at 1500 bar [ 38 , 40 ] . nanoedge - like approach employing either sonication or hph as the annealing step led to similar mean particle sizes ( 259 nm and 212 nm , resp . ) . in general , the particle size reduction effectiveness depends on several factors : the technology and equipment employed , as well as the physicochemical characteristics of the drug , such as solid state behavior , hardness , porosity , and morphology . a technique that produces amorphous drugs and/or highly brittle , porous , and friable structures can also lead to smaller particle sizes after the comminution step . the particle size reduction performances of standard and selected combinative processes with glibenclamide as a model compound are compared in figure 2 . the graphic description shows the superior particle size reduction effectiveness of the combinative technologies regarding the process length to achieve a nanosuspension and the smallest final mean particle size . the hph and wbm standard techniques achieved a final mean particle size of 772 nm and 191 nm at the end of their respective processes ( after 20 cycles of hph and 24 hours of wbm ) . however , these processes presented a slower particle size reduction progress than the combinative methods . when the h 96 technology ( black columns ) was employed , the nanosuspension had a mean particle size of about 200 nm after one cycle of hph . at this point , the standard hph presented a mean particle size of 1417 nm . additionally , the standard wbm presented a mean particle size of 840 nm after one hour of milling . this mean particle size result of 200 nm after only one cycle of hph was markedly improved than with standard hph ( 772 nm ) and almost the same as standard wbm ( 191 nm ) till the end of these processes . finally , the h 96 process achieved a final mean particle size of 164 nm after 20 hph cycles . when the h 96 technology was employed with wbm as the top - down step ( white columns ) , the nanosuspension had a mean particle size of 269 nm after only one hour of milling and 160 nm after 24 hours of processing . both results were also markedly improved compared to the standard approaches . in the case of processing glibenclamide with the h 42 technology ( grey columns ) , the nanosuspension had a mean particle size of 384 nm after one cycle of hph and a final mean particle size of 236 nm after 20 cycles of homogenization . again , these particle size results were improved compared to the standard techniques employing untreated drug material . in general , the combinative particle size reduction processes perform faster than the standard methods to produce nanosuspensions and achieve smaller final mean particle sizes . the application of nanotechnology in pharmaceutical development has great potential for the formulation of poorly water - soluble compounds . this approach has already proven to be successful with a steadily increasing number of marketed products . universal , efficient , and easily applicable processes are the most suitable technologies for the formulation of poorly soluble drugs . the combinative processes lead , in general , to faster top - down process steps , improved physical stability , and smaller particle sizes than the standard comminution processes such as high pressure homogenization or wet bead milling . the small particle sizes have a direct impact on the dissolution rate and bioavailability of poorly soluble drugs after oral , topic , and i.v . more research needs to be performed , however , to solve the technical challenges of the different technologies in order to achieve improved particle size reduction effectiveness and better formulations for new , problematic compounds . in the future , it is expected that more screenings will be performed employing the principle of design of experiments to systematically analyze the critical factors for the production of nanosuspensions . in this way , it will be possible to establish optimal process parameters to achieve final mean particle sizes below 100 nm for a wide variety of compounds .

nanosizing is a suitable method to enhance the dissolution rate and therefore the bioavailability of poorly soluble drugs . the success of the particle size reduction processes depends on critical factors such as the employed technology , equipment , and drug physicochemical properties . high pressure homogenization and wet bead milling are standard comminution techniques that have been already employed to successfully formulate poorly soluble drugs and bring them to market . however , these techniques have limitations in their particle size reduction performance , such as long production times and the necessity of employing a micronized drug as the starting material . this review article discusses the development of combinative methods , such as the nanoedge , h 96 , h 69 , h 42 , and ct technologies . these processes were developed to improve the particle size reduction effectiveness of the standard techniques . these novel technologies can combine bottom - up and/or top - down techniques in a two - step process . the combinative processes lead in general to improved particle size reduction effectiveness . faster production of drug nanocrystals and smaller final mean particle sizes are among the main advantages . the combinative particle size reduction technologies are very useful formulation tools , and they will continue acquiring importance for the production of drug nanocrystals .

1. Introduction 2. Precipitation Processes (Bottom-Up) 3. Comminution Processes (Top-Down) 4. Combinative Technologies 5. Technologies Evaluation: Comparison between Combinative Processes 6. Performance Comparison between Combinative and Standard Technologies 7. Conclusion

standard techniques employed for the production of drug nanocrystals are high pressure homogenization and wet bead milling . however , they have drawbacks , such as long processing times and the necessity of employing a micronized drug as the starting material . next - generation technologies involve combinative particle size reduction methods to improve the particle size reduction effectiveness of the standard techniques . there are basically two approaches to produce drug nanocrystals , with a third one ( the combinative approach ) being a combination of the first two techniques . this approach is called bottom - up , as the size of the particles is increased . this approach is called top - down , as the size of already existing particles is decreased . the third approach involves combinations of bottom - up and/or top - down steps to improve the particle size reduction effectiveness of the single - unit processes . the first step is usually a bottom - up process employed as a drug pretreatment to obtain a brittle , friable starting material for a subsequent comminution step . combinations of two different top - down steps ( such as bead milling followed by high pressure homogenization ) have also been developed [ 5 , 6 ] . the special features of this bottom - up technology are the production of crystalline drug nanoparticles and the ability for large - scale production [ 12 , 13 ] . both bottom - up technologies can also be employed to modify drug materials to make them more suitable ( i.e. the forces producing the particle size reduction include shear forces , and particle collision produced by the movement of the milling material inside the chamber . the nanocrystal technology is regarded as a successful technology : the first product containing drug nanocrystals ( rapamune by wyeth pharmaceuticals in 2000 ) came to the market only 10 years after the development of the technology [ 16 , 17 ] . the hph technique is a high - energy disintegration process that employs high pressure to reduce the particle size of drug particles in liquid media with surfactants for stabilization purposes . one interesting feature of the nanopure technology is that the oil dispersions can be employed to subsequently fill capsules as the final dosage form . in summary , the known limitations of the standard processes ( wbm , hph ) for the production of drug nanocrystals are the necessity of a micronized drug as the starting material and the long runtimes for the top - down equipment . the combinative particle size reduction techniques have been developed to overcome these drawbacks and to improve the particle size reduction effectiveness of the standard processes . nowadays , five combinative methods are known : nanoedge ( microprecipitation followed by a high - energy step such as hph ) , h 69 ( microprecipitation immediately followed by hph , also called cavi - precipitation ) , h 42 ( spray - drying followed by hph ) , h 96 ( freeze - drying followed by hph ) , and the ct combinative technology ( media milling followed by hph ) . the nanoedge technology from baxter is the first combinative particle size reduction method developed for the production of drug nanosuspensions . then , the drug particles are reduced in size and transformed to the more stable crystalline state after employing a high - energy annealing step , such as high pressure homogenization [ 24 , 25 ] . the objective of the annealing step is to improve the thermodynamic stability of the nanosuspensions by preventing the crystal growth of the precipitated particles to the micrometer range . the change to the more stable form is produced by the high - energy input of the top - down step , which promotes the formation of low - energy , more stable structures , either by enhancing the crystallinity of the particles ( reordering of the lattice structure ) or by rearrangement of the stabilizing system at the surface of the drug nanocrystals . the fast microprecipitation improves the particle size reduction effectiveness of the top - down step due to induced friable material , drug crystal defects , and dendritic morphology . the top - down process is usually high pressure homogenization but other techniques such as sonication or microfluidization can also be employed [ 25 , 26 ] . the nanoedge platform has been employed to formulate poorly soluble anticancer drugs such as paclitaxel to improve the plasma concentration and thereby the pharmacologic efficacy . with another poorly soluble , poorly bioavailable anticancer drug described as b , a linear relationship was found between the decreasing in nanosuspension particle size ( particle sizes of 4001000 nm , administered at 300 mg / kg ) and the increase of oral bioavailability measured from the plasma of rats . functionalized polyethylene glycols were employed as surfactants to minimize the opsonization of the drug nanocrystals , which had a mean particle size of 200 nm [ 27 , 28 ] . sonication for one minute at 10000 hertz ( hz ) and 400 watts ( w ) employed as the annealing step resulted in drug nanoparticles with a mean particle size of 177 nm . in this case , hph was used as the annealing step , achieving a final mean particle size of 581 nm . canada ) was employed for the high - energy step to process the three drug macrosuspensions , which resulted in final mean particle sizes of 400 nm for carbamazepin , 640 nm for prednisolone and 930 nm for nabumetone . the drug particles were obtained in the amorphous state and the best final mean particle size of 131 nm was obtained by homogenizing the drug suspension for 20 cycles at 1000 bar . the results showed a final mean particle size of 259 nm with the sonication method and 212 nm with the hph technique . additionally , low pressure could be maintained ( 500 bar ) , as higher pressures ( or a higher number of homogenization cycles ) did not improve the particle size reduction . however , this result was only slightly improved compared to the precipitation process without sonication ( 176 nm mean particle size ) . the precipitation process modified the high crystallinity of the starting material leading to amorphous drug nanoparticles [ 35 , 36 ] . different surfactants were screened for the precipitation step and the best stabilization results were achieved with hpmc k3 , which led to a mean particle size of 702 nm after sonication . it combines a microprecipitation step involving organic solvents , followed by high pressure homogenization for particle size reduction . the difference is that with the h 69 technology , the cavitation takes place at the same time as the particle formation ( cavi - precipitation ) or at most two seconds thereafter . the nonsolvent is added to the solvent in a controlled manner using , for example , an infuser device such as the perfusor from b. braun melsungen , germany . the top - down step not only reduces the particle size but also stabilizes the drug nanocrystals with the energy application . the drugs hydrocortisone acetate ( hca ) and omeprazol were processed employing the h 69 process , achieving mean particle sizes of 787 nm and 921 nm , respectively , after 20 cycles of homogenization at 1500 bar . in the case of resveratrol , a final mean particle size of 150 nm could be achieved in the case of resveratrol . the reduction of the time between precipitation and the top - down step and the proper selection of the organic solvent to dissolve the drug prior to the precipitation step were identified as critical factors in obtaining the smallest drug nanocrystals . the h 42 process this combinative technology combines spray - drying ( sd ) as a precipitation and pretreatment step , followed by hph for particle size reduction . the organic solvent is eliminated during the bottom - up step , which differentiates this technology from the nanoedge and h 69 processes.in the first unit operation ( sd ) , the poorly soluble compound is dissolved in organic solvents . surfactants such as poloxamer or sugars such as mannitol can be added to the drug solution to improve the results of the drying step . the h 42 combinative technology has advantages such as relatively short processing times during sd , solvent - free dry intermediates , and small drug nanocrystals after a reduced number of hph cycles . in this case , the improved reduction effectiveness was not linked to a change in the solid state behavior of the drug , but to the enhanced friability of the starting material . the micronized , unmodified hca led to a final mean particle size of 551 nm under the same process conditions . small amounts of the surfactant positively impacted the characteristics of the spray - dried powders , such as flowability and millability . , 1 : 1 drug / surfactant ratio ) negatively impacted the powders ' characteristics and the subsequent particle size reduction effectiveness . the effect of these parameters on the solid state behavior and morphology of the drug , as well as on the particle size reduction effectiveness of the top - down step , was analyzed . both solid state modification leading to an amorphous drug and the morphology change due to the precipitation process positively impacted the particle size reduction effectiveness of the top down - step . however , it was difficult to establish a link between the dc and the smallest drug particle sizes with resveratrol as the model drug . this process involves freeze - drying ( fd ) as a bottom - up and pretreatment step , followed by hph for particle size reduction . the bottom - up step eliminates the organic solvent content , just as with the h 42 technology . the aim of the drug pretreatment is to modify the starting material to improve the particle size reduction effectiveness of the hph [ 5 , 41 ] . mixtures of organic solvents can also be implemented to improve the performance of the lyophilization process . dmso contributed to the process with the necessary dissolving force for glibenclamide , and tba was added to the solution to improve the characteristics of the freeze - dried cakes [ 43 , 44 ] . the h 96 technology is especially suitable to process thermolabile or expensive drugs due to the low temperatures and the high yields of the fd . the freeze - dried drug powder was processed to a nanosuspension employing a micron lab 40 homogenizer for five cycles at 1500 bar producing drug nanocrystals of a 62 nm mean particle size . the snap - freezing or instant freezing with liquid nitrogen was necessary to achieve this very low particle size , as slowly freezing the drug solution resulted in bigger particle sizes after the top - down step ( 186 nm ) . in addition to the ultrasmall particle size , the process became extremely cost - effective by reducing the number of homogenization cycles from 20 with the standard technique to only one cycle at 1500 bar with the combinative technology . another drug processed with the h 96 process was cyclosporine a. this compound was dissolved in a 1 : 1 ethanol : dmso mixture , freeze - dried employing the snap - freezing technique , and further homogenized for 15 cycles at 1500 bar . further experiments employing glibenclamide revealed a relationship between the crystallization conditions and the particle size reduction effectiveness of the top - down step . the different ratios of a dmso - tba mixture ( 90 : 10 to 10 : 90 v / v ) and the drug concentration during the bottom - up process modified the solid state behavior of the drug as well as its morphology . however , when a design of experiments for the assessment of the critical crystallization factors was employed , it was found that solvent mixtures containing a high tba proportion ( i.e. in another study also employing glibenclamide as a model drug , the comminution effectiveness of the wbm and hph processes when employing lyophilized drug as a starting material was compared . the drug concentration was kept constant at 5% for both bottom - up and top - down steps . the ct process combines a low - energy pearl milling step , followed by high pressure homogenization for particle size reduction . the subsequent homogenization process improves the homogeneity of the nanosuspension by reducing the particle size and the amount of larger crystals . special features of the production of nanoparticulated hesperidin employing the ct process were the reduction of the homogenization cycles ( from 20 to five ) and of the necessary pressure ( from 1500 bar to 1000 bar ) to achieve a nanosuspension . employing apigenin , the ct technology led to a final mean particle size of 275 nm after only one homogenization cycle at 300 bar using an avestin c50 homogenizer . however , the homogenization produced a narrowing of the particle size distribution , manifested through a decreasing polydispersity index . the particle size , crystallinity , and physical stability of the nanosuspension were maintained when up - scaling the process , which is necessary for industrial production . the combinative particle size reduction processes have been presented as a part of the new enabling technologies . a schematic description of the standard particle size reduction processes ( left side ) and the combinative technologies ( right side ) is shown in figure 1 . employing the combinative methods , combinative processes such as the h 42 , h 96 , h 69 , and the nanoedge technologies enable the direct processing of a drug solution after synthesis without previously performing a crystallization step . however , as the h 69 and nanoedge technologies involve the precipitation of particles in liquid media that usually contain organic solvents , these nanosuspensions are not ready to be used further ( see figure 1 ) . on the contrary , when employing the h 42 and h 96 technologies , the organic solvent necessary to dissolve the poorly soluble drugs is eliminated during the bottom - up step . in this manner , the nanosuspensions produced with the dried intermediates can be directly used or down - streamed for the production of solid dosage forms [ 5 , 18 , 51 ] . when comparing the production length of the processes , the nanoedge and the h 69 techniques are relatively fast due to the rapid precipitation step . however , the organic solvent content of the nanosuspensions needs to be removed when employing these technologies , and so they lose the advantage of producing nanosuspensions in a fast process . regarding the particle size reduction effectiveness , the h 96 , h 69 , and h 42 technologies are the processes achieving the smallest particle sizes for a variety of drugs ( table 1 ) . in the case of amphotericin b , final mean particle sizes of 62 nm and 172 nm were achieved employing the h 96 and h 42 technologies , respectively [ 39 , 41 ] . in the case of glibenclamide , final mean particle sizes of 164 nm and 236 nm were achieved employing the h 96 and h 42 technologies , respectively . the factors influencing the particle size reduction effectiveness were the porosity and the crystallinity of the drug powders . the h 96 technology produced , under optimized conditions , glibenclamide powders with 1% dc , which subsequently led to the low mean particle size of 164 nm . in the case of ibuprofen , better particle size results these results were 170 nm with the h 69 process and 636 nm with the h 42 technology . however , the homogenizing equipment was different : the avestin c5 for 10 cycles at 1200 bar in the case of the h 69 process and the micron lab 40 for 20 cycles at 1500 bar in the case of the h 42 process [ 38 , 39 ] . however , this approach led to bigger mean particle sizes than with the other combinative approaches ( 702 nm ) . with hca as a model drug , the best results were achieved with the h 42 process with a final mean particle size of 281 nm after 20 cycles at 1500 bar . however , the h 96 process achieved a final mean particle size of 414 nm after 10 cycles at 1500 bar . finally , the h 69 process produced with hca a nanosuspension with a mean particle size of 787 nm after 20 cycles at 1500 bar . in the case of resveratrol as a model compound , the h 69 combinative processes led to a final mean particle size of 150 nm and the h 42 process achieved a final mean particle size of 200 nm . however , the homogenization conditions were different : the h 69 process was performed with an avestin c5 for 10 cycles at 1200 bar and the h 42 technology was performed with a micron lab 40 for 1 cycle at 1500 bar [ 38 , 40 ] . in general , the particle size reduction effectiveness depends on several factors : the technology and equipment employed , as well as the physicochemical characteristics of the drug , such as solid state behavior , hardness , porosity , and morphology . the particle size reduction performances of standard and selected combinative processes with glibenclamide as a model compound are compared in figure 2 . the graphic description shows the superior particle size reduction effectiveness of the combinative technologies regarding the process length to achieve a nanosuspension and the smallest final mean particle size . the hph and wbm standard techniques achieved a final mean particle size of 772 nm and 191 nm at the end of their respective processes ( after 20 cycles of hph and 24 hours of wbm ) . however , these processes presented a slower particle size reduction progress than the combinative methods . at this point , the standard hph presented a mean particle size of 1417 nm . finally , the h 96 process achieved a final mean particle size of 164 nm after 20 hph cycles . when the h 96 technology was employed with wbm as the top - down step ( white columns ) , the nanosuspension had a mean particle size of 269 nm after only one hour of milling and 160 nm after 24 hours of processing . in the case of processing glibenclamide with the h 42 technology ( grey columns ) , the nanosuspension had a mean particle size of 384 nm after one cycle of hph and a final mean particle size of 236 nm after 20 cycles of homogenization . again , these particle size results were improved compared to the standard techniques employing untreated drug material . in general , the combinative particle size reduction processes perform faster than the standard methods to produce nanosuspensions and achieve smaller final mean particle sizes . universal , efficient , and easily applicable processes are the most suitable technologies for the formulation of poorly soluble drugs . the combinative processes lead , in general , to faster top - down process steps , improved physical stability , and smaller particle sizes than the standard comminution processes such as high pressure homogenization or wet bead milling . the small particle sizes have a direct impact on the dissolution rate and bioavailability of poorly soluble drugs after oral , topic , and i.v . more research needs to be performed , however , to solve the technical challenges of the different technologies in order to achieve improved particle size reduction effectiveness and better formulations for new , problematic compounds . in the future , it is expected that more screenings will be performed employing the principle of design of experiments to systematically analyze the critical factors for the production of nanosuspensions . in this way , it will be possible to establish optimal process parameters to achieve final mean particle sizes below 100 nm for a wide variety of compounds .

eight caucasian women who had undergone a standardized laparoscopic rygbp ( 20 ) and seven caucasian , age - matched , normal - weight , healthy controls participated in our study ( table 1 ) . eligibility criteria for the rygbp group included the following : history of t2 dm with duration > 6 months and using pharmacological treatment before surgery ; complete remission of t2 dm at the time of evaluation ; and postsurgical follow - up period 24 months . t2 dm remission was defined as fasting plasma glucose < 100 mg / dl plus glycated hemoglobin ( hba1c ) < 6.0% in the absence of active pharmacological therapy and lasting at least 1 year ( 21 ) . normal glucose tolerance in the control group was established based on a fasting plasma glucose and hba1c in the normal range . all study participants had stable body weight for at least 1 month before the studies . the study was approved by the hospital ethics committee and written informed consent was obtained from all the participants . characteristics of the study subjects subjects attended the research facility at 8:30 a.m. after an overnight fast on two occasions , in random order , and separated by at least 72 h. on admission , a canula was inserted into a forearm for blood sample collection and another one was inserted in the opposite forearm for infusion of synthetic exendin-(939 ) ( clinalfa basic , bachem , germany ) or saline . after withdrawal of baseline blood samples ( time 30 min ) , subjects received either an intravenous bolus of synthetic exendin-(939 ) ( 7,500 pmol / kg ) in 1 min followed by continuous infusion ( 750 pmol / kg / min ) for the remainder of the study or saline ( up to 120 min ) . at 30 min , subjects ingested a standardized liquid meal ( slm ; 250 ml , 398 kcal , 50% carbohydrates , 35% fat , 15% protein ; isosource energy ; novartis , switzerland ) over 5 min . subjects were maintained in the recumbent position with the backside of the bed inclined at 30 degrees throughout the test . venous samples were obtained every 10 min ( from time 30 to time 120 min ) for the measurement of plasma glucose , insulin , and c - peptide . glucagon , glp-1 , and gip were assessed in blood samples obtained at baseline , every 10 min from 0 to 40 min , and every 20 min thereafter . blood samples for the measurement of c - peptide , glucagon , glp-1 , and gip were obtained in chilled edta tubes containing 500 units of aprotinin per milliliter of blood . the area under the curve ( auc ) for glucose , insulin , c - peptide , glucagon , and total glp-1 after the ingestion of the slm were calculated using the trapezoidal method . the insulinogenic index was calculated as the increment of insulin ( mu / l ) or c - peptide ( ng / ml ) between time 0 and time 30 divided by the change in plasma glucose ( mg / dl ) in the same time frame . insulin sensitivity was estimated from the homeostasis model assessment of insulin resistance ( homa - ir ) according to the formula : homa - ir = [ insulin ( mu / l ) glucose ( mmol / l ) / 22.5 ] . plasma glucose and insulin levels were measured respectively using a glucose oxidase method ( bayer diagnostics , munich , germany ) and a monoclonal immunoradiometric assay ( medgenix diagnostics , fleunes , belgium ) as previously described ( 22,23 ) . plasma c - peptide was measured by ria ( millipore , billerica , ma ) . human plasma total glp-1 and glucagon were measured by radioimmunoassays ( glucagon - like peptide [ total ] ria kit , and glucagon ria kit ; millipore ) , and gip was measured with an elisa ( human gip [ total ] elisa ; millipore ) as previously reported ( 22,23 ) . the glucagon assay uses an antibody that is specific for pancreatic glucagon , with < 0.1% cross - reactivity to oxytomodulin and no cross - reactivity with the exendin-(939 ) used in our study ( data not shown ) . the sample size of our study was powered ( error < 0.1 ) to detect an enlargement of the glucose excursion after the slm of 75.0% in the rygbp group with an error < 0.05 . unpublished observations from our group have shown a 75.4% larger auc0120 of glucose after the same slm used in this study when subjects with t2 dm before rygbp but with either sustained remission or t2 dm relapse were compared . all variables followed gaussian distribution ( p > 0.05 ) ; consequently , parametric test were used in statistical analysis . parameters obtained in studies with exendin-(939 ) or saline were compared within each group using paired t test . eight caucasian women who had undergone a standardized laparoscopic rygbp ( 20 ) and seven caucasian , age - matched , normal - weight , healthy controls participated in our study ( table 1 ) . eligibility criteria for the rygbp group included the following : history of t2 dm with duration > 6 months and using pharmacological treatment before surgery ; complete remission of t2 dm at the time of evaluation ; and postsurgical follow - up period 24 months . t2 dm remission was defined as fasting plasma glucose < 100 mg / dl plus glycated hemoglobin ( hba1c ) < 6.0% in the absence of active pharmacological therapy and lasting at least 1 year ( 21 ) . normal glucose tolerance in the control group was established based on a fasting plasma glucose and hba1c in the normal range . all study participants had stable body weight for at least 1 month before the studies . the study was approved by the hospital ethics committee and written informed consent was obtained from all the participants . subjects attended the research facility at 8:30 a.m. after an overnight fast on two occasions , in random order , and separated by at least 72 h. on admission , a canula was inserted into a forearm for blood sample collection and another one was inserted in the opposite forearm for infusion of synthetic exendin-(939 ) ( clinalfa basic , bachem , germany ) or saline . after withdrawal of baseline blood samples ( time 30 min ) , subjects received either an intravenous bolus of synthetic exendin-(939 ) ( 7,500 pmol / kg ) in 1 min followed by continuous infusion ( 750 pmol / kg / min ) for the remainder of the study or saline ( up to 120 min ) . at 30 min , subjects ingested a standardized liquid meal ( slm ; 250 ml , 398 kcal , 50% carbohydrates , 35% fat , 15% protein ; isosource energy ; novartis , switzerland ) over 5 min . subjects were maintained in the recumbent position with the backside of the bed inclined at 30 degrees throughout the test . venous samples were obtained every 10 min ( from time 30 to time 120 min ) for the measurement of plasma glucose , insulin , and c - peptide . glucagon , glp-1 , and gip were assessed in blood samples obtained at baseline , every 10 min from 0 to 40 min , and every 20 min thereafter . blood samples for the measurement of c - peptide , glucagon , glp-1 , and gip were obtained in chilled edta tubes containing 500 units of aprotinin per milliliter of blood . the area under the curve ( auc ) for glucose , insulin , c - peptide , glucagon , and total glp-1 after the ingestion of the slm were calculated using the trapezoidal method . the insulinogenic index was calculated as the increment of insulin ( mu / l ) or c - peptide ( ng / ml ) between time 0 and time 30 divided by the change in plasma glucose ( mg / dl ) in the same time frame . insulin sensitivity was estimated from the homeostasis model assessment of insulin resistance ( homa - ir ) according to the formula : homa - ir = [ insulin ( mu / l ) glucose ( mmol / l ) / 22.5 ] . plasma glucose and insulin levels were measured respectively using a glucose oxidase method ( bayer diagnostics , munich , germany ) and a monoclonal immunoradiometric assay ( medgenix diagnostics , fleunes , belgium ) as previously described ( 22,23 ) . plasma c - peptide was measured by ria ( millipore , billerica , ma ) . human plasma total glp-1 and glucagon were measured by radioimmunoassays ( glucagon - like peptide [ total ] ria kit , and glucagon ria kit ; millipore ) , and gip was measured with an elisa ( human gip [ total ] elisa ; millipore ) as previously reported ( 22,23 ) . the glucagon assay uses an antibody that is specific for pancreatic glucagon , with < 0.1% cross - reactivity to oxytomodulin and no cross - reactivity with the exendin-(939 ) used in our study ( data not shown ) . the sample size of our study was powered ( error < 0.1 ) to detect an enlargement of the glucose excursion after the slm of 75.0% in the rygbp group with an error < 0.05 . unpublished observations from our group have shown a 75.4% larger auc0120 of glucose after the same slm used in this study when subjects with t2 dm before rygbp but with either sustained remission or t2 dm relapse were compared . all variables followed gaussian distribution ( p > 0.05 ) ; consequently , parametric test were used in statistical analysis . parameters obtained in studies with exendin-(939 ) or saline were compared within each group using paired t test . the effects of exendin-(939 ) on the glucose response to slm in subjects in the rygbp and control groups are shown in fig . 1 and table 2 . before exendin-(939 ) or saline infusion ( time 30 min ) , plasma glucose was not significantly different either within or between study groups . exendin-(939 ) infusion resulted in a slight , albeit significant , increase in plasma glucose before meal ingestion ( time 0 min ) in both groups ( rygbp : p = 0.001 ; control : p = 0.053 ) relative to before the initiation of the test . blood glucose response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a ) and rygbp ( b ) subjects . data are presented as mean se . * p < 0.05 relative to the saline condition . effect of slm ingestion on plasma glucose and hormonal secretion in studies with or without exendin-(939 ) as shown in table 2 , infusion of exendin-(939 ) resulted in a significant enlargement of the auc of glucose after meal intake ( auc glucose0120 ) in both study groups ( rygbp : p = 0.013 ; control : p = 0.020 ) . a similar relative increase of the auc glucose0120 in the exendin-(939 ) day relative to the saline day was found when the two groups were compared ( rygbp : 9.94 8.4% ; control : 10.84 8.8% ; p = 0.884 ) . in the rygbp group , the 2-h plasma glucose after slm intake was larger after exendin-(939 ) infusion ( 116.5 22.3 mg / dl ) as compared with that in the saline study ( 78.4 15.1 mg / dl ; p < 0.001 ) . however , the 2-h plasma glucose was < 200 mg / dl in all study participants in the rygbp group . two subjects in the rygbp group presented 2-h plasma glucose between 140 and 200 mg / dl ( respectively , 141 mg / dl and 143 mg / dl ) . a distinct temporal pattern of the glucose response was found in the two study groups in each experimental condition . during saline studies , patients in the rygbp group showed an earlier ( p = 0.001 ) and higher glucose peak ( p = 0.001 ) as compared with the control group . as a result , the auc of glucose during the first hour after slm ingestion ( auc glucose060 ) was larger in the rygbp group ( rygbp : 10.28 1.41 mg dl min 10 ; control : 6.82 0.50 mg dl min 10 ; p < 0.001 ) . in contrast , plasma glucose levels were lower from 90 min onwards in the rygbp group as compared with controls ( fig . 1 ; p < 0.05 for the time points of 90 , 100 , 110 , and 120 min ) . nonetheless , no significant difference was found when the auc of glucose during the second hour after ingestion of the slm ( auc glucose60120 ) was compared between the two groups on the saline day ( rygbp : 6.59 1.57 mg dl min 10 ; control : 7.99 1.67 mg dl min 10 ; p = 0.120 ) . only one subject in the surgical group presented with plasma glucose levels < 60 mg / dl from 90 min onwards in the absence of symptoms on the day of saline infusion . the differences between the two study groups in the plasma glucose response to slm on glp-1 action blockade are illustrated in fig . 1 . as shown in table 2 , in the rygbp group exendin-(939 ) infusion did not result in a significant change in peak glucose ( p = 0.297 ) , time to peak glucose ( p = 0.104 ) , or the auc glucose060 ( 10.15 1.15 10 mg dl min ; p = 0.597 ) as compared with the saline study . peak glucose ( p = 0.090 ) and the auc glucose060 ( 8.02 0.92 mg dl min ; p = 0.056 ) were larger , albeit not significantly , after exendin-(939 ) as compared with saline infusion in the control group . throughout the second hour after the slm , exendin-(939 ) infusion resulted in higher plasma glucose levels ( p < 0.05 for the time points of 90 , 100 , 110 , and 120 min ) and larger auc glucose60120 in the rygbp group ( 8.42 1.62 10 mg dl min ; p = 0.003 ) but not in the control group as compared with the saline day . as shown in table 2 , fasting plasma insulin did not differ between the two study groups or within each study group before the initiation of the tests performed in the two experimental conditions . fasting c - peptide levels did not differ between the saline and exendin-(939 ) studies within each group ( rygbp group : p = 0.593 ; control group : p = 0.868 ) , although the surgical group presented a significantly higher fasting c - peptide as compared with the control group on both study days ( table 2 ) . during fasting , glp-1 action blockade had no effect on insulin or c - peptide plasma levels neither in the rygbp group ( insulin : p = 0.582 ; c - peptide : p = 0.130 ) nor in the control group ( insulin : p = 0.963 ; c - peptide : p = 0.609 ) . in the saline condition , and despite comparable homa - ir ( table 1 ) , the insulin ( auc insulin0120 , p = 0.001 ) and c - peptide ( auc c - peptide0120 , p = 0.001 ) responses ensuing after slm ingestion were larger in the rygbp group . corresponding to the glucose curve , differences in the insulin response between the two study groups were accounted for differences during the first hour ( auc insulin060 : 11.68 4.33 mu l min 10 for rygbp compared with 2.11 1.57 mu l min 10 for control ; p < 0.001 ; auc c - peptide060 : 422.5 91.5 ng ml min for rygpb and 239.7 54.7 ng ml min for control ; p < 0.001 ) but not during the second hour ( auc insulin60120 : rygbp compared with controls , p = 0.205 ; auc c - peptide60120 : rygbp compared with controls , p = 0.055 ) after the slm challenge . in the rygbp group , exendin-(939 ) infusion resulted in blunted insulin response to slm ( fig . 2 and table 2 ) , with a mean reduction of the auc0120 of insulin and c - peptide of 52.1 10.9% and 24.1 8.7% , respectively . the insulin - calculated ( p = 0.009 ) and c - peptide calculated ( p = 0.017 ) insulinogenic indices were reduced in the rygbp group during exendin-(939 ) infusion . in the control group , exendin-(939 ) yielded no significant effect on insulin ( auc insulin0120 : 12.2 35.2% ) or c - peptide ( auc insulin0120 : 8.5 22.6% ) postprandial response ( fig . 2 and table 2 ) . insulin and c - peptide response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a , b ) and rygbp ( c , d ) subjects . * p < 0.05 relative to the saline condition . as shown in table 2 , no differences were found in baseline plasma glucagon levels between the 2 study days in the rygbp group . unexpectedly , slightly but statistically elevated fasting glucagon levels were found in the control group before saline infusion as compared with previous to exendin-(939 ) infusion ( p = 0.001 ) . as shown in fig . 3 , in the saline infusion condition the slm challenge resulted in slight decrease in glucagon plasma concentration ( incremental auc glucagon0120 : 0.31 1.17 ( pg dl min 10 ) . this physiological response was blunted by exendin-(939 ) infusion ( incremental auc glucagon0120 : 0.99 1.52 pg dl min 10 ; p = 0.062 ) . in contrast , a paradoxical increase in the glucagon response was observed in the rygbp group during the saline and exendin-(939 ) conditions [ incremental auc glucagon0120 : 6.35 2.96 pg dl min 10 for saline and 7.34 3.60 pg dl min 10 for exendin-(939 ) ] . despite glucose tolerance being similar , the auc glucagon0120 was larger in the surgical group in both study days ( table 2 ) . glucagon , glp-1 , and gip response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a c ) and rygbp ( d f ) subjects . fasting glp-1 plasma levels did not differ between or within the two study groups in both experimental conditions ( table 2 ) . as expected , glp-1 response to slm was larger in the rygbp group as compared with the control group on the saline day ( p < 0.001 ) ( fig . exendin-(939 ) resulted in a significant increase in the glp-1 response to slm intake in the control group ( p = 0.005 ) but not in the rygbp group ( p = 0.358 ) . nonetheless , in that experimental condition the glp-1 response to the meal challenge also was larger in the rygbp group ( p = 0.001 ) . infusion resulted in an earlier gip response but no change in the auc0120 as compared with the saline condition . the early ( auc060 ) response of gip was larger ( p = 0.014 ) in the rygbp group as compared with the control group , but it was not changed on infusion of exendin-(939 ) . the effects of exendin-(939 ) on the glucose response to slm in subjects in the rygbp and control groups are shown in fig . 1 and table 2 . before exendin-(939 ) or saline infusion ( time 30 min ) , plasma glucose was not significantly different either within or between study groups . exendin-(939 ) infusion resulted in a slight , albeit significant , increase in plasma glucose before meal ingestion ( time 0 min ) in both groups ( rygbp : p = 0.001 ; control : p = 0.053 ) relative to before the initiation of the test . blood glucose response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a ) and rygbp ( b ) subjects effect of slm ingestion on plasma glucose and hormonal secretion in studies with or without exendin-(939 ) as shown in table 2 , infusion of exendin-(939 ) resulted in a significant enlargement of the auc of glucose after meal intake ( auc glucose0120 ) in both study groups ( rygbp : p = 0.013 ; control : p = 0.020 ) . a similar relative increase of the auc glucose0120 in the exendin-(939 ) day relative to the saline day was found when the two groups were compared ( rygbp : 9.94 8.4% ; control : 10.84 8.8% ; p = 0.884 ) . in the rygbp group , the 2-h plasma glucose after slm intake was larger after exendin-(939 ) infusion ( 116.5 22.3 mg / dl ) as compared with that in the saline study ( 78.4 15.1 mg / dl ; p < 0.001 ) . however , the 2-h plasma glucose was < 200 mg / dl in all study participants in the rygbp group . two subjects in the rygbp group presented 2-h plasma glucose between 140 and 200 mg / dl ( respectively , 141 mg / dl and 143 mg / dl ) . a distinct temporal pattern of the glucose response was found in the two study groups in each experimental condition . during saline studies , patients in the rygbp group showed an earlier ( p = 0.001 ) and higher glucose peak ( p = 0.001 ) as compared with the control group . as a result , the auc of glucose during the first hour after slm ingestion ( auc glucose060 ) was larger in the rygbp group ( rygbp : 10.28 1.41 mg dl min 10 ; control : 6.82 0.50 mg dl min 10 ; p < 0.001 ) . in contrast , plasma glucose levels were lower from 90 min onwards in the rygbp group as compared with controls ( fig . 1 ; p < 0.05 for the time points of 90 , 100 , 110 , and 120 min ) . nonetheless , no significant difference was found when the auc of glucose during the second hour after ingestion of the slm ( auc glucose60120 ) was compared between the two groups on the saline day ( rygbp : 6.59 1.57 mg dl min 10 ; control : 7.99 1.67 mg dl min 10 ; p = 0.120 ) . only one subject in the surgical group presented with plasma glucose levels < 60 mg / dl from 90 min onwards in the absence of symptoms on the day of saline infusion . the differences between the two study groups in the plasma glucose response to slm on glp-1 action blockade are illustrated in fig . 1 . as shown in table 2 , in the rygbp group exendin-(939 ) infusion did not result in a significant change in peak glucose ( p = 0.297 ) , time to peak glucose ( p = 0.104 ) , or the auc glucose060 ( 10.15 1.15 10 mg dl min ; p = 0.597 ) as compared with the saline study . peak glucose ( p = 0.090 ) and the auc glucose060 ( 8.02 0.92 mg dl min ; p = 0.056 ) were larger , albeit not significantly , after exendin-(939 ) as compared with saline infusion in the control group . throughout the second hour after the slm , exendin-(939 ) infusion resulted in higher plasma glucose levels ( p < 0.05 for the time points of 90 , 100 , 110 , and 120 min ) and larger auc glucose60120 in the rygbp group ( 8.42 1.62 10 mg dl min ; p = 0.003 ) but not in the control group as compared with the saline day . as shown in table 2 , fasting plasma insulin did not differ between the two study groups or within each study group before the initiation of the tests performed in the two experimental conditions . fasting c - peptide levels did not differ between the saline and exendin-(939 ) studies within each group ( rygbp group : p = 0.593 ; control group : p = 0.868 ) , although the surgical group presented a significantly higher fasting c - peptide as compared with the control group on both study days ( table 2 ) . during fasting , glp-1 action blockade had no effect on insulin or c - peptide plasma levels neither in the rygbp group ( insulin : p = 0.582 ; c - peptide : p = 0.130 ) nor in the control group ( insulin : p = 0.963 ; c - peptide : p = 0.609 ) . in the saline condition , and despite comparable homa - ir ( table 1 ) , the insulin ( auc insulin0120 , p = 0.001 ) and c - peptide ( auc c - peptide0120 , p = 0.001 ) responses ensuing after slm ingestion were larger in the rygbp group . corresponding to the glucose curve , differences in the insulin response between the two study groups were accounted for differences during the first hour ( auc insulin060 : 11.68 4.33 mu l min 10 for rygbp compared with 2.11 1.57 mu l min 10 for control ; p < 0.001 ; auc c - peptide060 : 422.5 91.5 ng ml min for rygpb and 239.7 54.7 ng ml min for control ; p < 0.001 ) but not during the second hour ( auc insulin60120 : rygbp compared with controls , p = 0.205 ; auc c - peptide60120 : rygbp compared with controls , p = 0.055 ) after the slm challenge . in the rygbp group , exendin-(939 ) infusion resulted in blunted insulin response to slm ( fig . 2 and table 2 ) , with a mean reduction of the auc0120 of insulin and c - peptide of 52.1 10.9% and 24.1 8.7% , respectively . the insulin - calculated ( p = 0.009 ) and c - peptide calculated ( p = 0.017 ) insulinogenic indices were reduced in the rygbp group during exendin-(939 ) infusion . in the control group , exendin-(939 ) yielded no significant effect on insulin ( auc insulin0120 : 12.2 35.2% ) or c - peptide ( auc insulin0120 : 8.5 22.6% ) postprandial response ( fig . 2 and table 2 ) . insulin and c - peptide response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a , b ) and rygbp ( c , d ) subjects . as shown in table 2 , no differences were found in baseline plasma glucagon levels between the 2 study days in the rygbp group . unexpectedly , slightly but statistically elevated fasting glucagon levels were found in the control group before saline infusion as compared with previous to exendin-(939 ) infusion ( p = 0.001 ) . as shown in fig . 3 , in the saline infusion condition the slm challenge resulted in slight decrease in glucagon plasma concentration ( incremental auc glucagon0120 : 0.31 1.17 ( pg dl min 10 ) . this physiological response was blunted by exendin-(939 ) infusion ( incremental auc glucagon0120 : 0.99 1.52 pg dl min 10 ; p = 0.062 ) . in contrast , a paradoxical increase in the glucagon response was observed in the rygbp group during the saline and exendin-(939 ) conditions [ incremental auc glucagon0120 : 6.35 2.96 pg dl min 10 for saline and 7.34 3.60 pg dl min 10 for exendin-(939 ) ] . despite glucose tolerance being similar , the auc glucagon0120 was larger in the surgical group in both study days ( table 2 ) . glucagon , glp-1 , and gip response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a c ) and rygbp ( d f ) subjects . * p < 0.05 relative to the saline condition . fasting glp-1 plasma levels did not differ between or within the two study groups in both experimental conditions ( table 2 ) . as expected , glp-1 response to slm was larger in the rygbp group as compared with the control group on the saline day ( p < 0.001 ) ( fig . exendin-(939 ) resulted in a significant increase in the glp-1 response to slm intake in the control group ( p = 0.005 ) but not in the rygbp group ( p = 0.358 ) . nonetheless , in that experimental condition the glp-1 response to the meal challenge also was larger in the rygbp group ( p = 0.001 ) . exendin-(939 ) infusion resulted in an earlier gip response but no change in the auc0120 as compared with the saline condition . the early ( auc060 ) response of gip was larger ( p = 0.014 ) in the rygbp group as compared with the control group , but it was not changed on infusion of exendin-(939 ) . our data show that in the long - term after rygbp , blocking the action of glp-1 with exendin-(939 ) results in limited deterioration of the glucose response to a mixed meal in subjects with remission of t2 dm antedating surgery . thus , our data suggest that the dramatic increase in glp-1 secretion observed during the long - term after rygbp surgery is not the key determinant of the resolution of t2 dm after this type of surgery . previous studies implicating glp-1 as mechanism for the improvement in glucose tolerance after rygbp have relied on association . rygbp has been associated with a large glp-1 response to meal intake that parallels diabetes remission ( 7 ) , or that is larger than that observed after other surgical techniques ( 9 ) or dietary interventions ( 8) associated with smaller ameliorations of glucose tolerance . furthermore , it has been reported that per oral feeding versus feeding through a gastrostomy catheter inserted in the gastric remnant in rygbp - operated patients is associated with an exaggerated glp-1 response along with better glucose tolerance ( 24 ) . however , because association studies are not well suited to prove causality , to assess the degree to which increased glp-1 release in rygbp improved glucose tolerance we performed a mixed - meal tolerance test in the presence or absence of exendin-(939 ) . if antagonizing glp-1 receptors had been critical for the maintenance of a normal glucose tolerance in our rygbp - operated cohort , then meal ingestion would have resulted in plasma glucose levels in the diabetic range or , alternatively , in a much larger deterioration of the glucose response to slm as compared with normal - weight healthy controls . the 2-h plasma glucose after the slm in our cohort was much lower than the threshold of 200 mg / dl used for the diagnosis of diabetes , and only two out the eight rygbp patients presented with plasma glucose slightly higher than 140 mg / dl , which defines normal glucose tolerance . moreover , deterioration of glucose excursions after meal ingestion in the surgical group was comparable with that in controls . using hyperglycemic clamp combined with slm , salehi et al . ( 18 ) evaluated the effects of glp-1 action blockade on insulin secretion in a cohort of subjects who had undergone rygbp . at variance with our series , noteworthy , the glucose infusion rate after meal ingestion had to be decreased 13% with exendin-(939 ) infusion as compared with saline infusion to maintain the glucose clamped at the predefined target level . on the other hand , deane et al . ( 13 ) reported a 10% deterioration of glucose tolerance associated with exendin-(939 ) infusion after the ingestion of a solid meal in a series of eight healthy subjects . thus , the data reported herein concord with those in previous studies in humans using different experimental approaches . we acknowledge that by not controlling for changes in blood glucose , our study is not optimal to disentangle the relative contributions of glucose and glp-1 in the control of postprandial glycemia after rygb ( 25 ) . nonetheless , comparison of the glucose and hormonal responses between the control and surgical groups in our study strongly suggests that rygbp is associated with profound changes in the mechanisms involved in the control of postprandial glucose after the ingestion of the slm . under normal physiological conditions , insulin secretion , glucagon secretion , and gastric emptying are considered important in the control of glycemic excursions after meal intake ( 15,26 ) . noteworthy , it has been demonstrated that in addition to stimulating insulin release in a glucose - dependent manner , glp-1 action results in the inhibition of glucagon secretion and delayed gastric gastric emptying ( 27 ) . thus , the comparison of the glucose and hormonal responses between our two study groups in the absence or presence of exendin-(939 ) may help us illustrate differences in these mechanisms under normal living conditions . the earlier and higher peak glucose observed in the rygbp group as compared with controls during the saline infusion condition in our study is compatible with the previously demonstrated increased rate of exogenous glucose absorption into the systemic circulation after this type of surgery ( 28 ) . this finding most likely reflects a rapid emptying rate of the liquid meal from the gastric remnant after rygbp ( 29,30 ) . furthermore , the lack of an effect of exendin-(939 ) infusion on the time to peak glucose and the peak glucose concentration after meal intake despite profound effects on the insulin response further supports the importance of the rate of glucose absorption as determinant of plasma glucose concentration in the early period after the ingestion of a liquid meal in rygbp operated subjects ( 28 ) . in contrast , exendin-(939 ) infusion resulted in a tendency toward larger peak glucose and larger early glucose curve in our control subjects consistent with accelerated gastric emptying as a result of the absence of the physiological glp-1 action on gastric motility . the relevance of gastric emptying in the control of postprandial glucose excursions in healthy subjects recently has been challenged ( 15 ) . however , pharmacological deceleration of gastric emptying has been associated with smaller postprandial glucose excursions in the presence of decreased insulin secretion ( 31 ) . conversely , studies have shown that blockade of glp-1 action in healthy volunteers is associated with parallel acceleration of gastric emptying and larger glucose excursions occurring during the first 60 min after meal ingestion ( 13,16 ) . data on the insulin response after the slm challenge in our study support the relevance of glp-1 as determinant of insulin secretion after rygbp ( 3,18 ) . as previously shown , meal ingestion resulted in profound -cell stimulation in the rygbp group as compared with the control group ( 22,28,32 ) . the enlarged insulin response during the saline condition could be accounted for by the discussed increased rate of glucose absorption and an enlarged secretion and potentiated insulinotropic action of glp-1 as compared with the nonsurgical controls ( 27,33 ) . nonetheless , the early blunting of the insulin response , despite no significant change in plasma glucose throughout the first 90 min after meal challenge , emphasizes glp-1 as determinant of the insulin response in the rygbp group . interestingly , the percent decreases in the auc insulin ( 52% ) and c - peptide ( 24% ) in our surgical group are similar to those reported by salehi et al . ( 18 ) ( insulin 51% ; c - peptide 32% ) in rygbp - operated subjects in an experimental setting consisting of a meal tolerance test combined with a hyperglycemic clamp . admittedly , the -cell function data reported herein refer to peripheral insulin concentration . thus , because insulin clearance was not taken into account , true insulin secretion rate could not be calculated ( 18 ) . ( 18 ) , the effect of exendin-(939 ) on the estimated insulin secretion rate ( 33% ) was similar to that derived from insulin and c - peptide concentrations . importantly , as discussed , the blunted insulin response secondary to glp-1 action blockade did not affect the early ( 060 min ) glucose response to meal intake . furthermore , despite profound inhibition , reduced insulin response resulted in limited deterioration of glucose tolerance at 120 min of meal ingestion . thus , it could be argued that the insulin response pattern observed in the rygbp subjects is a result of the deregulation of the normal delivery of nutrients into the gut ( 26,27 ) . the increased insulin response results in limited impact on the ensuing glucose tolerance and may rather result in increased risk of hypoglycemia late after meal ingestion ( 18,32 ) . at variance to our finding in the rygbp group , and as previously reported , we failed to find a significant change in the insulin response to meal intake in the control group on blockade of glp-1 action ( 15 ) . a lower contribution of enteral factors to insulin secretion in control subjects in comparison with rygbp - operated subjects the lack of a descent in the insulin response in the control group in our study could be accounted for , at least in part , by the increased plasma glucose concentration . the enlarged glucagon response in the exendin-(939 ) condition observed in our control group is concordant with the glucanostatic role of glp-1 ( 27 ) . in contrast , in the rygbp group , meal ingestion resulted in marked elevation of plasma glucagon , despite exaggerated glp-1 response and in the absence of hypoglycemia throughout the meal test . a paradoxical glucagon response after meal intake in rygbp patients previously has been reported both during the short - term and the long - term after surgery after nutrient challenge with different composition , and in subjects with or without a history of t2 dm preceding the surgical procedure ( 8,18,23,33,34 ) . it has been shown that rygbp surgery does not alter the inhibitory effects of hyperglycemia on the -cells ( 18 ) . alternatively , it has been proposed that changes in other gut hormones such as gip with glucanotropic action could be involved ( 23 ) . ( 15 ) recently have demonstrated that glp-1 inhibition of postprandial glucagon secretion is a significant contributor to normal glucose homeostasis after a mixed oral meal in healthy volunteers . thus , our data clearly show the correction of the hyperglucagonemia that has been shown to contribute importantly to diabetic hyperglycemia ( 35 ) is not the mechanism by which glucose intolerance is corrected after rygbp . first , a larger bmi in the surgical group could have detrimentally influenced glp-1 action as compared with lean nonsurgical controls ( 36 ) . however , it has been proposed that the negative impact of a larger bmi on the incretin effect is accounted for by impaired insulin sensitivity , and homa - ir was comparable between our study groups ( 37 ) . second , although current bariatric surgery series mainly comprise females , further studies are needed to extrapolate our findings to the male gender . third , our observation period of 120 min after meal intake resulted in glucose returning to baseline values in the rygbp group but not in the control group . however , previous studies have not shown further amplification of the effect of exendin-(939 ) on the glucose and insulin responses to meal intake beyond the 120-min observation period in healthy controls ( 13,15,16 ) . thus , we deem unlikely that prolonging our observation period to 180 min would have changed our results appreciably . although , it could be argued that exendin-(939 ) dosage was insufficient to induce a significant blockade of glp-1 action , it previously has been shown that exendin-(939 ) at lower doses ( 600 pmol kg min ) blocked the effect of supraphysiological doses of glp-1 on insulin and c - peptide response to a nutrient challenge by 92% and 86% ( 25 ) . noteworthy , because we elected to study subjects during the long - term after surgery , it could be argued that our data do not discard glp-1 as a significant contributor to the rapid amelioration of glucose tolerance ensuing after rygbp ( 35 ) . however , we elected our study period to avoid the confounding effects of marked energy restriction and weight loss occurring early after surgery . finally , it is worth mentioning that , as previously reported by others , exendin-(939 ) infusion resulted in increased plasma glucose concentration before the ingestion of the meal challenge ( 38 ) . furthermore , exendin-(939 ) infusion resulted in enlarged glp-1 response to a meal challenge in healthy controls . it has been suggested that basal levels of endogenous glp-1 , continuously secreted in the absence of nutrient ingestion , convey a physiologically important endocrine signal to islet glp-1 receptor ( 38 ) . however , effects of exendin-(939 ) beyond the blockade of glp-1 receptor can not be ruled out ( 39 ) . in summary , we have examined the effects of the blockade of glp-1 action on the glucose response to an slm in subjects with sustained remission of t2 dm present before rygbp . our data show the blockade of glp-1 receptor by means of exendin-(939 ) results in limited deterioration of the glucose response to meal intake . hence , our data suggest the resolution of t2 dm after rygbp may be explained by mechanisms beyond enhancement of glp-1 action .

objectiveglucagon like peptide-1 ( glp-1 ) has been suggested as a major factor for the improved glucose tolerance ensuing after roux - en - y gastric bypass ( rygbp ) surgery . we examined the effect of blocking endogenous glp-1 action on glucose tolerance in subjects with sustained remission of type 2 diabetes mellitus ( t2 dm ) present before rygbp.research design and methodsblood glucose , insulin , c - peptide , glucagon , glp-1 , and glucose - dependent insulinotropic peptide levels were measured after a meal challenge with either exendin-(939 ) ( a glp-1r antagonist ) or saline infusion in eight subjects with sustained remission of t2 dm after rygbp and seven healthy controls.resultsinfusion of exendin-(939 ) resulted in marginal deterioration of the 2-h plasma glucose after meal intake in rygbp subjects [ saline 78.4 15.1 mg / dl compared with exendin-(939 ) 116.5 22.3 mg / dl ; p < 0.001 ] . furthermore , glucose response to meal intake was similarly enlarged in the two study groups [ percent change in the area under the curve of glucose exendin-(939 ) infusion versus saline infusion : controls 10.84 8.8% versus rygbp 9.94 8.4% ; p = 0.884 ] . in the rygbp group , the blockade of the enlarged glp-1 response to meal intake resulted in reduced insulin ( p = 0.001 ) and c - peptide ( p < 0.001 ) , but no change in glucagon ( p = 0.258 ) responses.conclusionsthe limited deterioration of glucose tolerance on blockade of glp-1 action in our study suggests the resolution of t2 dm after rygbp may be explained by mechanisms beyond enhancement of glp-1 action .

RESEARCH DESIGN AND METHODS Subjects Experimental procedures Assays Statistical analysis RESULTS Glucose response to an SLM Insulin response to an SLM Glucagon, GLP-1, and GIP response to an SLM CONCLUSIONS

blood samples for the measurement of c - peptide , glucagon , glp-1 , and gip were obtained in chilled edta tubes containing 500 units of aprotinin per milliliter of blood . the area under the curve ( auc ) for glucose , insulin , c - peptide , glucagon , and total glp-1 after the ingestion of the slm were calculated using the trapezoidal method . the insulinogenic index was calculated as the increment of insulin ( mu / l ) or c - peptide ( ng / ml ) between time 0 and time 30 divided by the change in plasma glucose ( mg / dl ) in the same time frame . blood samples for the measurement of c - peptide , glucagon , glp-1 , and gip were obtained in chilled edta tubes containing 500 units of aprotinin per milliliter of blood . the area under the curve ( auc ) for glucose , insulin , c - peptide , glucagon , and total glp-1 after the ingestion of the slm were calculated using the trapezoidal method . the insulinogenic index was calculated as the increment of insulin ( mu / l ) or c - peptide ( ng / ml ) between time 0 and time 30 divided by the change in plasma glucose ( mg / dl ) in the same time frame . exendin-(939 ) infusion resulted in a slight , albeit significant , increase in plasma glucose before meal ingestion ( time 0 min ) in both groups ( rygbp : p = 0.001 ; control : p = 0.053 ) relative to before the initiation of the test . effect of slm ingestion on plasma glucose and hormonal secretion in studies with or without exendin-(939 ) as shown in table 2 , infusion of exendin-(939 ) resulted in a significant enlargement of the auc of glucose after meal intake ( auc glucose0120 ) in both study groups ( rygbp : p = 0.013 ; control : p = 0.020 ) . a similar relative increase of the auc glucose0120 in the exendin-(939 ) day relative to the saline day was found when the two groups were compared ( rygbp : 9.94 8.4% ; control : 10.84 8.8% ; p = 0.884 ) . in the rygbp group , the 2-h plasma glucose after slm intake was larger after exendin-(939 ) infusion ( 116.5 22.3 mg / dl ) as compared with that in the saline study ( 78.4 15.1 mg / dl ; p < 0.001 ) . however , the 2-h plasma glucose was < 200 mg / dl in all study participants in the rygbp group . during saline studies , patients in the rygbp group showed an earlier ( p = 0.001 ) and higher glucose peak ( p = 0.001 ) as compared with the control group . as a result , the auc of glucose during the first hour after slm ingestion ( auc glucose060 ) was larger in the rygbp group ( rygbp : 10.28 1.41 mg dl min 10 ; control : 6.82 0.50 mg dl min 10 ; p < 0.001 ) . as shown in table 2 , in the rygbp group exendin-(939 ) infusion did not result in a significant change in peak glucose ( p = 0.297 ) , time to peak glucose ( p = 0.104 ) , or the auc glucose060 ( 10.15 1.15 10 mg dl min ; p = 0.597 ) as compared with the saline study . peak glucose ( p = 0.090 ) and the auc glucose060 ( 8.02 0.92 mg dl min ; p = 0.056 ) were larger , albeit not significantly , after exendin-(939 ) as compared with saline infusion in the control group . throughout the second hour after the slm , exendin-(939 ) infusion resulted in higher plasma glucose levels ( p < 0.05 for the time points of 90 , 100 , 110 , and 120 min ) and larger auc glucose60120 in the rygbp group ( 8.42 1.62 10 mg dl min ; p = 0.003 ) but not in the control group as compared with the saline day . fasting c - peptide levels did not differ between the saline and exendin-(939 ) studies within each group ( rygbp group : p = 0.593 ; control group : p = 0.868 ) , although the surgical group presented a significantly higher fasting c - peptide as compared with the control group on both study days ( table 2 ) . during fasting , glp-1 action blockade had no effect on insulin or c - peptide plasma levels neither in the rygbp group ( insulin : p = 0.582 ; c - peptide : p = 0.130 ) nor in the control group ( insulin : p = 0.963 ; c - peptide : p = 0.609 ) . in the saline condition , and despite comparable homa - ir ( table 1 ) , the insulin ( auc insulin0120 , p = 0.001 ) and c - peptide ( auc c - peptide0120 , p = 0.001 ) responses ensuing after slm ingestion were larger in the rygbp group . corresponding to the glucose curve , differences in the insulin response between the two study groups were accounted for differences during the first hour ( auc insulin060 : 11.68 4.33 mu l min 10 for rygbp compared with 2.11 1.57 mu l min 10 for control ; p < 0.001 ; auc c - peptide060 : 422.5 91.5 ng ml min for rygpb and 239.7 54.7 ng ml min for control ; p < 0.001 ) but not during the second hour ( auc insulin60120 : rygbp compared with controls , p = 0.205 ; auc c - peptide60120 : rygbp compared with controls , p = 0.055 ) after the slm challenge . the insulin - calculated ( p = 0.009 ) and c - peptide calculated ( p = 0.017 ) insulinogenic indices were reduced in the rygbp group during exendin-(939 ) infusion . in the control group , exendin-(939 ) yielded no significant effect on insulin ( auc insulin0120 : 12.2 35.2% ) or c - peptide ( auc insulin0120 : 8.5 22.6% ) postprandial response ( fig . insulin and c - peptide response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a , b ) and rygbp ( c , d ) subjects . unexpectedly , slightly but statistically elevated fasting glucagon levels were found in the control group before saline infusion as compared with previous to exendin-(939 ) infusion ( p = 0.001 ) . glucagon , glp-1 , and gip response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a c ) and rygbp ( d f ) subjects . as expected , glp-1 response to slm was larger in the rygbp group as compared with the control group on the saline day ( p < 0.001 ) ( fig . exendin-(939 ) resulted in a significant increase in the glp-1 response to slm intake in the control group ( p = 0.005 ) but not in the rygbp group ( p = 0.358 ) . nonetheless , in that experimental condition the glp-1 response to the meal challenge also was larger in the rygbp group ( p = 0.001 ) . the early ( auc060 ) response of gip was larger ( p = 0.014 ) in the rygbp group as compared with the control group , but it was not changed on infusion of exendin-(939 ) . exendin-(939 ) infusion resulted in a slight , albeit significant , increase in plasma glucose before meal ingestion ( time 0 min ) in both groups ( rygbp : p = 0.001 ; control : p = 0.053 ) relative to before the initiation of the test . blood glucose response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a ) and rygbp ( b ) subjects effect of slm ingestion on plasma glucose and hormonal secretion in studies with or without exendin-(939 ) as shown in table 2 , infusion of exendin-(939 ) resulted in a significant enlargement of the auc of glucose after meal intake ( auc glucose0120 ) in both study groups ( rygbp : p = 0.013 ; control : p = 0.020 ) . a similar relative increase of the auc glucose0120 in the exendin-(939 ) day relative to the saline day was found when the two groups were compared ( rygbp : 9.94 8.4% ; control : 10.84 8.8% ; p = 0.884 ) . in the rygbp group , the 2-h plasma glucose after slm intake was larger after exendin-(939 ) infusion ( 116.5 22.3 mg / dl ) as compared with that in the saline study ( 78.4 15.1 mg / dl ; p < 0.001 ) . however , the 2-h plasma glucose was < 200 mg / dl in all study participants in the rygbp group . during saline studies , patients in the rygbp group showed an earlier ( p = 0.001 ) and higher glucose peak ( p = 0.001 ) as compared with the control group . as a result , the auc of glucose during the first hour after slm ingestion ( auc glucose060 ) was larger in the rygbp group ( rygbp : 10.28 1.41 mg dl min 10 ; control : 6.82 0.50 mg dl min 10 ; p < 0.001 ) . as shown in table 2 , in the rygbp group exendin-(939 ) infusion did not result in a significant change in peak glucose ( p = 0.297 ) , time to peak glucose ( p = 0.104 ) , or the auc glucose060 ( 10.15 1.15 10 mg dl min ; p = 0.597 ) as compared with the saline study . peak glucose ( p = 0.090 ) and the auc glucose060 ( 8.02 0.92 mg dl min ; p = 0.056 ) were larger , albeit not significantly , after exendin-(939 ) as compared with saline infusion in the control group . throughout the second hour after the slm , exendin-(939 ) infusion resulted in higher plasma glucose levels ( p < 0.05 for the time points of 90 , 100 , 110 , and 120 min ) and larger auc glucose60120 in the rygbp group ( 8.42 1.62 10 mg dl min ; p = 0.003 ) but not in the control group as compared with the saline day . fasting c - peptide levels did not differ between the saline and exendin-(939 ) studies within each group ( rygbp group : p = 0.593 ; control group : p = 0.868 ) , although the surgical group presented a significantly higher fasting c - peptide as compared with the control group on both study days ( table 2 ) . during fasting , glp-1 action blockade had no effect on insulin or c - peptide plasma levels neither in the rygbp group ( insulin : p = 0.582 ; c - peptide : p = 0.130 ) nor in the control group ( insulin : p = 0.963 ; c - peptide : p = 0.609 ) . in the saline condition , and despite comparable homa - ir ( table 1 ) , the insulin ( auc insulin0120 , p = 0.001 ) and c - peptide ( auc c - peptide0120 , p = 0.001 ) responses ensuing after slm ingestion were larger in the rygbp group . corresponding to the glucose curve , differences in the insulin response between the two study groups were accounted for differences during the first hour ( auc insulin060 : 11.68 4.33 mu l min 10 for rygbp compared with 2.11 1.57 mu l min 10 for control ; p < 0.001 ; auc c - peptide060 : 422.5 91.5 ng ml min for rygpb and 239.7 54.7 ng ml min for control ; p < 0.001 ) but not during the second hour ( auc insulin60120 : rygbp compared with controls , p = 0.205 ; auc c - peptide60120 : rygbp compared with controls , p = 0.055 ) after the slm challenge . the insulin - calculated ( p = 0.009 ) and c - peptide calculated ( p = 0.017 ) insulinogenic indices were reduced in the rygbp group during exendin-(939 ) infusion . in the control group , exendin-(939 ) yielded no significant effect on insulin ( auc insulin0120 : 12.2 35.2% ) or c - peptide ( auc insulin0120 : 8.5 22.6% ) postprandial response ( fig . insulin and c - peptide response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a , b ) and rygbp ( c , d ) subjects . unexpectedly , slightly but statistically elevated fasting glucagon levels were found in the control group before saline infusion as compared with previous to exendin-(939 ) infusion ( p = 0.001 ) . glucagon , glp-1 , and gip response to a standardized meal test with saline infusion ( open squares ) or exendin-(939 ) ( black squares ) in control ( a c ) and rygbp ( d f ) subjects . as expected , glp-1 response to slm was larger in the rygbp group as compared with the control group on the saline day ( p < 0.001 ) ( fig . exendin-(939 ) resulted in a significant increase in the glp-1 response to slm intake in the control group ( p = 0.005 ) but not in the rygbp group ( p = 0.358 ) . nonetheless , in that experimental condition the glp-1 response to the meal challenge also was larger in the rygbp group ( p = 0.001 ) . the early ( auc060 ) response of gip was larger ( p = 0.014 ) in the rygbp group as compared with the control group , but it was not changed on infusion of exendin-(939 ) . our data show that in the long - term after rygbp , blocking the action of glp-1 with exendin-(939 ) results in limited deterioration of the glucose response to a mixed meal in subjects with remission of t2 dm antedating surgery . rygbp has been associated with a large glp-1 response to meal intake that parallels diabetes remission ( 7 ) , or that is larger than that observed after other surgical techniques ( 9 ) or dietary interventions ( 8) associated with smaller ameliorations of glucose tolerance . if antagonizing glp-1 receptors had been critical for the maintenance of a normal glucose tolerance in our rygbp - operated cohort , then meal ingestion would have resulted in plasma glucose levels in the diabetic range or , alternatively , in a much larger deterioration of the glucose response to slm as compared with normal - weight healthy controls . the 2-h plasma glucose after the slm in our cohort was much lower than the threshold of 200 mg / dl used for the diagnosis of diabetes , and only two out the eight rygbp patients presented with plasma glucose slightly higher than 140 mg / dl , which defines normal glucose tolerance . furthermore , the lack of an effect of exendin-(939 ) infusion on the time to peak glucose and the peak glucose concentration after meal intake despite profound effects on the insulin response further supports the importance of the rate of glucose absorption as determinant of plasma glucose concentration in the early period after the ingestion of a liquid meal in rygbp operated subjects ( 28 ) . nonetheless , the early blunting of the insulin response , despite no significant change in plasma glucose throughout the first 90 min after meal challenge , emphasizes glp-1 as determinant of the insulin response in the rygbp group . ( 18 ) , the effect of exendin-(939 ) on the estimated insulin secretion rate ( 33% ) was similar to that derived from insulin and c - peptide concentrations . at variance to our finding in the rygbp group , and as previously reported , we failed to find a significant change in the insulin response to meal intake in the control group on blockade of glp-1 action ( 15 ) . a paradoxical glucagon response after meal intake in rygbp patients previously has been reported both during the short - term and the long - term after surgery after nutrient challenge with different composition , and in subjects with or without a history of t2 dm preceding the surgical procedure ( 8,18,23,33,34 ) . although , it could be argued that exendin-(939 ) dosage was insufficient to induce a significant blockade of glp-1 action , it previously has been shown that exendin-(939 ) at lower doses ( 600 pmol kg min ) blocked the effect of supraphysiological doses of glp-1 on insulin and c - peptide response to a nutrient challenge by 92% and 86% ( 25 ) . furthermore , exendin-(939 ) infusion resulted in enlarged glp-1 response to a meal challenge in healthy controls . in summary , we have examined the effects of the blockade of glp-1 action on the glucose response to an slm in subjects with sustained remission of t2 dm present before rygbp . our data show the blockade of glp-1 receptor by means of exendin-(939 ) results in limited deterioration of the glucose response to meal intake . hence , our data suggest the resolution of t2 dm after rygbp may be explained by mechanisms beyond enhancement of glp-1 action .

the development of oral ingestion in mammalian infants proceeds in a distinct and invariant sequence : suckling from a nipple is followed by a transition to independent feeding . conventional wisdom is that early ingestion ( suckling ) is dominated by reflexes and by centrally generated behavior patterns that give rise to a more complex , varied , and volitional form of ingestion ( feeding ) . put simply , our thesis is that in every mammal there are at least two , separable ingestion systems , namely , the suckling system and the feeding system . each component of this dual ingestion system can be differentiated by its evolutionary origin , because mammalian ingestion evolved twice . similarly , each component of this dual ingestion system can be differentiated developmentally because oral feeding develops twice in mammals . as such , proper and appropriate developmental care is aimed at protecting , supporting , and facilitating the development of two separable and specialized feeding systems the development of suckling and the development of independent feeding . to the parents , nurses , therapists , and physicians who care for prematurely born infants , indeed , infants younger than 32 weeks postconceptional age are often incapable of orally grasping a nipple and sucking , to say nothing of coordinating sucking with breathing and swallowing [ 14 ] . caregivers of infants born preterm then provide life - sustaining support while anticipating the best time to initiate oral ingestion ( suckling ) and then applying a variety of techniques to facilitate the further development of ingestion [ 57 ] so that the infant can be discharged home where continued development of feeding behavior will occur . in the present paper , we follow the terminological rules suggested by hall et al . for discussing the processes involved in milk transfer from mother to offspring . suckling refers to the behavior of the young that contribute to the receipt of milk from a nipple or teat . sucking consists of the oral motor movements that typically produce the intraoral pressures that help express milk from the nipple ( see [ 8 , 9 ] for a more complete discussion and definitions ) . in addition , feeding refers to the oral ingestion of nutritive substances other than milk , usually but not necessarily in solid or near - solid forms that typically are chewed . finally , ingestion is used to encompass all forms of oral intake and includes both suckling and feeding . we will briefly describe the evolution of lactation and mammalian ingestion and its relationship to suckling and feeding in the human . we will then summarize a set of developmental analyses from nonhuman research that compare a variety of perspectives on suckling and feeding behavior . finally , we will identify some implications for a research agenda and for improving outcomes in children who are born preterm . the vocabulary of geological timescales will be used to frame and discuss the evolution of mammals , the origins of their lactation , and the evolution of suckling offspring . we will take a rapid and abbreviated trip through geological time , sampling representative evidence that constitutes some of the fundamental understanding of mammalian origins . within the timeline of the story this is uncertain territory , but we can draw liberally from a remarkable synthesis of paleontological , physiological , histological , and comparative data , recently proffered by oftedal [ 10 , 11 ] and others that provides a coherent and exciting picture of when and how lactation may have emerged to become a unique , universal , and defining feature of mammalian life . table 1 shows some standard divisions [ 12 , 13 ] arranged chronologically from the oldest ( bottom ) to most recent ( top ) . at the base of table 1 is the cambrian period , over 500 million years ago ( mya ) , the period yielding the oldest known fossils of vertebrates . cambrian vertebrates were aquatic , eel - like creatures , reflected today in the jawless hagfish . these early forms proliferated and differentiated , giving rise to new types of aquatic life , such as the armoured fishes of the silurian and devonian periods . parental care was unknown , so the offspring emerged from the egg case ready to live and feed independently . by the carboniferous period ( 360 mya ) vertebrate life found its way from the aquatic medium into the air , some became amphibious , and others adopted fully terrestrial habits . locomotion and diet evolved along with many changes in skeletal structures , appendages , and skull structure . during the pennsylvanian , around 300 mya , there appeared a developmental innovation that proved to be a pivotal point in vertebrate evolution the amniote egg , distinguished by additional extraembryonic membranes as well as outer layers that improved gas exchange , utilization of nutrients , and water retention . such features made it possible for the amniote - producing tetrapods to move into previously untapped habitats and accomplish successful reproduction and development . the pennsylvanian fossil record contains a number of different amniotes that branched into separate lines , which differ by the presence and number of skull windows ( fenestrae ) . one of these amniotes led to the branches comprising the turtles and another to the diapsids that would diversify into today 's squamate species , which include all lizards and snakes . this is the line that would become the predecessor of all the mammals on earth . the importance for our discussion of the proliferation of different amniote lines is that it shows that mammals did not evolve from reptiles . rather , mammals and reptiles evolved from a common ancestry , the earliest amniotes , from which they diverged onto separate evolutionary paths more than 300 mya , denoted by the i. in figure 1 . at the demarcation of the amniota in the figure this is a noteworthy point because , as some readers will recall , early synapsids have been traditionally termed mammal - like reptiles ( [ 14 , 16 ] ) , a vestige of an earlier habit of calling all the early amniotes ( both synapsid and sauropsid ) reptiles . recognizing the evolutionary distinction between synapsid and sauropsid helps to clarify that neither mammals nor mammary glands evolved from a reptile - like ancestor with a scaly , nonglandular epidermis and calcified eggshells . with the clarity of retrospection , we can see the trajectory from early synapsids to mammals . beginning in the late pennsylvanian period , the synapsids underwent multiple evolutionary radiations and extinctions . ( figure 1 ) ; depicts some of the new forms that appeared and disappeared along the path from the early amniotes to the synapsida and then to the therapsida and cynodontia . these were eras of robust speciation for the synapsids , which became the dominant forms of animal life during the permian and triassic periods until the dinosaurs ascended to ruling status in the late triassic . cynodonts were among the enduring groups of synapsid ( figure 2 ) ; they became abundant during the triassic [ 17 , 18 ] . for example , respiratory turbinals , structures associated exclusively with conservation of respiratory water and the rapid respiratory rates needed to oxygenate endothermy , are found in the nasal cavity of cynodonts . cynodont fossils also present vascularized fibrolamellar bone , suggesting rapid rates of bone growth and remodeling . in addition , there is present in cynodont fossils a secondary hard palate that enables nasal breathing while holding prey in the mouth which is helpful , if not necessary for a predator . accompanying all these features are modifications of skull , jaw , and teeth that are consistent with handling the high levels of food intake needed to support elevated metabolic rates . together , such observations lead paleontologists to date the advent of mammals as around 200 mya . although true mammals appear to have existed during the jurassic and cretaceous periods , they were not widely distributed or abundant until the tertiary , after the catastrophic ending to the age of dinosaurs . the evolutionary path from basal synapsids to true mammals contains many transformative changes : anatomical additions , deletions , and reshaping . all of these changes reflected and contributed to modifications in physiology and function . among the most fundamental was the gradual evolution of endothermy , which was accomplished across the mid - permian to the triassic . related to the metabolic changes associated with endogenous heat production were changes in reproductive strategy , most notably the production of smaller eggs that were retained inside the mother 's body . longer periods of egg retention are associated with the eventual evolution of viviparity [ 10 , 14 , 17 , 19 , 20 ] . we have rapidly recited an evolutionary trajectory from the basal synapsids to the advent of mammals . during the 200 million years between the appearance of amniotic eggs and placental mammals , similarly , there began the oral ingestion of maternal secretions , and this behavior gradually evolved , probably in concert with the evolving maternal lactational system , to become suckling specializations that we see in contemporary mammals . in terms of origins , however , oftedal makes the exciting and nonintuitive assertion that lactation actually predated mammals ! as we will see , the origin of lactation is obscured by our knowledge of its eventual role as the nutritive foundation for mammalian newborns . we note that originally , vertebrate evolution was based on the production of independent offspring . the calcium - based structures that define the vertebrate body - spine , skeletal appendages for walking and running , thoracic rib arrangements to enable the rapid breathing needed to support sustained exercise , skulls for the evolving brain - also produced the jaws , and teeth that enabled dietary diversification . the dentition of early ancestors tells us that they were capable feeders as growing juveniles . we can see this illustrated in the basal synapsids whose teeth were replaced continuously [ 17 , 21 ] , suggesting that members of every age group could feed independently . indeed , such feeding independence was an imperative , for these were offspring which experienced no parental care , representative of many highly conserved and prevalent reproductive strategies that have persisted to the present . according to oftedal 's analysis and synthesis [ 10 , 11 ] , mammary gland secretions first evolved in synapsids to provide moisture to their eggs . that is , lactation arose as a means of protecting against dessication rather than providing nutrition . oftedal buttresses this novel hypothesis with a thorough analysis of the physiological characteristics of parchment shell eggs , the type most agree was produced by the synapsids and which are still produced by contemporary monotremes . oftedal 's provocative suggestion would place the evolution of lactation in the permian , some 300 mya . of course , the hydration of eggs through mammary secretion is not lactation as we know it today , but it is a beginning . oftedal [ 10 , 11 ] argues persuasively that lactation began well before the appearance of species we would classify as mammals . he traces the origins of lactation to reproductive strategies used in the triassic and jurassic , focusing on some of the mammaliaformes of that time . these were small insectivores which came in a range of sizes from about 3 g ( comparable to contemporary shrews ) to 25 g ( mice ) to 500 g ( large rats ) . their skeletal structures suggests that they were agile climbers ( an advantage in a world dominated by dinosaurs ) and probably nocturnal and elusive in their habits . these suggestions link with more specific observations supporting the idea that these early mammaliaformes were endothermic . halos of dense fur have been found surrounding fossils of small eutherian mammals from the early cretaceous . this is a potentially vital link to the origins of lactation , for there are a variety of proposed pathways that link endothermy to both parental behavior and lactation . the presence of epipubic bones in these small mammaliaformes is used as evidence for maternal care of immature offspring . in addition to their role in locomotion , the sexually dimorphic epipubic bones can provide support to developing young in a marsupial pouch or to suckling young in pouchless marsupials . epipubic bones are ancient appearing in some cynodonts and in many mesozoic mammaliaformes [ 22 , 2426 ] . oftedal projects a scenario for these small , insectivorous mammaliaformes in which the mammary patch secretions used for egg hydration became the basis of a nutrient medium for hatchlings ; lactose was probably contributed by apocrine - like glands , some of which may been present in early synapsids . by the late triassic , cynodonts were likely secreting a nutrient - rich milk , enabling a decline in egg size and an increase in altriciality of the young at hatching . there are a host of details that support this compelling facet of evolution , but they are not vital to our discussion here . one of the central themes , however , is key : birth of immature offspring begins to develop in these early premammalian forms . dentition is again relevant , as it was earlier when we discussed the production of independent offspring . collectively , the fossils of these small , presumably dependent offspring tell us that they were diphyodonts animals with two successive sets of teeth , the first the demands of diphyodonty include a period of dependent feeding , as well as substantial acquisition of calcium , phosphorous , and other nutrients for skeletal growth prior to independent feeding ; therefore , lactation or some facultative form of nutrient provision is required . thus , with the evolution of small , immature offspring comes the addition of a new feeding relationship and a new mode of oral ingestion . it is noteworthy that the three taxonomic clades that comprise the existing mammals , that is , the monotremes ( platypus and echidnas ) , metatherians ( marsupials ) , and eutherians ( placental mammals ) , first appear in the fossil record in the cretaceous , but may have diverged earlier , in the jurassic ( figure 1 ) . it is generally accepted that oviparity ( egg - laying ) is ancestral to viviparity ( live - bearing ) . it has been estimated that among reptiles , viviparity has evolved independently from a prior period of oviparity more than 100 times and never has a case of the reverse been observed . evolutionarily , the progression seems to involve sequential increases in the duration of egg retention , until a condition is reached in which the egg essentially hatches internally , thus resulting in viviparity . clearly , we have drawn extensively on oftedal 's creative and scholarly efforts , for they provide a broad base of evidence for the early origins of lactation and suckling . they explore the idea of a development sequence in which the mammary gland evolved from simple skin glands that secreted mucuous containing a variety of antimicrobial molecules for the protection of damaged skin and only subsequently evolved nutritional components to nourish hatchlings or newborn mammals . for our purposes , the validity of oftedal 's early hydrational origins hypothesis versus that of vorbach et al in fact , the central ideas in these two innovative perspectives are not mutually exclusive and may prove to be jointly true . the essential insight , and the value of reflecting on the grand picture of mammalian evolution , is to recognize that oral ingestion evolved at least twice . we have considered many tens of millions of years of evolutionary history during which the many and various forms of animal life produced and reproduced offspring that hatched from eggs ( fertilized externally or internally ) into a world in which parental care was nonexistent and from which they had to feed independently . thus , the sensory apparatus , motor systems , digestive capabilities , and nervous systems of all these juvenile forms were such that they were capable of immediate independent feeding . there is ample evidence of such life forms today , all descendants of ancestors common to the taxa that subsequently evolved altriciality and parental care . thus , brain - body systems for independent feeding is the ancestral state . lactation and other parental secretions that can provision the young evolved subsequently . it was accompanied , probably facilitated by coevolutionary pressures on altriciality of offspring as well as novel adaptations in the offspring . undoubtedly , some of the features that were or became specializations for suckling can be traced directly to adult adaptations , such as the bony secondary palate . this feature of certain synapsids , posited to have been selected as a facultative feature for predatory feeding , is an essential precondition for breathing while sucking . evolution , then , created and honed a constellation of specializations in increasingly altricial offspring that enabled exploitation of hydrational , nutritive , and immunological provisioning by the parents . these specializations were added to an existing system for feeding and they were added during relatively early phases of development , rather than tagged onto the end of an existing developmental sequence . in the language of heterochrony , the addition of a new feature at an early stage of development is termed pre - displacement , [ 29 , 30 ] and this would be the case for the addition of suckling to a preexisting system for independent feeding . the result , then , is the evolution of two feeding systems the ancestral feeding system and the specialized , limited , and more modern addition of a suckling system . in this way , oral feeding is not unitary and should be appreciated in terms of its evolution as having two separable heritages . to support and enrich the assertion that oral ingestion is not a unitary process , we turn to years of research , mostly from the field of developmental psychobiology . that discipline offers a broad body of well - controlled studies of the ontogeny of ingestive behavior , primarily focused on norway rat pups . we apply that literature here , treating the rat as a decent representative of mammalian development with perhaps special applicability to the development of other generalist , omnivorous feeders - including humans . rat development has proven value as a source of insight and understanding of human development , especially in key areas of sensory , motor , and physiological development ( e.g. , [ 3136 ] ) . we will make the case that suckling and feeding are separable systems by systematically comparing them . the interested reader may consult hall and williams ' seminal discussion in which they compare suckling and feeding in rats . suckling by infant rats and other nonhuman species is dependent on the sense of smell . teicher and blass washed the nipples and ventrum of anesthetized rat mothers with a fat soluble solvent or with water . pups presented to the ventrum of a dam washed with the fat soluble solution did not attach to any of her 12 nipples , whereas pups presented with a water - washed control dam readily attached . impressively , when the distillate of the wash fluids was applied to the dams ' nipples , nipple attachment and suckling were reinstated , thus making it clear that the critical olfactory cue(s ) had been removed and replaced and eliminating explanations such as masked odors or aversive residues . the dramatic finding that nipple attachment in the infant rat is wholly dependent on olfactory cues was further supported by the effects of anosmia on pups ' suckling and weight gain [ 3941 ] . whereas anosmia can eliminate suckling in rats in fact , pups made anosmic at a weanling age , when they will both suckle and feed independently , will fail to attach to nipple but will readily eat . ] asked a basic and blunt question , is suckling a neuromuscular precursor to chewing , or are suckling and chewing independent systems ? they used electromyogram ( emg ) recordings from superficial masseter , anterior digastric , sternohyoideus , and genioglossus muscles during suckling and chewing by rat pups at six ages , ranging from postnatal day ( pnd ) 6 to 21 . suckling behavior by rats consists of nipple attachment , rhythmic sucking , and the stretch response to milk letdown . chewing emgs were present by pnd 12 and are adult - like by pnd 1821 . emg patterns during nipple attachment and adult chewing were similar , but the other elements of sucking differed from chewing . thus , at the neuromuscular level , there are discontinuities between the sucking and feeding patterns as well as at least one emg continuity between the forms of ingestion . we can also look into the brain and examine the central circuits that correspond to the muscles that suck and chew , again noting the rhythmic components of both sucking and adult chewing . here the data come from guinea pig , a species born at a relatively well - formed stage of development . the infants both suckle and , within the first postnatal week , also chew solid food . the rhythmic patterns for guinea pig oral ingestion originate from central pattern generators in the frontal cortex of the brain [ 43 , 44 ] . it was shown that there exist at least two separate pattern generators , one for sucking and one for chewing ; during the transition from suckling to chewing , there is a corresponding structural and functional transition of active neurons . it is possible to dissociate the suckling and feeding systems in the developmental profiles of some of the digestive enzymes that comprise the postingestion physiology of the infant and weanling . the open circles in figure 2 illustrate levels of intestinal lactase in the suckling rat , which are high throughout the first two postnatal weeks ( when the rat is an obligate suckler ) and then begin a precipitous decline during weaning which , in this species , lasts until about pnd 30 [ 33 , 48 , 49 ] . in contrast , the figure 's triangles , broken line , and filled circles show levels of enzymes that help digest solid foods at different levels of the oro - gastrointestinal system , ) the coordinated changes in digestive enzymes in figure 2 beautifully illustrate the developmental dissociation of the suckling and feeding systems . there are a host of endogenous hormones and peptides that modulate food intake in weanling age or older rats , but do not exert similar effects on the suckling animals . for instance , exogenous insulin is known to increase food intake and weight gain in adults , but it did not have similar effects in suckling pups . likewise , a 7-day treatment regime of ghrelin was found to increase body weight of 7-week - old rats , but the same regime applied to suckling pups did not change their weights . these intriguing findings alone would be suspect because there may not have been adequate experimental measures to ensure that the maternal milk supply was sufficiently abundant to allow pups to increase their intake and accumulate additional body mass . it was also shown , however , that intraperitoneal or intraventricular administration of leptin ( a putative satiety factor ) decreases food intake in 28-day - old pups but does not affect suckling intake . similarly , cholecystokinin inhibits food intake in adults but does not alter suckling in 5- or 10-day - old pups . in a particularly broad and inventive study , hall and rosenblatt equipped rat pups of various ages with an oral cannula through which they could infuse controlled amounts of milk , thus by - passing the limits of maternal milk supply . cannulated pups attach to and suckle from the nipples of anesthetized dams , so this experimental preparation is highly versatile : the experimenter can provide measured amounts of milk directly into the infant 's mouth even if the pup is attached to a nipple . a small amount of milk infused into the mouth of a pup attached to a nipple elicits the age - typical behavioral responses to a natural letdown . pups readily ingested and reacted as though the dam had a milk letdown . using this preparation , it was shown that infant rats pnd 10 and younger do not inhibit intake when milk is delivered while pups are on - nipple . this is not an artifact of the cannulation preparation , however , because by pnd 15 , pups show satiety responses to the same type of milk transfer . friedman concluded that under natural conditions with an awake , active lactating dam , it is maternal milk supply that limits the pups ' intake . preloads of milk or other forms of calories did not reduce milk intake by 10-day - old pups . interestingly , friedman reported that preloading the pups with water did reduce their intake which pointed his investigation toward hydrational cues , rather than nutritive controls of early suckling . rat pups at pnd 10 did not alter their suckling to an osmotic challenge , but they did increase their milk intake in response to hypovolemia induced by a subcutaneous injection of polyethylene glycol , a hyperoncotic colloid . our thesis is further buttressed by contrasting results on suckling and feeding obtained with pharmacological manipulations . adult rats and humans show dose - related decreases in hunger and food intake to amphetamine . in rat pups , however , amphetamine does not decrease intake [ 52 , 57 ] . suckling rat pups given amphetamine and presented with an anesthetized dam actually attached to nipples more readily and suckled more vigorously than did controls [ 37 , 57 ] . milk intake was not part of this study because the anesthetized dam did not deliver milk , but it was striking to see a drug that dramatically diminishes an adult 's interest in food actually augment the appetitive elements of suckling pups . there is an additional series of studies that provides data suggesting differential controls on suckling and feeding by the neurotransmitter serotonin ( 5-hydroxytryptamine , or 5-ht ) . drugs that block 5-ht receptors , such as methysergide , stimulate weanling age pups to suckle . that is , they increase sucking in pups that are developmentally disinclined to attach to nipples and suck . an exciting observation in relation to our interest in dissociation of dual ingestion systems is that 5-ht antagonists cause weanling pups to prefer to suckle rather than to eat food . furthermore , the more age - typical preference for feeding can be reinstated by treatment with a 5-ht agonist . there are remarkably few data on the development of brain mechanisms for suckling and feeding . historically , much attention was directed at hypothalamic control of hunger and satiety and the involvement of ascending pathways in and around the medial forebrain bundle , but such studies have largely yielded to investigations of neurotransmitters and systemic factors as noted earlier in our review . electrolytic lesions in a variety of ingestion - related brain regions have generally had similar - appearing disruptive effects on both sucking and feeding , but such work has not been pursued developmentally in great detail . other forays into questions of central mechanisms for suckling versus feeding are similarly incomplete but intriguing . bignall and schramm reported a developmental study of ingestion by mesencephalic animals , kittens decerebrated within one week of birth and followed up to two months of age . they reported that the abolishment of suckling behavior was the only discernible immediate deficit and that the ingestion of solid foods and lapping of milk developed at weaning age . there are many limitations to their study , including the small sample size ( n = 5 ) and the fact that the surgical transection was complete in only two of the animals . nevertheless , they reported that suckling was permanently abolished in all the animals , but by maintaining them on gavage feeding to weaning age , feeding behavior emerged on schedule . the open circles depict the initial presence of suckling , its maintenance through infancy , and then its gradual decline and disappearance . the closed circles depict the gradual emergence and establishment of feeding . typically , the two processes are synchronized ( as depicted in the figure ) , so much so that many reasonable and thoughtful observers see the synchrony simply as the continuous development of a single system of oral ingestion . in this context , then , it was stunning when pfister et al . raised rats among litters of pups that were periodically replaced so that the subject rat was always living with suckling age pups and the mother . these subjects weaned onto solid food , but continued ( even into sexual maturity ) to suckle along with their preweanling counterparts ! this dramatic ( and bizarre ) experiment tells us at least two things pertinent to the present thesis . first , the developmental dissociation of suckling dissolution from the emergence of feeding provides an important demonstration that suckling does not become feeding ; the odd social context provided by these experimenters kept suckling behavior intact while the pups ' feeding system separately emerged and matured . second , it is possible to manipulate separately the ontogeny of suckling and the ontogeny of feeding , adding another type of evidence that the systems are separable . the independence of suckling and feeding systems was echoed in a different type of experiment , the results of which strongly suggest that feeding in rats can develop without prior experience or practice of suckling behavior . hall devised a preparation whereby a cannula is installed in the stomach of a newborn rat so that it can be raised alone , typically in a container that is kept in a warm , moist environment where a milk formula is infused directly into the stomach and experimenters periodically stimulate urination and defecation . pup - in - a - cup technique has been used for a variety of preliminary investigations including questions about of the role of suckling experience as a precursor to feeding . pups raised in isolation from the mother and fed via the gastric cannula pnd 18 were then tested for their ability to feed . remarkably , when these suckling - deprived animals were given access to solid food on pnd 19 , the latency to begin eating was identical to normally reared pups . moreover , the suckling - deprived pups performed well and even equivalently to suckled pups with compensatory feeding after 24 hr food deprivation , with compensatory drinking after water deprivation , in response to dietary adulteration , and they demonstrated equivalent food motivation by learning to press a bar for food and water as rapidly as did normal rats . in contrast to the feeding system 's apparent independence from prior suckling experience , the suckling system appears to depend on stimulation and experience . after as little as two days of the artificial rearing regime , including the suspension of suckling experience , pups no longer display the ability or willingness to suckle . in this way , the psychobiology literature contains many examples and much knowledge of how both suckling and feeding are experience - sensitive and also how the two systems interact . here we chose to emphasize findings that elucidate the ways in which the systems are separable , for these aspects are relatively novel and can lead us to new approaches to solve old problems . one of the most rapid and enduring associations that rat pups can learn are conditioned taste aversions . such learning occurs when a distinct and novel taste is followed by a treatment that causes nausea or gastrointestinal malaise , typically induced by a dose of a nonlethal toxin such as lithium chloride ( licl ) . in one part of a series of experiments [ 51 , 64 , 65 ] , rat pups were equipped with the type of oral cannula described earlier that enabled precise amounts of flavored milk to be delivered by an experimenter . when milk containing a novel flavor was pulsed into a pup 's mouth and this flavor experience was paired with licl - induced illness , the pup would subsequently avoid ingesting a food ( powdered rat chow ) containing the illness - associated flavor . remarkably , if the pup was attached to a dam 's nipple when the same flavored milk was delivered to the same site of the tongue in the same amount , on the same schedule , and paired with licl - induced illness , the pup did not react in any way that indicated its memory of a taste aversion . a variety of control studies established that the key variable was whether the pup was in a suckling context . like the painful shock study described earlier , the suckling context redefines the pup 's learning contingencies and negative associations are not formed . this type of surprising finding has been discussed in a variety of ways [ 51 , 66 , 67 ] but for present purposes , it is a view of a learning mechanism that is functionally defined by whether it occurs in the context of suckling or of feeding . a neonatal rat pup , at pnd 3 , deprived of nutrition for nearly a day and placed alone in a warm , humid chamber with a puddle of milk or wet mash on the floor will , under such conditions , orally ingest milk from the puddle . it is argued that this remarkable phenomenon is a case of regulated , independent ingestion because the behavior ceases when the pup ingests about 5% of its body weight ; impressively , the amount the pup ingests is diminished by a preload of milk . recall that rat pups 10 days old and younger are demonstrably nonregulatory when they are tested on nipple . a popular interpretation of independent ingestion by an isolated 3-day - old infant rat is that it demonstrates that there exists a system for independent feeding in the neonatal brain that is normally inaccessible , further evidence of a dual ingestive system in a developing mammal . treating oral ingestion as an aspect of behavioral biology makes it obligatory to consider its the evolutionary and developmental aspects . within a biological framework , we expect the ontogeny of oral ingestion in humans to be consistent with that of other mammalian species , and that human infants also possess dual ingestive systems . the dual ingestive system thesis is novel , particularly in clinical domains , so it is understandable that there are few data such as those known for rodent development . nevertheless , it is possible to note several lines of evidence that are consistent with our assertion that dual ingestive systems are a conserved feature of mammalian development that pertains to all mammalian species , including humans . human infants , like the offspring of every mammal , begin postnatal life as monophagous organisms ( those that ingest only one substance ) , deriving all nutrition , water , and electrolytes from a single source mother 's milk . suckling is the singular mode of ingestion while in the developmental phase of monophagy . as in other newborn mammals , feeding , the ingestion of nonmilk foods including solids and boluses of soft substances , typically has a gradual onset after suckling is established . in human offspring , as in most mammalian young , suckling continues to be displayed even after the onset of feeding . in fact , the developmental duration of overlap of the two modes of ingestion can be considerable , depending on a variety of contextual variables , including food availability and cultural traditions . thus , human infants may suckle exclusively for six months and then both suckle and feed for another six months or more . a host of morphological changes and muscular developments underlie age - related modifications of the tongue , and oral - pharyngeal complex helps differentiate the movements that comprise sucking and chewing in human infants ( see for a useful review ) . similarly , the neural systems that mediate sucking ( and coordinate breathing and swallowing ) are separable from many of those that mediate feeding ( see ) . in such ways , as we have seen in analyses of nonhuman ingestive development , suckling does not transform into feeding ; both modes of ingestion are expressed separately within the same developmental phases . that the absolute duration during which the two behaviors exist in an infant can vary greatly also suggests that feeding develops independently of suckling in humans . understandably , experimental studies involving some of the more dramatic manipulations imposed on laboratory species are not conducted with humans . provide nonexperimental evidence that achieving competence with nipple feeding depends on the amount and timing of prior experience . such observations connect importantly to our general knowledge that suckling is a complete , adaptive , integrated behavior with its own functional integrity that is shaped by experience and context . although we emphasize that suckling and feeding are separable along many dimensions , the two behaviors are not separate . the prevalence of feeding disorders among children who sustained disrupted suckling experiences is an important reminder that we must pursue deeper understanding of the connections between suckling and feeding . menella and beauchamp 's studies ( e.g. , [ 7173 ] ) showing that flavor experiences during suckling or even in utero can affect feeding preferences in later life indicate that there are experiential lines of continuity between the two ingestive systems . lactation and suckling are unique to mammals and universal among them , thus squarely placing this aspect of human reproduction and development in a broad biological context and directing our attention to evolutionary history , for this is the source of insights into comparative questions . when we examine the evolution of lactation and oral ingestion we discover beautiful continuities , as we should with any feature shared by diverse species with common ancestors . the evolutionary story tells us that infant suckling is a new invention that followed the advent of live birth and immature offspring . our evolutionary ancestry is from externally delivered eggs which hatched we saw that suckling was subsequently invented and therefore added to the pre - existing systems for independent , oral ingestion . when we examined the development of oral ingestion in a living relative of ours a laboratory rodent we discovered a swath of evidence for two ingestive systems , one for sucking and one for feeding . the perspective on the evolution and development of oral ingestion , as outlined here , has value and validity for practitioners and researchers concerned with oral ingestion in human infants . the perspective we have introduced can increase and sharpen our awareness of the developmental structure of oral ingestion [ 13 , 7 ] . this alone can help define areas where knowledge is needed and help prioritize what knowledge we should apply . as such , we are better able to treat suckling as a complete behavior , not as practice for a subsequent behavior or as a precursor to feeding , but as a complex , ontogenetically complete behavior with an adaptive integrity to be attained and then followed by developmental dissolution . with the adoption of such a view , a new set of research questions can be articulated and take on a new importance . when suckling is seen as a developmental goal , the precursors of suckling become germane , especially to clinicians who work with premature infants whose suckling is undeveloped . for example , swallowing might now be viewed as an important behavioral precursor to the onset and development of suckling . we know that fetuses swallow and that the behavior serves a variety of immediate and prospective functions . how often , when , and how much do fetuses swallow ? it seems likely that an infant , prematurely thrust into an environment where nutrients are delivered to its stomach via nasogastric tube and pulmonary breathing is not only required but is assisted with forms of pressurized airflow , will adopt patterns of swallowing different than that of a same - aged fetus . we would probably serve the infant well to understand this in greater detail . within the new framework , chemical cues ( tastes and smells ) that contribute to state , activity , orientation , arousal , and calming [ 7577 ] and that can initiate digestive processes now have magnified potential . through the lens of the dual ingestive systems , we are compelled to ask if there is important information in knowledge of the tastes and odors of amniotic fluid and vernix and glandular secretions of mothers and fathers . can we use such odors to augment state regulation in the infant or to stimulate ingestion ? can we better see the natural contingencies for learning when a mother brings her baby to the breast , stimulating its senses and coaxing from it a series of new acts that are rewarded with external and internal sensations ? in the context of suckling as a complete developmental system , it is possible to envision how odors might be used in the nicu to recreate some of the key contingencies and associations and facilitate the preterm infant 's developmental course . our thesis provides a framework that can help order and organize experience into elements that can separately contribute to suckling and later to feeding . one kind of experience may have similar or different effects on either or both of the two kinds of ingestion . the experimental literature can show how to recognize and to differentiate among such kinds of effects . these are but a small sample of some of the practical implications and promises for improved practice that could evolve from the thesis we have introduced in this paper . there is need for further research , so that we might better help infants develop suckling as part of an integrated array of adaptive competencies that will enable discharge from nicu to home [ 3 , 4 , 6 , 7 ] . as with any new formulation , but only by adopting and applying these ideas and pursuing with research their value and utility will we be able to make the most of them and understand the extent to which and how clinical practice and , most importantly , the outcomes on babies and their families , can be improved .

traditionally , the development of oral feeding is viewed as a continuous , unitary process in which reflex - dominated sucking behavior gives rise to a more varied and volitional feeding behavior . in contrast , we consider the thesis that the infant develops two separable ingestive systems , one for suckling and one for feeding . first , we apply an evolutionary perspective , recognizing that suckling - feeding is a universal , mammalian developmental sequence . we find that in mammalian evolution , feeding systems in offspring were established prior to the evolution of lactation , and therefore suckling is a separable feature that was added to feeding . we next review an experimental literature that characterizes suckling and feeding as separable in terms of their topography , sensory controls , physiological controls , neural substrates , and experience - based development . together , these considerations constitute a view of dual ingestive systems . the thesis , then , is that suckling is not a simple precursor of feeding but is a complete behavior that emerges , forms , and then undergoes a dissolution that overlaps with the emergence of independent feeding . this thesis guides us to focus differently on the challenges of properly managing and facilitating oral ingestion in infants , especially those born preterm , prior to the developmental onset of suckling .

1. Introduction 2. The Evolutionary Context of Lactation and Suckling Young 3. Suckling and Feeding as Separable Systems 4. Do Human Infants Possess Dual Ingestion Systems? 5. How Does the Thesis of Dual Ingestion Systems Relate to the Development of Oral Feeding in the NICU?

the development of oral ingestion in mammalian infants proceeds in a distinct and invariant sequence : suckling from a nipple is followed by a transition to independent feeding . conventional wisdom is that early ingestion ( suckling ) is dominated by reflexes and by centrally generated behavior patterns that give rise to a more complex , varied , and volitional form of ingestion ( feeding ) . put simply , our thesis is that in every mammal there are at least two , separable ingestion systems , namely , the suckling system and the feeding system . as such , proper and appropriate developmental care is aimed at protecting , supporting , and facilitating the development of two separable and specialized feeding systems the development of suckling and the development of independent feeding . to the parents , nurses , therapists , and physicians who care for prematurely born infants , indeed , infants younger than 32 weeks postconceptional age are often incapable of orally grasping a nipple and sucking , to say nothing of coordinating sucking with breathing and swallowing [ 14 ] . caregivers of infants born preterm then provide life - sustaining support while anticipating the best time to initiate oral ingestion ( suckling ) and then applying a variety of techniques to facilitate the further development of ingestion [ 57 ] so that the infant can be discharged home where continued development of feeding behavior will occur . in addition , feeding refers to the oral ingestion of nutritive substances other than milk , usually but not necessarily in solid or near - solid forms that typically are chewed . finally , ingestion is used to encompass all forms of oral intake and includes both suckling and feeding . we will briefly describe the evolution of lactation and mammalian ingestion and its relationship to suckling and feeding in the human . we will then summarize a set of developmental analyses from nonhuman research that compare a variety of perspectives on suckling and feeding behavior . finally , we will identify some implications for a research agenda and for improving outcomes in children who are born preterm . the vocabulary of geological timescales will be used to frame and discuss the evolution of mammals , the origins of their lactation , and the evolution of suckling offspring . within the timeline of the story this is uncertain territory , but we can draw liberally from a remarkable synthesis of paleontological , physiological , histological , and comparative data , recently proffered by oftedal [ 10 , 11 ] and others that provides a coherent and exciting picture of when and how lactation may have emerged to become a unique , universal , and defining feature of mammalian life . locomotion and diet evolved along with many changes in skeletal structures , appendages , and skull structure . one of these amniotes led to the branches comprising the turtles and another to the diapsids that would diversify into today 's squamate species , which include all lizards and snakes . with the clarity of retrospection , we can see the trajectory from early synapsids to mammals . beginning in the late pennsylvanian period , the synapsids underwent multiple evolutionary radiations and extinctions . ( figure 1 ) ; depicts some of the new forms that appeared and disappeared along the path from the early amniotes to the synapsida and then to the therapsida and cynodontia . among the most fundamental was the gradual evolution of endothermy , which was accomplished across the mid - permian to the triassic . longer periods of egg retention are associated with the eventual evolution of viviparity [ 10 , 14 , 17 , 19 , 20 ] . we have rapidly recited an evolutionary trajectory from the basal synapsids to the advent of mammals . during the 200 million years between the appearance of amniotic eggs and placental mammals , similarly , there began the oral ingestion of maternal secretions , and this behavior gradually evolved , probably in concert with the evolving maternal lactational system , to become suckling specializations that we see in contemporary mammals . in terms of origins , however , oftedal makes the exciting and nonintuitive assertion that lactation actually predated mammals ! as we will see , the origin of lactation is obscured by our knowledge of its eventual role as the nutritive foundation for mammalian newborns . we note that originally , vertebrate evolution was based on the production of independent offspring . the calcium - based structures that define the vertebrate body - spine , skeletal appendages for walking and running , thoracic rib arrangements to enable the rapid breathing needed to support sustained exercise , skulls for the evolving brain - also produced the jaws , and teeth that enabled dietary diversification . oftedal 's provocative suggestion would place the evolution of lactation in the permian , some 300 mya . of course , the hydration of eggs through mammary secretion is not lactation as we know it today , but it is a beginning . this is a potentially vital link to the origins of lactation , for there are a variety of proposed pathways that link endothermy to both parental behavior and lactation . collectively , the fossils of these small , presumably dependent offspring tell us that they were diphyodonts animals with two successive sets of teeth , the first the demands of diphyodonty include a period of dependent feeding , as well as substantial acquisition of calcium , phosphorous , and other nutrients for skeletal growth prior to independent feeding ; therefore , lactation or some facultative form of nutrient provision is required . thus , with the evolution of small , immature offspring comes the addition of a new feeding relationship and a new mode of oral ingestion . it is noteworthy that the three taxonomic clades that comprise the existing mammals , that is , the monotremes ( platypus and echidnas ) , metatherians ( marsupials ) , and eutherians ( placental mammals ) , first appear in the fossil record in the cretaceous , but may have diverged earlier , in the jurassic ( figure 1 ) . evolutionarily , the progression seems to involve sequential increases in the duration of egg retention , until a condition is reached in which the egg essentially hatches internally , thus resulting in viviparity . clearly , we have drawn extensively on oftedal 's creative and scholarly efforts , for they provide a broad base of evidence for the early origins of lactation and suckling . the essential insight , and the value of reflecting on the grand picture of mammalian evolution , is to recognize that oral ingestion evolved at least twice . thus , the sensory apparatus , motor systems , digestive capabilities , and nervous systems of all these juvenile forms were such that they were capable of immediate independent feeding . there is ample evidence of such life forms today , all descendants of ancestors common to the taxa that subsequently evolved altriciality and parental care . thus , brain - body systems for independent feeding is the ancestral state . this feature of certain synapsids , posited to have been selected as a facultative feature for predatory feeding , is an essential precondition for breathing while sucking . evolution , then , created and honed a constellation of specializations in increasingly altricial offspring that enabled exploitation of hydrational , nutritive , and immunological provisioning by the parents . these specializations were added to an existing system for feeding and they were added during relatively early phases of development , rather than tagged onto the end of an existing developmental sequence . in the language of heterochrony , the addition of a new feature at an early stage of development is termed pre - displacement , [ 29 , 30 ] and this would be the case for the addition of suckling to a preexisting system for independent feeding . the result , then , is the evolution of two feeding systems the ancestral feeding system and the specialized , limited , and more modern addition of a suckling system . in this way , oral feeding is not unitary and should be appreciated in terms of its evolution as having two separable heritages . to support and enrich the assertion that oral ingestion is not a unitary process , we turn to years of research , mostly from the field of developmental psychobiology . we apply that literature here , treating the rat as a decent representative of mammalian development with perhaps special applicability to the development of other generalist , omnivorous feeders - including humans . rat development has proven value as a source of insight and understanding of human development , especially in key areas of sensory , motor , and physiological development ( e.g. we will make the case that suckling and feeding are separable systems by systematically comparing them . the interested reader may consult hall and williams ' seminal discussion in which they compare suckling and feeding in rats . pups presented to the ventrum of a dam washed with the fat soluble solution did not attach to any of her 12 nipples , whereas pups presented with a water - washed control dam readily attached . impressively , when the distillate of the wash fluids was applied to the dams ' nipples , nipple attachment and suckling were reinstated , thus making it clear that the critical olfactory cue(s ) had been removed and replaced and eliminating explanations such as masked odors or aversive residues . the dramatic finding that nipple attachment in the infant rat is wholly dependent on olfactory cues was further supported by the effects of anosmia on pups ' suckling and weight gain [ 3941 ] . asked a basic and blunt question , is suckling a neuromuscular precursor to chewing , or are suckling and chewing independent systems ? they used electromyogram ( emg ) recordings from superficial masseter , anterior digastric , sternohyoideus , and genioglossus muscles during suckling and chewing by rat pups at six ages , ranging from postnatal day ( pnd ) 6 to 21 . it was shown that there exist at least two separate pattern generators , one for sucking and one for chewing ; during the transition from suckling to chewing , there is a corresponding structural and functional transition of active neurons . it is possible to dissociate the suckling and feeding systems in the developmental profiles of some of the digestive enzymes that comprise the postingestion physiology of the infant and weanling . in contrast , the figure 's triangles , broken line , and filled circles show levels of enzymes that help digest solid foods at different levels of the oro - gastrointestinal system , ) the coordinated changes in digestive enzymes in figure 2 beautifully illustrate the developmental dissociation of the suckling and feeding systems . cannulated pups attach to and suckle from the nipples of anesthetized dams , so this experimental preparation is highly versatile : the experimenter can provide measured amounts of milk directly into the infant 's mouth even if the pup is attached to a nipple . this is not an artifact of the cannulation preparation , however , because by pnd 15 , pups show satiety responses to the same type of milk transfer . our thesis is further buttressed by contrasting results on suckling and feeding obtained with pharmacological manipulations . there is an additional series of studies that provides data suggesting differential controls on suckling and feeding by the neurotransmitter serotonin ( 5-hydroxytryptamine , or 5-ht ) . an exciting observation in relation to our interest in dissociation of dual ingestion systems is that 5-ht antagonists cause weanling pups to prefer to suckle rather than to eat food . furthermore , the more age - typical preference for feeding can be reinstated by treatment with a 5-ht agonist . there are remarkably few data on the development of brain mechanisms for suckling and feeding . they reported that the abolishment of suckling behavior was the only discernible immediate deficit and that the ingestion of solid foods and lapping of milk developed at weaning age . there are many limitations to their study , including the small sample size ( n = 5 ) and the fact that the surgical transection was complete in only two of the animals . nevertheless , they reported that suckling was permanently abolished in all the animals , but by maintaining them on gavage feeding to weaning age , feeding behavior emerged on schedule . the open circles depict the initial presence of suckling , its maintenance through infancy , and then its gradual decline and disappearance . typically , the two processes are synchronized ( as depicted in the figure ) , so much so that many reasonable and thoughtful observers see the synchrony simply as the continuous development of a single system of oral ingestion . in this context , then , it was stunning when pfister et al . first , the developmental dissociation of suckling dissolution from the emergence of feeding provides an important demonstration that suckling does not become feeding ; the odd social context provided by these experimenters kept suckling behavior intact while the pups ' feeding system separately emerged and matured . second , it is possible to manipulate separately the ontogeny of suckling and the ontogeny of feeding , adding another type of evidence that the systems are separable . the independence of suckling and feeding systems was echoed in a different type of experiment , the results of which strongly suggest that feeding in rats can develop without prior experience or practice of suckling behavior . pup - in - a - cup technique has been used for a variety of preliminary investigations including questions about of the role of suckling experience as a precursor to feeding . remarkably , when these suckling - deprived animals were given access to solid food on pnd 19 , the latency to begin eating was identical to normally reared pups . moreover , the suckling - deprived pups performed well and even equivalently to suckled pups with compensatory feeding after 24 hr food deprivation , with compensatory drinking after water deprivation , in response to dietary adulteration , and they demonstrated equivalent food motivation by learning to press a bar for food and water as rapidly as did normal rats . in contrast to the feeding system 's apparent independence from prior suckling experience , the suckling system appears to depend on stimulation and experience . in this way , the psychobiology literature contains many examples and much knowledge of how both suckling and feeding are experience - sensitive and also how the two systems interact . here we chose to emphasize findings that elucidate the ways in which the systems are separable , for these aspects are relatively novel and can lead us to new approaches to solve old problems . in one part of a series of experiments [ 51 , 64 , 65 ] , rat pups were equipped with the type of oral cannula described earlier that enabled precise amounts of flavored milk to be delivered by an experimenter . remarkably , if the pup was attached to a dam 's nipple when the same flavored milk was delivered to the same site of the tongue in the same amount , on the same schedule , and paired with licl - induced illness , the pup did not react in any way that indicated its memory of a taste aversion . this type of surprising finding has been discussed in a variety of ways [ 51 , 66 , 67 ] but for present purposes , it is a view of a learning mechanism that is functionally defined by whether it occurs in the context of suckling or of feeding . it is argued that this remarkable phenomenon is a case of regulated , independent ingestion because the behavior ceases when the pup ingests about 5% of its body weight ; impressively , the amount the pup ingests is diminished by a preload of milk . a popular interpretation of independent ingestion by an isolated 3-day - old infant rat is that it demonstrates that there exists a system for independent feeding in the neonatal brain that is normally inaccessible , further evidence of a dual ingestive system in a developing mammal . within a biological framework , we expect the ontogeny of oral ingestion in humans to be consistent with that of other mammalian species , and that human infants also possess dual ingestive systems . nevertheless , it is possible to note several lines of evidence that are consistent with our assertion that dual ingestive systems are a conserved feature of mammalian development that pertains to all mammalian species , including humans . human infants , like the offspring of every mammal , begin postnatal life as monophagous organisms ( those that ingest only one substance ) , deriving all nutrition , water , and electrolytes from a single source mother 's milk . suckling is the singular mode of ingestion while in the developmental phase of monophagy . as in other newborn mammals , feeding , the ingestion of nonmilk foods including solids and boluses of soft substances , typically has a gradual onset after suckling is established . in human offspring , as in most mammalian young , suckling continues to be displayed even after the onset of feeding . in fact , the developmental duration of overlap of the two modes of ingestion can be considerable , depending on a variety of contextual variables , including food availability and cultural traditions . that the absolute duration during which the two behaviors exist in an infant can vary greatly also suggests that feeding develops independently of suckling in humans . such observations connect importantly to our general knowledge that suckling is a complete , adaptive , integrated behavior with its own functional integrity that is shaped by experience and context . although we emphasize that suckling and feeding are separable along many dimensions , the two behaviors are not separate . the prevalence of feeding disorders among children who sustained disrupted suckling experiences is an important reminder that we must pursue deeper understanding of the connections between suckling and feeding . when we examine the evolution of lactation and oral ingestion we discover beautiful continuities , as we should with any feature shared by diverse species with common ancestors . the evolutionary story tells us that infant suckling is a new invention that followed the advent of live birth and immature offspring . our evolutionary ancestry is from externally delivered eggs which hatched we saw that suckling was subsequently invented and therefore added to the pre - existing systems for independent , oral ingestion . when we examined the development of oral ingestion in a living relative of ours a laboratory rodent we discovered a swath of evidence for two ingestive systems , one for sucking and one for feeding . the perspective on the evolution and development of oral ingestion , as outlined here , has value and validity for practitioners and researchers concerned with oral ingestion in human infants . the perspective we have introduced can increase and sharpen our awareness of the developmental structure of oral ingestion [ 13 , 7 ] . as such , we are better able to treat suckling as a complete behavior , not as practice for a subsequent behavior or as a precursor to feeding , but as a complex , ontogenetically complete behavior with an adaptive integrity to be attained and then followed by developmental dissolution . with the adoption of such a view , a new set of research questions can be articulated and take on a new importance . when suckling is seen as a developmental goal , the precursors of suckling become germane , especially to clinicians who work with premature infants whose suckling is undeveloped . for example , swallowing might now be viewed as an important behavioral precursor to the onset and development of suckling . it seems likely that an infant , prematurely thrust into an environment where nutrients are delivered to its stomach via nasogastric tube and pulmonary breathing is not only required but is assisted with forms of pressurized airflow , will adopt patterns of swallowing different than that of a same - aged fetus . through the lens of the dual ingestive systems , we are compelled to ask if there is important information in knowledge of the tastes and odors of amniotic fluid and vernix and glandular secretions of mothers and fathers . in the context of suckling as a complete developmental system , it is possible to envision how odors might be used in the nicu to recreate some of the key contingencies and associations and facilitate the preterm infant 's developmental course . our thesis provides a framework that can help order and organize experience into elements that can separately contribute to suckling and later to feeding . as with any new formulation , but only by adopting and applying these ideas and pursuing with research their value and utility will we be able to make the most of them and understand the extent to which and how clinical practice and , most importantly , the outcomes on babies and their families , can be improved .

aeromonas hydrophila is typically an opportunistic bacterial pathogen that is ubiquitous in freshwater environments and is responsible for diseases in different species , including amphibians , reptiles , fish , and mammals ( 1 ) . motile aeromonas septicemia ( mas ) caused by mesophilic a. hydrophila affects a wide variety of primarily freshwater fish species , including carp , tilapia , perch , catfish , and salmon ( 2 ) . epidemic disease outbreaks in fish caused by a. hydrophila , resulting in millions of dollars of lost revenue , have been reported worldwide but have not occurred in the united states until recently ( 3 , 4 ) . in the summer of 2009 , an outbreak of mas in commercially raised catfish caused by highly virulent a. hydrophila ( vah ) began in western alabama ( 5 ) . since the initial outbreak during the summer of 2009 , this outbreak has spread to other , adjacent states , including mississippi and arkansas ( 6 ) . to date , this epidemic of mas outbreaks is responsible for an estimated loss of more than $ 12 million in catfish aquaculture operations in the southeastern united states . our comparative and functional genomic analyses demonstrated that a. hydrophila isolates from recent epidemic outbreaks in the united states are highly clonal but genomically distinct from a. hydrophila historical isolates from diseased catfish in the united states , and these epidemic isolates contain a large number of genomic regions predicted to be acquired through lateral genetic transfer ( 7 ) . several episodes of epidemic outbreaks caused by a. hydrophila beginning in the late 1980s and within the last 10 years have been reported in farmed carp in china ( 8 , 9 ) . catfish and chinese carp epidemics caused by a. hydrophila , including the timing of the epidemics , which primarily occurred during the summer ; the extensive mortality observed in seemingly healthy fish ; and the highly clonal nature of the epidemic isolates ( 5 , 8) . nonnative species can endanger the native or endemic species by disturbing the trophic levels of the native fish community ( 10 ) and pose a potential threat of the emergence of infectious diseases ( 11 ) . because of the globalization of the aquaculture and ornamental fish trade industries , fish transport provides a gateway for the introduction of exotic species that could introduce new pathogens that can devastate populations of indigenous species ( 12 ) . for instance , spring viremia of carp ( svc ) , a viral disease historically affecting fish in europe , the middle east , and russia , has been found in carp in the united states since first being reported in 2002 ( 13 ) . the chinese origin of the svc viral isolates in the united states was thought to be mediated through importation of ornamental fish ( 14 ) . the emergence of several other viral diseases of fish and shrimp reported worldwide has been attributed to the dissemination of infected fish or eggs as a carrier of nonindigenous pathogens ( 15 ) . over the past several decades , asian carp have been imported into the united states , including grass carp ( ctenopharyngodon idella ) , bighead carp ( hypophthalmichthys nobilis ) , silver carp ( hypophthalmichthys molitrix ) , and black carp ( mylopharyngodon piceus ) , as well as countless species of live ornamental fish sold in the united states . grass carp were imported to the united states from east asia beginning in 1963 by the u.s . fish and wildlife service ( 16 ) and have been extensively used in polyculture of catfish and in reservoirs for aquatic weed control . as a result of the great flood of 1993 , silver carp were released in large numbers into the mississippi river drainage system and currently threaten the ecological balance of the u.s . the origin of the clonal a. hydrophila isolates responsible for the ongoing epidemic mas outbreak in united states - farmed channel catfish is unknown . we used a phylogenomic approach to study the molecular epidemiology of the bacterial isolates responsible for this epidemic outbreak . catfish and chinese carp isolates have a recent common ancestor . to determine the evolutionary relationships of the recent virulent a. hydrophila ( vah ) isolates , a gyrb - based phylogenetic analysis was conducted using a total of 264 aeromonas strains downloaded from the aeromonas multilocus sequence typing ( mlst ) database ( http://www.pubmlst.org/aeromonas ) ( 18 ) and including other strains of u.s . and non - u.s . the a. hydrophila phylogenetic analysis revealed a coherent and well - supported clade that included all vah strains ( see fig . interestingly , the only strain retrieved from the genbank database that was affiliated with vah strains was a. hydrophila strain zc1 , which was isolated from a diseased grass carp in guangdong province , china , from ponds that had experienced an epidemic outbreak of hemorrhagic septicemia ( 19 ) . in order to identify any other isolates that were affiliated with epidemic strains , we screened a. hydrophila strain collections available in the united states that were isolated from fish and other hosts and identified one additional a. hydrophila strain , s04 - 690 , which was affiliated with vah strains and strain zc1 ( see fig . strain s04 - 690 was isolated in 2004 from a diseased catfish obtained from a commercial aquaculture pond located in washington county , mississippi , which experienced a single event of mas outbreak that killed thousands of catfish but importantly did not result in a widespread epidemic outbreak in the surrounding regions or subsequent outbreaks in following years within the affected farm . since our gyrb - based phylogeny suggests that chinese carp isolate zc1 is highly similar to vah isolates and a 2004 mississippi isolate of catfish origin , we evaluated the evolutionary relationships of additional chinese carp isolates of epidemic origin in hubei province , china ( see table s1 in the supplemental material ) ( 4 ) . the gyrb phylogeny constructed using the neighbor - joining method revealed that all of the chinese carp epidemic isolates , including isolate zc1 , consistently group together as a single clade along with the recent u.s . epidemic isolates of catfish origin that included mississippi isolate s04 - 690 ( see fig . in contrast , we found that a. hydrophila isolates of nonepidemic origin are widely spread through different clades ( see fig . catfish ) formed a monophyletic clade with 100% sequence identities and with 100% bootstrap support for the vah - associated lineage . this suggests that a clonal a. hydrophila complex descended from a common ancestor responsible for epidemic outbreaks of fish disease in china and the united states . to provide a more refined phylogenetic placement of the u.s a. hydrophila isolates , we used a multilocus sequence - based phylogeny that included 3,751 bp of concatenated nucleotide sequences from six housekeeping genes : gyrase subunits a and b ( gyra and gyrb ) , rna polymerase sigma factor ( rpod ) , bacterial dna recombination protein ( reca ) , heat shock protein ( dnaj ) , and dna polymerase iii tau and gamma subunits ( dnax ) ( see table s2 in the supplemental material ) . our analysis included a. hydrophila vah catfish isolates , mississippi catfish isolate s04 - 690 , chinese carp isolate zc1 ( 19 ) , and nonepidemic aeromonas isolates of u.s . origin ( see table s1 ) , yielding a phylogenetic tree with strong bootstrap support for the terminal nodes ( fig . 1 ) . this multilocus phylogeny demonstrated that the chinese carp isolate zc1 clustered together with the alabama and mississippi catfish isolates as a monophyletic clade ( fig . 1 ) ; in contrast , the nonepidemic a. hydrophila isolates are highly divergent ( fig . 1 ) . in addition to this multilocus sequence - based phylogeny , we searched the aeromonas pubmlst database ( http://www.pubmlst.org/aeromonas ) ( 18 ) using gyrb , grol , glta , metg , ppsa , and reca gene - specific sequences of isolates ml09 - 119 , s04 - 690 , and zc1 , and we found that these three isolates belong to sequence type ( st ) st251 , which until this study contained only aeromonas hydrophila isolate xs91 - 4 - 1 . interestingly , this highly virulent chinese isolate , aeromonas hydrophila xs91 - 4 - 1 , was obtained from a diseased silver loweye carp from an epidemic outbreak in 1991 ( 20 ) . taken together , these findings demonstrate that chinese carp isolates xs91 - 4 - 1 and zc1 along with u.s . we also constructed a phylogenetic tree using the neighbor - joining method that demonstrates that u.s . catfish isolates ml09 - 119 and s04 - 690 and chinese carp isolates xs91 - 4 - 1 and zc1 cluster together in a monophyletic clade ( data not shown ) . the internal color ranges refer to the geographical origin of the isolates , while the external ring indicates the biological source . our multilocus phylogeny based on two different sets of housekeeping genes as well as mlst typing is concordant with the gyrb - based phylogeny that demonstrated that the chinese carp and u.s . catfish isolates cluster together as a clonal group and share identical alleles of all 10 housekeeping genes . therefore , these findings strongly support the hypothesis that the recent a. hydrophila isolates responsible for catfish epidemics in the united states emerged due to the rapid spread of a clonal group of pathogenic isolates that share an ancestor with a. hydrophila isolates identified from diseased carp from asia . these data , while supporting the close phylogenetic affiliation of u.s . catfish and asian carp isolates , do not provide any indication of the relative timing of the evolutionary changes that have occurred in these a. hydrophila populations , nor did these analyses indicate the relative virulence of these strains within carp or catfish . channel catfish and grass carp were experimentally challenged to compare the virulence of strains ml09 - 119 , zc1 , and al06 - 06 , with survival rates ranging from 0.02 0.04 to 0.98 0.04 . a. hydrophila isolate al06 - 06 was included in the challenge experiment in this study as a reference strain that is typical of a. hydrophila strains that have been historically isolated from stressed fish prior to the advent of the mas epidemic and has shown reduced mortality ( 20% ) in channel catfish relative to that observed from ml09 - 119 ( 7 ) . in this study , we observed that isolate ml09 - 119 was significantly more virulent than either zc1 or al06 - 06 and that zc1 was more virulent than al06 - 06 ( p < 0.0001 ) ( fig . additionally , overall comparisons between fish species show that channel catfish were more susceptible to a. hydrophila than were grass carp ( p = 0.0126 ) ; however , this observation occurred only for vah strain ml09 - 119 ( fig . 2 ) . these data suggest that ml09 - 119 has evolved increased virulence and that channel catfish appear to be more susceptible . percent survival of channel catfish or grass carp after challenge with a. hydrophila strain ml09 - 119 , zc1 , or al06 - 06 . significant differences in survival were observed for bacterial strains ml09 - 119 , zc1 , and al06 - 06 ( p < 0.0001 ) and between fish species in the ml09 - 119 group ( p = 0.0126 ) . no interaction effect between variables of bacterial strains and fish species was observed ( pfish species bacterial strains = 0.1002 ) . the difference in survival between channel catfish and grass carp injected with ml09 - 119 was significant ( p = 0.0069 ) . no significant differences in survival between channel catfish and grass carp intraperitoneally injected with zc1 or al06 - 06 were observed , but there was a significant difference between strains zc1 and al06 - 06 in both channel catfish ( p = 0.0075 ) and grass carp ( p = 0.0089 ) . one of the phenotypic traits common among all vah strains is the ability to utilize myo - inositol as a sole carbon source ( 7 ) . we therefore tested the chinese carp isolate zc1 and the mississippi catfish isolate s04 - 690 for this phenotype and found that these strains could also utilize myo - inositol as a sole carbon source and had growth curves similar to those observed for vah strains , whereas isolate al06 - 06 , used as a negative control , did not utilize myo - inositol as a sole carbon source ( data not shown ) . the comparison of the 17.5-kb myo - inositol utilization cluster of vah isolates with that of chinese carp isolate zc1 ( accession no . previously , we observed a consistent correlation between the presence of an epidemic - specific genetic marker and myo - inositol utilization capacity of all tested vah strains ( 7 ) . to our knowledge , the utilization of myo - inositol as a sole carbon source has not been reported for any other a. hydrophila isolates obtained from diseased fish or any other environmental or clinical origin in the united states ( 1 ) . however , myo - inositol - utilizing aeromonas isolates are prevalent in east and southeast asia ( 21 ) . this is further evidence in support of an asian origin for the epidemic a. hydrophila strains in the united states . catfish isolates made it impossible to differentiate them based on housekeeping gene divergence . to systematically investigate the emergence of the recent vah isolates , we sequenced the genomes of the chinese carp isolate zc1 and the mississippi catfish isolate s04 - 690 . the genomic comparison of both of these isolates with the highly virulent recent vah isolates and other nonepidemic disease isolates was conducted to understand the genomic changes among these strains and provide finer resolution for the phylogenetic relationships among these strains . reference mappings of the illumina sequence reads from the genomes of chinese carp isolate zc1 and mississippi catfish isolate s04 - 690 were conducted against the 55 genomic regions unique among vah strains ( 7 ) , demonstrating that the genomes of chinese carp isolate zc1 and mississippi catfish isolate s04 - 690 share 53 and 54 of these 55 epidemic - associated regions , respectively ( fig . these 55 epidemic - associated genomic regions ( 336,469 bp in total ) , present within the genome of all sequenced vah isolates , are comprised of prophage elements , pathogenicity islands , metabolic islands , fitness islands , and genes of unknown functions ( 7 ) . these epidemic - associated unique regions are considered horizontally acquired regions of the a. hydrophila genome since they were obtained by excluding core genomes of the 11 a. hydrophila isolates described previously ( 7 ) . though it has not been demonstrated experimentally , the analysis of the open reading frames ( orfs ) present within these unique regions suggests their potential roles in pathogenesis ( 7 ) . the c18r2 ( 11,731 bp ) and c26r1 ( 20,780 bp ) epidemic - associated unique regions of a recent vah isolate are absent in chinese isolate zc1 ( fig . the c26r1 region , the same region absent in zc1 , is the only epidemic - associated unique region of a recent vah isolate absent in the genome of mississippi isolate s04 - 690 ( fig . , we reported that epidemic - associated unique regions c18r2 and c26r1 contained two different genomic islands ( gis ) , namely , gi6 and gi12 , respectively ( 7 ) , which suggested their possible acquisition through horizontal gene transfer . the comparison of genomic islands present within the genomes of chinese carp and u.s . catfish isolates demonstrated that most of the genomic islands , except for gi5 , gi6 , and gi12 , which are found consistently within the genomes of vah isolates are also present within the genome of strain zc1 ( fig . we also observed that the majority of the genomic islands , except for gi12 , were present in mississippi isolate s04 - 690 ( fig . these shared genomic islands with identical synteny and dna sequences of high similarity are located in the same positions within the genomes of zc1 , s04 - 690 , and ml09 - 119 isolates ( data not shown ) . no additional gis were predicted within the genome of s04 - 690 , whereas 4 additional gis were predicted within the genome of zc1 ( data not shown ) . our analysis of gi12 , a vah - specific gi absent within the genomes of zc1 and s04 - 690 , predicts the presence of a fitness island with a large number of orfs involved in dna modification functions ( 7 ) . gi5 and gi6 are observed in vah isolates ( 7 ) but absent in zc1 and are predicted to encode 15 and 8 proteins of unknown functions , respectively . since genomic islands of bacterial species are highly variable and frequently used to distinguish strains due to their acquisition through horizontal gene transfer ( 22 ) , the sharing of a large number of identical genomic islands has led to the hypothesis that u.s . vah isolates originated from asia and that the mississippi isolate s04 - 690 represents an intermediate strain between the asian carp and alabama catfish isolates . the commonality of a large number of genomic islands also indicates that the horizontal acquisition of those genomic islands occurred in their common ancestor prior to the diversification of the chinese carp isolate zc1 , mississippi catfish isolate s04 - 690 , and alabama catfish vah isolates . reference alignment showing the maps of the orthologous genetic regions ( blue arrow ) of chinese carp isolate zc1 ( a ) or mississippi catfish isolate s04 - 690 ( b ) specific to the epidemic - associated genetic regions of vah isolates ( red arrow ) . the scales for the percent conservation and average coverage of the illumina sequences from both the isolates aligned with epidemic - associated genetic regions of vah isolates are shown on the left axis . the black horizontal line illustrates the continuous alignment of the sequences , and the discontinuous black line indicates the interruption of the sequence alignment . the average coverages of the zc1 and s04 - 690 sequences mapped against epidemic - associated genetic regions of vah isolates are 87 and 115 , respectively . reference alignment showing the maps of the orthologous genetic regions ( blue arrow ) of isolates s04 - 690 ( a ) and zc1 ( b ) specific to the gis of vah isolates ( red arrow ) . the scales for the percent conservation and average coverage of the illumina sequences from both the isolates aligned with gis of vah isolates are shown on the left axis . , this analysis did not provide sufficient phylogenetic resolution to differentiate between these isolates . to further refine the evolutionary relationships of the u.s . catfish and chinese carp isolates , we constructed a phylogenetic tree based on the 303,863 bp of concatenated sequences from epidemic - specific regions shared among vah isolates ( 7 ) , zc1 , and s04 - 690 . we observed that vah epidemic isolates form a distinct monophyletic clade that is divergent from the s04 - 690 strain ( see fig . the evolutionary history inferred from this phylogenetic tree suggests a common ancestor between all of these strains , with s04 - 690 being most closely related to vah isolates . epidemic isolates from asia with the mississippi catfish isolate as a representative intermediate in this evolutionary model . finally , we conducted a pairwise comparison of the proteomes of 14 a. hydrophila isolates using a blastp matrix , an all - against - all blastp analysis , to determine the similarity among proteomes based on the number of conserved gene families ( 23 ) . similarly to our previous findings ( 7 ) , these results demonstrated that the vah strain proteomes are highly clonal and that the proteome of mississippi catfish isolate s04 - 690 shows a high degree of similarity to that of vah strains , with 99.5% similarity to the representative strain ml09 - 119 ( see fig . in contrast , the proteome of the chinese carp isolate zc1 shows 97.7% and 97.5% similarity to those of ml09 - 119 and s04 - 690 , respectively . these findings are concordant with the phylogenomic analysis ( see fig . s2 ) that suggests that s04 - 690 is an example of an intermediate strain in the evolutionary emergence of vah . we observed that the genomes of s04 - 690 , zc1 , and all of the vah isolates share > 97% similarities among themselves . in contrast , none of the genomes of nonepidemic u.s . catfish vah and mississippi isolates are a monophyletic group with a coherent genome and a discriminatory phenotype ( i.e. , myo - inositol utilization ) that clearly differentiate them as clonal , pathogenic strains that are distinct from a. hydrophila isolates that have been previously described in the united states . this study provides multiple lines of evidence that suggest that highly virulent a. hydrophila isolates responsible for epidemic outbreaks of mas in catfish in the southeastern united states have an asian origin . at this point , the exact means of introduction of these virulent isolates in the united states is not clear . there are several possible ways in which asian isolates might have been introduced in the united states : ( i ) the introduction of asian carp into the united states and their extensive use as biological control agents may have introduced an asian variant of a. hydrophila that served as a precursor for the emergence of highly virulent catfish isolates , ( ii ) transport and distribution of imported ornamental fish from asia may have introduced the isolates , or ( iii ) import of contaminated processed seafood products may have introduced asian isolates into the united states , since imported seafood is frequently contaminated with a. hydrophila ( 24 ) . given the widespread occurrence of this a. hydrophila lineage within different regions of china ( guangdong , hubei , jiangsu , and zhejiang provinces ) and the report of myo - inositol - utilizing a. hydrophila strains in eastern and southeastern asia in the years prior to the u.s . epidemic , the most likely scenario is that this pathogenic a. hydrophila lineage was introduced into the united states from fish imported from asia . additionally , highly virulent disease episodes have been documented since the late 1980s in china with a. hydrophila of this genotype ( 9 ) and no disease episodes like those observed on commercial catfish farms in the u.s . occurred until 2004 , with the onset of epidemic outbreaks occurring in 2009 . we hypothesize that a virulence factor(s ) encoded within genomic islands of the epidemic catfish isolates may contribute to the enhanced pathogenicity and/or host specificity of the vah isolates in catfish . further studies of the role and mechanisms of this virulence factor(s ) in a. hydrophila pathogenesis in catfish and carp will increase our understanding of the emergence of highly virulent bacterial pathogens and the contribution of geographically introduced nonnative species in the pandemic spread of pathogens facilitated by human activities . sequences for the myo - inositol utilization clusters of chinese carp isolate zc1 ( accession no . the raw sequence reads for strains zc1 and s04 - 690 were deposited in ncbi s short read archive under accession numbers srr1031999 and srr1032027 , respectively . sequences for the myo - inositol utilization clusters of chinese carp isolate zc1 ( accession no . the raw sequence reads for strains zc1 and s04 - 690 were deposited in ncbi s short read archive under accession numbers srr1031999 and srr1032027 , respectively . download text s1 , docx file , 0.1 mb gyrb phylogeny using sequences from 169 a. hydrophila isolates , including strains of asian and u.s the tree figure was generated using the interactive tree of life web application ( http://itol.embl.de ) ( 27 ) . download figure s1 , jpg file , 1.4 mb phylogenetic analysis based on the virulent a. hydrophila ( vah)-specific sequences . evolutionary analyses were conducted in mega5 with 1,000 repetitions to generate bootstrap support ( 28 ) . * , isolate pb10 - 118 was isolated from a diseased fish obtained from arkansas , usa ( 7 ) . download figure s2 , jpg file , 0.3 mb blastp matrix of 14 different a. hydrophila isolates . the proteomes of each of the a. hydrophila isolates were compared pairwise using all - against - all blastp as described previously ( 23 ) . this matrix illustrates the output from the pairwise comparison of conserved protein families of each of the isolates to each other . green represents the relative percent homology between proteomes , and red represents relative percent homology within the isolate s own proteome . the dense green pyramid on the right indicates greater homology ( > 97% ) between the proteomes of vah isolates from u.s . catfish epidemics , chinese carp isolate zc1 , and mississippi catfish isolate s04 - 690 . download figure s3 , jpg file , 11.7 mb aeromonas hydrophila isolates used for gyrb phylogeny .

abstractsince 2009 , catfish farming in the southeastern united states has been severely impacted by a highly virulent and clonal population of aeromonas hydrophila causing motile aeromonas septicemia ( mas ) in catfish . the possible origin of this newly emerged highly virulent a. hydrophila strain is unknown . in this study , we show using whole - genome sequencing and comparative genomics that a. hydrophila isolates from diseased grass carp in china and catfish in the united states have highly similar genomes . our phylogenomic analyses suggest that u.s . catfish isolates emerged from a. hydrophila populations of asian origin . furthermore , we identified an a. hydrophila strain isolated in 2004 from a diseased catfish in mississippi , prior to the onset of the major epidemic outbreaks in alabama starting in 2009 , with genomic characteristics that are intermediate between those of the asian and alabama fish isolates . investigation of a. hydrophila strain virulence demonstrated that the isolate from the u.s . catfish epidemic is significantly more virulent to both channel catfish and grass carp than is the chinese carp isolate . this study implicates the importation of fish or fishery products into the united states as the source of highly virulent a. hydrophila that has caused severe epidemic outbreaks in united states - farmed catfish and further demonstrates the potential for invasive animal species to disseminate bacterial pathogens worldwide .

Observation Nucleotide sequence accession numbers. SUPPLEMENTAL MATERIAL

motile aeromonas septicemia ( mas ) caused by mesophilic a. hydrophila affects a wide variety of primarily freshwater fish species , including carp , tilapia , perch , catfish , and salmon ( 2 ) . epidemic disease outbreaks in fish caused by a. hydrophila , resulting in millions of dollars of lost revenue , have been reported worldwide but have not occurred in the united states until recently ( 3 , 4 ) . in the summer of 2009 , an outbreak of mas in commercially raised catfish caused by highly virulent a. hydrophila ( vah ) began in western alabama ( 5 ) . to date , this epidemic of mas outbreaks is responsible for an estimated loss of more than $ 12 million in catfish aquaculture operations in the southeastern united states . our comparative and functional genomic analyses demonstrated that a. hydrophila isolates from recent epidemic outbreaks in the united states are highly clonal but genomically distinct from a. hydrophila historical isolates from diseased catfish in the united states , and these epidemic isolates contain a large number of genomic regions predicted to be acquired through lateral genetic transfer ( 7 ) . several episodes of epidemic outbreaks caused by a. hydrophila beginning in the late 1980s and within the last 10 years have been reported in farmed carp in china ( 8 , 9 ) . catfish and chinese carp epidemics caused by a. hydrophila , including the timing of the epidemics , which primarily occurred during the summer ; the extensive mortality observed in seemingly healthy fish ; and the highly clonal nature of the epidemic isolates ( 5 , 8) . because of the globalization of the aquaculture and ornamental fish trade industries , fish transport provides a gateway for the introduction of exotic species that could introduce new pathogens that can devastate populations of indigenous species ( 12 ) . for instance , spring viremia of carp ( svc ) , a viral disease historically affecting fish in europe , the middle east , and russia , has been found in carp in the united states since first being reported in 2002 ( 13 ) . the chinese origin of the svc viral isolates in the united states was thought to be mediated through importation of ornamental fish ( 14 ) . the emergence of several other viral diseases of fish and shrimp reported worldwide has been attributed to the dissemination of infected fish or eggs as a carrier of nonindigenous pathogens ( 15 ) . over the past several decades , asian carp have been imported into the united states , including grass carp ( ctenopharyngodon idella ) , bighead carp ( hypophthalmichthys nobilis ) , silver carp ( hypophthalmichthys molitrix ) , and black carp ( mylopharyngodon piceus ) , as well as countless species of live ornamental fish sold in the united states . grass carp were imported to the united states from east asia beginning in 1963 by the u.s . as a result of the great flood of 1993 , silver carp were released in large numbers into the mississippi river drainage system and currently threaten the ecological balance of the u.s . the origin of the clonal a. hydrophila isolates responsible for the ongoing epidemic mas outbreak in united states - farmed channel catfish is unknown . catfish and chinese carp isolates have a recent common ancestor . to determine the evolutionary relationships of the recent virulent a. hydrophila ( vah ) isolates , a gyrb - based phylogenetic analysis was conducted using a total of 264 aeromonas strains downloaded from the aeromonas multilocus sequence typing ( mlst ) database ( http://www.pubmlst.org/aeromonas ) ( 18 ) and including other strains of u.s . interestingly , the only strain retrieved from the genbank database that was affiliated with vah strains was a. hydrophila strain zc1 , which was isolated from a diseased grass carp in guangdong province , china , from ponds that had experienced an epidemic outbreak of hemorrhagic septicemia ( 19 ) . in order to identify any other isolates that were affiliated with epidemic strains , we screened a. hydrophila strain collections available in the united states that were isolated from fish and other hosts and identified one additional a. hydrophila strain , s04 - 690 , which was affiliated with vah strains and strain zc1 ( see fig . strain s04 - 690 was isolated in 2004 from a diseased catfish obtained from a commercial aquaculture pond located in washington county , mississippi , which experienced a single event of mas outbreak that killed thousands of catfish but importantly did not result in a widespread epidemic outbreak in the surrounding regions or subsequent outbreaks in following years within the affected farm . since our gyrb - based phylogeny suggests that chinese carp isolate zc1 is highly similar to vah isolates and a 2004 mississippi isolate of catfish origin , we evaluated the evolutionary relationships of additional chinese carp isolates of epidemic origin in hubei province , china ( see table s1 in the supplemental material ) ( 4 ) . the gyrb phylogeny constructed using the neighbor - joining method revealed that all of the chinese carp epidemic isolates , including isolate zc1 , consistently group together as a single clade along with the recent u.s . in contrast , we found that a. hydrophila isolates of nonepidemic origin are widely spread through different clades ( see fig . this suggests that a clonal a. hydrophila complex descended from a common ancestor responsible for epidemic outbreaks of fish disease in china and the united states . to provide a more refined phylogenetic placement of the u.s a. hydrophila isolates , we used a multilocus sequence - based phylogeny that included 3,751 bp of concatenated nucleotide sequences from six housekeeping genes : gyrase subunits a and b ( gyra and gyrb ) , rna polymerase sigma factor ( rpod ) , bacterial dna recombination protein ( reca ) , heat shock protein ( dnaj ) , and dna polymerase iii tau and gamma subunits ( dnax ) ( see table s2 in the supplemental material ) . our analysis included a. hydrophila vah catfish isolates , mississippi catfish isolate s04 - 690 , chinese carp isolate zc1 ( 19 ) , and nonepidemic aeromonas isolates of u.s . this multilocus phylogeny demonstrated that the chinese carp isolate zc1 clustered together with the alabama and mississippi catfish isolates as a monophyletic clade ( fig . 1 ) ; in contrast , the nonepidemic a. hydrophila isolates are highly divergent ( fig . in addition to this multilocus sequence - based phylogeny , we searched the aeromonas pubmlst database ( http://www.pubmlst.org/aeromonas ) ( 18 ) using gyrb , grol , glta , metg , ppsa , and reca gene - specific sequences of isolates ml09 - 119 , s04 - 690 , and zc1 , and we found that these three isolates belong to sequence type ( st ) st251 , which until this study contained only aeromonas hydrophila isolate xs91 - 4 - 1 . interestingly , this highly virulent chinese isolate , aeromonas hydrophila xs91 - 4 - 1 , was obtained from a diseased silver loweye carp from an epidemic outbreak in 1991 ( 20 ) . taken together , these findings demonstrate that chinese carp isolates xs91 - 4 - 1 and zc1 along with u.s . the internal color ranges refer to the geographical origin of the isolates , while the external ring indicates the biological source . our multilocus phylogeny based on two different sets of housekeeping genes as well as mlst typing is concordant with the gyrb - based phylogeny that demonstrated that the chinese carp and u.s . therefore , these findings strongly support the hypothesis that the recent a. hydrophila isolates responsible for catfish epidemics in the united states emerged due to the rapid spread of a clonal group of pathogenic isolates that share an ancestor with a. hydrophila isolates identified from diseased carp from asia . catfish and asian carp isolates , do not provide any indication of the relative timing of the evolutionary changes that have occurred in these a. hydrophila populations , nor did these analyses indicate the relative virulence of these strains within carp or catfish . channel catfish and grass carp were experimentally challenged to compare the virulence of strains ml09 - 119 , zc1 , and al06 - 06 , with survival rates ranging from 0.02 0.04 to 0.98 0.04 . a. hydrophila isolate al06 - 06 was included in the challenge experiment in this study as a reference strain that is typical of a. hydrophila strains that have been historically isolated from stressed fish prior to the advent of the mas epidemic and has shown reduced mortality ( 20% ) in channel catfish relative to that observed from ml09 - 119 ( 7 ) . in this study , we observed that isolate ml09 - 119 was significantly more virulent than either zc1 or al06 - 06 and that zc1 was more virulent than al06 - 06 ( p < 0.0001 ) ( fig . additionally , overall comparisons between fish species show that channel catfish were more susceptible to a. hydrophila than were grass carp ( p = 0.0126 ) ; however , this observation occurred only for vah strain ml09 - 119 ( fig . these data suggest that ml09 - 119 has evolved increased virulence and that channel catfish appear to be more susceptible . percent survival of channel catfish or grass carp after challenge with a. hydrophila strain ml09 - 119 , zc1 , or al06 - 06 . the difference in survival between channel catfish and grass carp injected with ml09 - 119 was significant ( p = 0.0069 ) . no significant differences in survival between channel catfish and grass carp intraperitoneally injected with zc1 or al06 - 06 were observed , but there was a significant difference between strains zc1 and al06 - 06 in both channel catfish ( p = 0.0075 ) and grass carp ( p = 0.0089 ) . one of the phenotypic traits common among all vah strains is the ability to utilize myo - inositol as a sole carbon source ( 7 ) . we therefore tested the chinese carp isolate zc1 and the mississippi catfish isolate s04 - 690 for this phenotype and found that these strains could also utilize myo - inositol as a sole carbon source and had growth curves similar to those observed for vah strains , whereas isolate al06 - 06 , used as a negative control , did not utilize myo - inositol as a sole carbon source ( data not shown ) . the comparison of the 17.5-kb myo - inositol utilization cluster of vah isolates with that of chinese carp isolate zc1 ( accession no . to our knowledge , the utilization of myo - inositol as a sole carbon source has not been reported for any other a. hydrophila isolates obtained from diseased fish or any other environmental or clinical origin in the united states ( 1 ) . this is further evidence in support of an asian origin for the epidemic a. hydrophila strains in the united states . to systematically investigate the emergence of the recent vah isolates , we sequenced the genomes of the chinese carp isolate zc1 and the mississippi catfish isolate s04 - 690 . reference mappings of the illumina sequence reads from the genomes of chinese carp isolate zc1 and mississippi catfish isolate s04 - 690 were conducted against the 55 genomic regions unique among vah strains ( 7 ) , demonstrating that the genomes of chinese carp isolate zc1 and mississippi catfish isolate s04 - 690 share 53 and 54 of these 55 epidemic - associated regions , respectively ( fig . these epidemic - associated unique regions are considered horizontally acquired regions of the a. hydrophila genome since they were obtained by excluding core genomes of the 11 a. hydrophila isolates described previously ( 7 ) . the c26r1 region , the same region absent in zc1 , is the only epidemic - associated unique region of a recent vah isolate absent in the genome of mississippi isolate s04 - 690 ( fig . the comparison of genomic islands present within the genomes of chinese carp and u.s . catfish isolates demonstrated that most of the genomic islands , except for gi5 , gi6 , and gi12 , which are found consistently within the genomes of vah isolates are also present within the genome of strain zc1 ( fig . we also observed that the majority of the genomic islands , except for gi12 , were present in mississippi isolate s04 - 690 ( fig . since genomic islands of bacterial species are highly variable and frequently used to distinguish strains due to their acquisition through horizontal gene transfer ( 22 ) , the sharing of a large number of identical genomic islands has led to the hypothesis that u.s . vah isolates originated from asia and that the mississippi isolate s04 - 690 represents an intermediate strain between the asian carp and alabama catfish isolates . the commonality of a large number of genomic islands also indicates that the horizontal acquisition of those genomic islands occurred in their common ancestor prior to the diversification of the chinese carp isolate zc1 , mississippi catfish isolate s04 - 690 , and alabama catfish vah isolates . reference alignment showing the maps of the orthologous genetic regions ( blue arrow ) of chinese carp isolate zc1 ( a ) or mississippi catfish isolate s04 - 690 ( b ) specific to the epidemic - associated genetic regions of vah isolates ( red arrow ) . to further refine the evolutionary relationships of the u.s . catfish and chinese carp isolates , we constructed a phylogenetic tree based on the 303,863 bp of concatenated sequences from epidemic - specific regions shared among vah isolates ( 7 ) , zc1 , and s04 - 690 . finally , we conducted a pairwise comparison of the proteomes of 14 a. hydrophila isolates using a blastp matrix , an all - against - all blastp analysis , to determine the similarity among proteomes based on the number of conserved gene families ( 23 ) . similarly to our previous findings ( 7 ) , these results demonstrated that the vah strain proteomes are highly clonal and that the proteome of mississippi catfish isolate s04 - 690 shows a high degree of similarity to that of vah strains , with 99.5% similarity to the representative strain ml09 - 119 ( see fig . in contrast , the proteome of the chinese carp isolate zc1 shows 97.7% and 97.5% similarity to those of ml09 - 119 and s04 - 690 , respectively . we observed that the genomes of s04 - 690 , zc1 , and all of the vah isolates share > 97% similarities among themselves . in contrast , none of the genomes of nonepidemic u.s . , myo - inositol utilization ) that clearly differentiate them as clonal , pathogenic strains that are distinct from a. hydrophila isolates that have been previously described in the united states . this study provides multiple lines of evidence that suggest that highly virulent a. hydrophila isolates responsible for epidemic outbreaks of mas in catfish in the southeastern united states have an asian origin . at this point , the exact means of introduction of these virulent isolates in the united states is not clear . there are several possible ways in which asian isolates might have been introduced in the united states : ( i ) the introduction of asian carp into the united states and their extensive use as biological control agents may have introduced an asian variant of a. hydrophila that served as a precursor for the emergence of highly virulent catfish isolates , ( ii ) transport and distribution of imported ornamental fish from asia may have introduced the isolates , or ( iii ) import of contaminated processed seafood products may have introduced asian isolates into the united states , since imported seafood is frequently contaminated with a. hydrophila ( 24 ) . given the widespread occurrence of this a. hydrophila lineage within different regions of china ( guangdong , hubei , jiangsu , and zhejiang provinces ) and the report of myo - inositol - utilizing a. hydrophila strains in eastern and southeastern asia in the years prior to the u.s . epidemic , the most likely scenario is that this pathogenic a. hydrophila lineage was introduced into the united states from fish imported from asia . additionally , highly virulent disease episodes have been documented since the late 1980s in china with a. hydrophila of this genotype ( 9 ) and no disease episodes like those observed on commercial catfish farms in the u.s . occurred until 2004 , with the onset of epidemic outbreaks occurring in 2009 . we hypothesize that a virulence factor(s ) encoded within genomic islands of the epidemic catfish isolates may contribute to the enhanced pathogenicity and/or host specificity of the vah isolates in catfish . further studies of the role and mechanisms of this virulence factor(s ) in a. hydrophila pathogenesis in catfish and carp will increase our understanding of the emergence of highly virulent bacterial pathogens and the contribution of geographically introduced nonnative species in the pandemic spread of pathogens facilitated by human activities . sequences for the myo - inositol utilization clusters of chinese carp isolate zc1 ( accession no . download text s1 , docx file , 0.1 mb gyrb phylogeny using sequences from 169 a. hydrophila isolates , including strains of asian and u.s the tree figure was generated using the interactive tree of life web application ( http://itol.embl.de ) ( 27 ) . download figure s1 , jpg file , 1.4 mb phylogenetic analysis based on the virulent a. hydrophila ( vah)-specific sequences . * , isolate pb10 - 118 was isolated from a diseased fish obtained from arkansas , usa ( 7 ) . download figure s2 , jpg file , 0.3 mb blastp matrix of 14 different a. hydrophila isolates . the proteomes of each of the a. hydrophila isolates were compared pairwise using all - against - all blastp as described previously ( 23 ) . this matrix illustrates the output from the pairwise comparison of conserved protein families of each of the isolates to each other . catfish epidemics , chinese carp isolate zc1 , and mississippi catfish isolate s04 - 690 . download figure s3 , jpg file , 11.7 mb aeromonas hydrophila isolates used for gyrb phylogeny .

from lower vertebrates to humans , it is known that locomotor activity is generated by spinal neuronal circuits referred to as the central pattern generator ( cpg ) . studies in lower vertebrates have been useful for modeling how the cpg performs in the absence of supraspinal and afferent feedback as well as determining the roles of neurotransmitters and afferent stimuli ( load , speed , and perturbations ) . within the context of spinal cord injury ( sci ) , central pattern generation has become a conceptual basis for locomotor training after injury [ 2 , 3 ] . in rats , cats , and humans , a large amount of spontaneous recovery can occur following sci , and this recovery is closely related to white matter sparing [ 46 ] . specifically , this recovery has mainly been attributed to spared fibers in the ventral and ventral lateral funiculi where the rubrospinal , reticulospinal , and vestibulospinal tracts are located [ 7 , 8 ] . yet , the recovery achieved via training is generally specific to the task practiced , for example , stand , step , or swim training [ 9 , 10 ] . the amount of activity imposed on the limbs is also a crucial component of locomotor rehabilitation . importantly , step training on a treadmill using body weight support should provide a high number of repetitions to facilitate motor learning . recovery potential therefore is a function of ( 1 ) amount of sparing / injury severity , ( 2 ) task specificity , and ( 3 ) amount of activity ( activity dependent plasticity ) . other aspects of step training or general exercise may also contribute to sci recovery , including environmental enrichment , intermittent hypoxia , and general improvements in body strength and psychological well - being . more studies are needed to determine the efficacy and mechanisms of training on functional outcomes ( including locomotion , allodynia , and autonomic functions ) after incomplete contusions in combination with appropriate control groups , kinematics , and overground locomotion ( for review see ) . in humans , complete cord transection is rare . therefore , a clinically relevant rat contusion model of sci may provide very useful information for the study and translation of locomotor training . research with spinally complete and incomplete rodents has affirmed central pattern generation and identified important aspects of training and sci locomotion [ 11 , 14 , 15 ] . while it is clear that training influences aspects of treadmill locomotion post - sci , basic research studies have yielded conflicting outcomes regarding overground locomotion [ 1619 ] . furthermore , very little is known about the efficacy of step training on nonlocomotor functions . we previously demonstrated that 60 minutes of training 7 days per week significantly improved both locomotor and nonlocomotor functions , such as open field locomotion , hindlimb kinematics , at - level mechanical allodynia , and bladder function in contused rats . in the current study , we ( 1 ) tested the efficacy of a 30-minute body weight supported treadmill step training paradigm for the recovery of overground locomotion using qualitative scoring ( bbb ) and quantitative ( kinematic ) locomotor tests , ( 2 ) determined the contribution of in - cage activity to spontaneous locomotor recovery versus training - induced recovery , and ( 3 ) examined bladder function and allodynia ( pain response to a nonnoxious stimulus ) . sci control groups ( nontrain , forelimb ) were identically handled , harnessed , and tested . sham ( laminectomy only ) animals provided a standard comparison throughout the study for each parameter . all animal procedures were performed according to the nih guidelines , and the protocols were reviewed and approved by the institutional animal use and care committee at the university of louisville , school of medicine . indianapolis , in ) approximately 60 to 70 days old weighing approximately 300 grams were individually housed in an animal room with a 12-hour light and dark cycle . one group ( n = 14 ) was quadrupedally trained ; a second group ( n = 10 ) served as nontrained controls ; a third group was forelimb trained ( n = 10 ) . training began at two weeks after sci for thirty minutes per day , six days per week and continued for six weeks . animals were anesthetized with an intraperitoneal dose of ketamine ( 80 mg / kg , ketoset , fort dodge laboratories , fort dodge , ia ) and xylazine ( 10 mg / kg , anased , lloyd laboratories , shenandoah , ia ) mixture . the following were administered subcutaneously : 0.5 ml of dual penicillin ( penject ; the butler company ; columbus , oh ) single dose perioperatively as a general prophylactic , 5 mg / kg gentamicin ( gentafuse , butler schein , dublin , oh ) once per day for 5 days to prevent bladder infections , 2.5 mg / kg ketoprofen ( ketofen , fort dodge laboratories , fort dodge , ia ) twice per day for two days to alleviate postsurgical pain , and 10 ml saline , as previously described . spinal clamps were applied to the t7 and t9 spinous processes to stabilize the spinal column . the muscle was closed with suture , the skin closed with michel clips , and topical antibiotic applied . animals were single housed on a 12 : 12 light : dark cycle . training paradigm . however , training interventions initiated too early after sci may be detrimental to recovery efforts by exacerbating secondary injury cascades . we initiated training at two weeks after sci after the majority of spontaneous recovery had occurred according to past experience with this injury severity ( based on locomotor scores and recovery of spontaneous bladder voiding ) . step training was performed on the exer3 treadmill system ( columbus instruments , columbus , oh ) customized with spring scales for body weight support . mission viejo , ca ) and hook - and - loop material and velcro straps . trained animals began quadrupedal step training 2 weeks after sci surgery , 6 days per week , 30 minutes per day , for 6 weeks , at 22 cm / s . trainers adjusted the body weight support as needed using manual assistance at the hip flexor region to facilitate proper plantar placement , for example , complete toe extension , no ankle rotation , and incorporation of forelimb - hindlimb coordination with minimal assistance . rats were encouraged to step independently as they began to gain consistent stepping and more stability without collapsing and dragging their hindlimbs . it is of note that animals were quadrupedally trained , as many studies utilize an upright bipedal training and testing position . forelimb animals were harnessed and walked with forelimbs on the treadmill while a custom 4 6 inch metal platform supported the hindlimbs just above the moving treadmill belt . nontrained animals were harnessed while a custom platform supported all limbs . for nontrained animals , one trained , one nontrained , and one forelimb trained animal could be harnessed simultaneously . sham animals were not exposed to the treadmill system and were handled four times per week ( minimum ) . rats were not required to complete the 30-minute session during the first week of training if they exhibited signs of stress , that is , porphyrin staining around the eyes or nose , irregular breathing , or excessive diarrhea . noxious stimuli were avoided during training sessions ; for example , if an animal had skin abrasion on a paw , animals ceased from training until the issue was resolved , as potentially noxious input may inhibit spinal learning [ 22 , 24 , 25 ] . an open field locomotor assessment , the basso - beattie - bresnahan ( bbb ) scale , was used to evaluate hindlimb function in the rats . once per week , each animal was placed in an open field and tested for 4 minutes by the same two scorers , who were presented with the trained , forelimb , nontrained , and sham animals in random order ( blinded to group ) . the bbb uses a 21-point scale for locomotion , which rates parameters , such as joint movements ( 08 ) , weight support ( 913 ) , and paw placement ( 1421 ) . intact animals demonstrate a locomotor score of 21 , whereas animals that exhibit complete paralysis of the hindlimbs are scored as 0 . after the six - week training period the hindquarters were shaved and the bony landmarks on the lateral side of the left and right hindlimbs were marked with permanent marker : iliac crest , greater trochanter , lateral malleolus , and metatarsophalangeal joint ( ilium , hip , ankle , and toe ) . each animal was then individually placed in a clear plexiglass runway ( in random order ) . as the animal passed from one end of the tank to the other darkened side , cameras positioned on the side and underneath the tank captured the angles and footfall patterns . 2d overground ( unassisted ) kinematics were analyzed using the maxtraq motion analysis system ( innovision systems , columbiaville , mi ) . the iliac crest , hip , ankle ( lateral malleolus ) , toe , and paw were digitized by a blinded observer , and the hip - ankle - toe ( hat ) and iliac crest - hip - ankle ( iha ) angles were marked to quantify the movement of the hindlimbs during overground stepping . four animals ( n = 3 forelimb ; n = 1 nontrain ) could not generate weight bearing steps ( bbb = 8 ; 8 weeks after sci ) ; these animals did not participate in the ri or psi tests . horizontal ladder walk . animals were additionally tested for fine locomotor control by crossing a horizontal ladder of metal bars ( columbus instruments , columbus , oh ) [ 28 , 29 ] . animals were tested for their ability to correctly place their hind paws while crossing the bars . animals with more severe deficits ( bbb score < 11 ) were not tested because their limbs drag across the rungs and have a falsely reduced error count . two blinded raters manually counted the number of footfall errors ( hind paw / limb slip or fall through the bars ) . after each crossing , the raters discussed and agreed on the error count . if , at the end of the crossing , the raters did not agree on error count within two errors , the trial was repeated . footfall error is reported as the mean of three high quality passes ( crossed with little or no hesitation and without changing direction ; hindlimbs did not drag ; raters agreed on error count ) . behavioral testing of sci rats for sensitivity to normally innocuous stimuli ( touch and gentle squeeze / pressure ) was performed using our published grading scale for the scoring of pain - like behavior to trunk stimulation in the rat . once per week , testing sessions commenced at approximately the same time of day ( 9 am before the start of training ) . the dorsolateral trunk ( t7t9 dermatomal level ) just above the t9 spinal injury level was tested bilaterally for at - level mechanical hypersensitivity to touch and gentle touch / squeeze . two baseline measurements ( at least 3 days apart ) were performed before injury for all rats . throughout the study , all allodynia testing was consistently performed by the same two experimenters , blinded to treatment groups . at the start of testing , the top of the cage was removed and the animal was allowed to acclimate to the environment for 2 minutes . while in its cage , each animal was stroked at the dorsolateral trunk five times bilaterally with a number 5 paintbrush ( 1.5 0.5 cm bristles ; average pressure , 15 g ) in an alternating rostral / caudal plane . an interstimulus interval of 1 min between sides was maintained . after each stroking stimulus , the presence / absence of any evoked responses that were indicative of pain was noted : ( 1 ) a freezing response ( stopping of normal activity and staying still in response to the stimulus ) , ( 2 ) escape ( any movement of the animal away from the stimulus probe ) , and ( 3 ) grabbing at or pushing away the stimulus probe with their forepaws . an animal must show an evoked pain - like behavioral response at least 60% of the time in a given session to be considered responsive to the testing stimulus ( i.e. , an animal responded to at least three of five stimuli / strokes per side ) . responses to brush , if present , were further assessed for threshold values using a set of semmes - weinstein monofilaments ( 20-filament set , 15 of which are in the range of 0.008 g to 15 g ; stoelting co. , wood dale , il ) . animals designated as responders to brush ( 15 g stimulus ) were then given a numeric score based on their associated responses to filament testing , receiving a minimum of 4 ( 4 = freeze , 5 = escape , and 6 = grab / push as the aggressiveness of the behavioral response increases , so does the score ) to a maximum of 10 ( see for scoring scale ) . depending on the behavioral response of the animal to brush , an initial filament stimulus was applied by pressing the tip of the filament into the dorsolateral trunk ( t7t9 ) until it bent . if the animal responded , a lower gram filament was applied to test the animal 's sensitivity . in between filament the process was repeated until the lowest gram filament that the animal responded to 60% of the time was determined . if the animal was not responsive to the initial probing stimulus , the next greatest gram filament ( and repeated if needed ) was used to determine the threshold of the animal 's sensitivity . for those animals not responsive to brush stroke ( i.e. , evoked pain - like behavioral response to less than 60% of the stimuli less than three of five strokes ) , a gentle squeeze / pressure test was conducted to determine if the animal had increased sensitivity to a stronger mechanical stimulus over a wider surface area ( which also normally does not provoke avoidance behaviors and is thus considered innocuous ) . in this instance , the animal 's skin is gently squeezed with a pair of modified adson tissue forceps ( 2.0 mm wide tips ) , which is equivalent to the 60 g semmes - weinstein monofilament . gentle squeeze / pressure was applied to the dorsolateral trunk ( t7t9 ) five times bilaterally , with an interstimulus squeeze interval of 1 min . as with touch - evoked agitation , any evoked pain - like responses were observed and documented ( 0 = no response , 1 = freeze , 2 = escape , and 3 = grab / push ) . after the testing session , animals were scored for their degree of at - level allodynia based on a 10-point scale , with 10 being the maximum score an animal could receive . scores from each weekly testing were documented for each animal and averaged together to obtain a mean weekly allodynia score for each group . transvesical catheter . after the last training session and all locomotor testing , a transvesical bladder catheter was implanted under 2% isoflurane ( similar to ) . briefly , the bladder was exposed via a midline abdominal incision through the skin and musculature . a purse - string suture ( 4 - 0 ethilon ) was placed in the urothelium of the bladder dome . pe-60 tubing ( the tip previously heated to form a collar ~2 mm from the end ) was inserted through the bladder dome within the suture limits and secured . the bladder was emptied , and the tubing was passed through the subcutaneous tissue and exteriorized behind the neck . after a 1.5-hour recovery period , the animal was placed in a darkened box for cystometric recordings . normal saline was infused into the bladder at a rate of 0.25 ml / min to evoke voiding contractions . urodynamic data ( voiding and nonvoiding events , voided volumes , and animal movements or spasms ) and experimenter notes were recorded on video for offline playback and analysis with datawave software ( http://www.dwavetech.com/ ) . cystometrogram ( cmg ) parameters are the mean of 5 consecutive cycles ( which were sampled approximately 15 minutes after the start of saline infusion ) . cmgs were analyzed for resting pressure ( mm hg ) , maximal amplitude of contraction ( mm hg ; peak pressure minus resting pressure ) , contraction time ( sec ) , and intercontraction interval ( sec ) . the in - cage activity of every animal was recorded using an opto - m3 infrared activity monitor ( columbus instruments , columbus , oh ) . a cradle equipped with infrared beams , spaced one inch apart , was placed around the cage so that the infrared beams shined across the cage near the floor . the data were collected as ambulatory ( number of two consecutive beam breaks ; i.e. , the animal was moving across the cage ) and total movements ( number of beam breaks ) during the active phase ( 6 pm6 am ) . each animal was deeply anesthetized with ketamine / xylazine and perfused transcardially with a solution of normal saline and heparin . the bladder was blunt dissected away from the prostate , blotted dry , and weighed . the spinal lesion area was removed and placed in 4% paraformaldehyde for at least 48 hours , followed by 30% sucrose / phosphate buffer solution with 1% sodium azide for at least 24 hours and until the tissue was cut on a cryostat ( leica cm 1850 ) at 18 m thickness and stained with both luxol fast blue and cresyl violet ( kluver - barrera method ) . the lesion area was quantified as previously described using spot advanced software ( diagnostic instruments , sterling heights , mi ) and the nikon e400 microscope . briefly , white matter was divided into four regions ( dorsal columns , dorsolateral funiculus , ventrolateral funiculus , and the ventral funiculus ) and each area was subdivided into left and right sides . medial edges of the dorsal horn , and the tips of the ventral horn were used as landmarks for the divisions . the percent of white matter sparing ( wms ) was determined by dividing the white matter remaining at the epicenter , .5 mm rostrally , and 1.0 mm rostrally by the average area of white matter present in intact sections . the intact area of white matter for a given region was estimated by averaging together measurements from 2 sections 2 mm rostral to the epicenter . sci animals were excluded from analysis if the recorded displacement of the impactor tip was less than 1.0 mm during the injury ( n = 3 ) ; these animals typically have a very mild injury . an additional 2 animals were sacrificed during the first two weeks following injury due to autophagia . this resulted in the following groups : train , n = 13 ; nontrain , n = 7 ; forelimb , n = 9 ; sham , n = 4 . one - way repeated measures analysis of variance ( anova ) ( fixed effects ) was performed for tests of within subject and between subject effects followed by bonferroni post hoc t - tests for the bbb , allodynia , and activity . one - way anova ( fixed effects ) was used for cystometry parameters , bladder weight , gait , and kinematics followed by bonferroni post hoc t - tests , significance level p < .05 . for the regularity index ( ri ) , post hoc tests approached significance and were followed by the mann - whitney u test . all groups demonstrated significant spontaneous recovery from 1 to 2 weeks after sci . at the initiation of training ( 2 weeks after sci ) all groups functionally displayed plantar paw placement with occasional weight support ( bbb scores : trained : 9.61 1.7 ; forelimb : 9.5 2.2 ; nontrained 9.3 2.0 ) . after 3 weeks of training ( 5 weeks after sci ) , trained animals ' bbb scores were significantly higher compared to pretraining and remained significantly higher through the rest of the study ( figure 1 ) . consistent weight supported stepping ( bbb 11 ) is a functional milestone on the bbb scale ( which makes each animal eligible to receive a subscore ) . a significantly higher proportion of animals in the trained group achieved consistent weight support . no differences were found between sham and trained animals after week 3 ( figure 2 ) . however , the trained group was also not significantly different from the forelimb or nontrain groups . forelimb and nontrain groups had significant proportions of animals unable to consistently weight - support as compared to sham throughout the study . kinematic measurements of overground locomotion revealed that quadrupedal - trained animals more closely approximated the hindlimb angular movement of shams ( no significant differences ) . however , the trained group was also not significantly different from the forelimb or nontrain groups . both nontrained and forelimb trained control groups had significantly larger angular excursions of the hip and ankle , as well as significantly larger ankle extension ( figure 3 ) . the trained animals recovered to bbb scores necessitating the assessment of forelimb - hindlimb coordination . importantly , although the bbb can be used to assess coordination , we also utilized a more objective assessment of coordination , the regularity index ( ri ) . the ri is a score of plantar footfall pattern and limb coordination , and the trained group scored significantly better than the nontrained group ( figure 4 ) . the plantar stepping index ( psi ) , which is a ratio of plantar hindlimb to forelimb steps , revealed no differences between trained and sham controls , while nontrained and forelimb controls scored significantly lower than shams ( figure 4 ) . however , the trained group psi was also not significantly different from the forelimb or nontrain groups . analyses of gait parameters did not detect differences between groups for stride length , stride time , base of support , or toe velocity ( data not shown ) . sham animals were able to cross the horizontal ladder with only one or two errors ( 1.1 0.51 ) . although trained animals displayed improvements in overground locomotion and limb coordination , their ability to control fine placement of the hind paw remained impaired . there were no differences between any sci group ( train 7.67 2.88 ; forelimb 8.43 2.44 ; nontrain 7.42 3.80 ) . quantitative measurements of sensitivity to mechanical stimuli were obtained on two separate occasions prior to injury and then once per week for 8 weeks after sci in the trained , nontrained , and forelimb groups . preinjury measurements revealed that none of the groups demonstrated allodynic behavior to innocuous stimuli and were equivalent at baseline . , all rats exhibited a moderate degree of evoked at - level allodynia consisting of either freezing , escaping , or grabbing ( with or without vocalization ) towards a stimulus filament ranging from 15 g to 0.008 g. these pain - related aversion behaviors persisted throughout the course of training for all sci groups ( allodynia score at 8 weeks after sci : train 5.72 1.44 ; forelimb 5.19 1.47 ; nontrain 5.23 2.33 ) ( figure 5 ) . we examined a total of 12 urodynamic parameters as well as bladder weights to identify possible treatment effects . shams were directly compared to sci groups for bladder weight but not for urodynamics , as the fill rate of .25 ml / min causes different physiological outcomes in a very small bladder compared to a bladder twice the normal size . no significant differences were observed between trained , nontrained , and forelimb groups ( one - way anova , p > .05 ) . bladder weights of all sci animals were larger than shams ( bladder weight in milligrams : sham 143.3 25.2 ; train 226.5 52.7 ; forelimb 287.2 78.9 ; nontrain 240.0 65.6 ; one - way anova p = .013 ) [ 38 , 39 ] . intercontraction interval ( ici , seconds ) was not different between groups ( train 130.5 88.8 ; nontrain 120.0 51.1 ; forelimb 127.2 77.4 ) . voiding efficiency was not different between groups ( train 89.5 9.8 ; nontrain 92.3 8.3 ; forelimb 86.1 15.2 ) . home cage activity was monitored during the active phase and revealed that while all groups significantly decreased activity with time , there were no significant differences between any group when measuring ambulatory or total in - cage movements . this finding suggests that home cage activity was not a significant contributor to improved locomotion in the trained sci group . histological assessment of the injury started with the epicenter and continued 1 mm rostrally . figure 7 shows representative sections of the contused cord at the epicenter and .5 mm and 1.0 mm rostrally from the injury as well as an intact section . there were no significant differences between any group when analyzing weight gain ( each group gained approximately 100 grams ) , injury parameters , white matter at the epicenter , .5 mm , or 1.0 mm ( table 1 ) . the white matter was further subdivided into ventral and ventral lateral funiculi and gray matter of the ventral horn was also calculated at the epicenter , .5 mm , and 1.0 mm . there were no differences detected between any group when analyzing the ventral portions of the spinal cord . these data suggest that functional differences observed between groups can not be attributed to differences in the amount of spared white or gray matter . the method of training used in this study facilitated additional recovery of the trained group ( beyond the substantial amount of spontaneous recovery that occurred during the first two weeks following sci [ 18 , 26 , 40 ] ) and is the first to show overground improvements using quantitative kinematic and gait analyses in addition to qualitative open field scoring for contused male rats . the differences in locomotor parameters of trained versus nontrained and forelimb controls could not be attributed to differences in home cage activity , similar to a study comparing spontaneous exercise and enriched environment in which the amount of activity did not correlate to locomotor recovery . additionally , like other studies using activity based therapies [ 16 , 18 , 42 ] , we found that training did not increase white matter sparing , even when analyzing subdivisions of the spinal cord , such as the ventrolateral funiculus . these results suggest that training reinforced or facilitated plasticity within the remaining pathways or lumbosacral circuits rather than promoting regeneration or sprouting of new pathways . after 6 weeks of training , almost all trained sci animals had achieved weight supported stepping and degrees of forelimb - hindlimb coordination . in contrast , while many animals in the sci control groups recovered consistent weight supported stepping , recovery was slower and some animals never regained the ability to weight - support . these data are consistent with stand training in transected cats , which increased the duration of hindlimb standing [ 43 , 44 ] . in rats with contusion or compression injury , treadmill training improved ankle extension and weight bearing during open field locomotion . compared to our previous findings , as little as 30 minutes of daily locomotor training can improve weight bearing locomotor ability in sci animals ( 60 minutes of daily training did not further improve bbb scores ) . many studies perform quantitative kinematic measurements on the treadmill ( bipedal and quadrupedal ) where the hindlimb is passively extended during stance , the trunk is supported by a harness , and partial body weight support is provided . here , we used overground kinematic and gait analyses to quantify the training effects on full weight bearing overground locomotion compared to sham and sci controls . ankle extension and ankle and hip excursion were normalized by training ( more similar to shams ) . coordination and plantar paw placement were also normalized ( ri and psi ) . in cats , treadmill training increases the recruitment of flexor motor pools compared to nontrained cats . trained sci cats and rats have also been shown to have greater paw lift and hip flexion , respectively , both allowing a reduction in paw dragging [ 6 , 15 , 45 ] . our results extend these studies and indicate that training facilitates normal step cycle trajectories during overground locomotion by decreasing ankle extension and excursion , increasing hip excursion , and increasing the plantar stepping index . step training that provides alternate limb loading and rhythmic repeated steps can promote neural activity and improve emg patterns and amplitudes [ 17 , 46 , 47 ] , which could explain how ankle extension ( during stance ) , plantar paw placement , and weight support improved with training . importantly , not all aspects of motor control improved with quadrupedal training . although training promoted the recovery of overground locomotion , training did not improve the ability to cross a horizontal ladder . , this task requires precision paw placement and relies on proprioception [ 49 , 50 ] . while our training paradigm improved gross locomotion ( weight bearing , coordination , etc . ) , fine proprioceptive movements remained impaired , possibly due to the task specificity of training . locomotor training has been reported as having beneficial effects on bladder function in clinical settings [ 5153 ] . indeed , we previously demonstrated that 60 min of training significantly improved bladder function . however , our results according to cystometry and bladder weights in this study and sci - induced polyuria in our previous study indicate that 30 minutes of training did not affect bladder function after sci . indeed , we found that , with 60 minutes of training , both quadrupedal and forelimb training improved the maximum bladder contraction amplitude and reduced sci - induced polyuria . with respect to sensory function , the emergence of neuropathic pain after sci significantly impacts patients ' quality of life and interferes with functional recovery [ 5658 ] . in the clinical setting , we previously demonstrated that 60 min of locomotor training resulted in a significant improvement in at - level allodynia scores compared to the nontrained group . this effect was observed after 3 weeks of training and remained consistent throughout the course of training . other rodent models of sci , employing varying degrees of locomotor training intensity and duration , also report attenuations of allodynic responses following either below - level to the plantar aspects of the paws [ 62 , 63 ] or at - level mechanical threshold testing . in this study , 30 minutes of step training was not sufficient to ameliorate the onset of trunk at - level allodynia after sci . differences across studies may be attributed to the spinal location ( cervical versus thoracic ) and type of injury model ( contusion versus compression ) , gender , magnitude , and intensity of training as well as the region being tested ( trunk versus paw ) . it is not clear whether step training induces a level of resistance to the glabrous plantar aspect of the paw , perhaps influencing mechanical withdrawal thresholds . overall , the degree of spared spinal pathways , spontaneous recovery , and the rhythmic weight bearing load during stepping , which may promote activation of cutaneous and proprioceptive afferents , may all be driving factors influencing improvements in tactile sensation . a comparison between males and females may also be warranted as the development of pain and the requirements for differential effective exercise protocols may be sex - dependent [ 6567 ] . enriched environments and spontaneous exercise , such as wheel running , have been shown to improve locomotor recovery after sci [ 41 , 42 , 68 ] . the possibility exists that exercise could promote post - sci recovery through more general mechanisms , such as through alterations in inflammatory pathways , a nursing effect through neurotrophins , or increased well - being . in this study , we utilized a control group to induce exercise without specific activation of lumbar circuitry . in fact , although not statistically different , the forelimb trained sci animals scored slightly worse on multiple locomotor parameters , including bbb score , ankle flexion and extension , and ladder errors . these findings suggest that step training improves locomotor recovery through direct activation of lumbar circuitry . in contrast , according to our finding that 60 minutes of forelimb training significantly altered some parameters of urinary tract function , other mechanisms must be responsible for these nonlocomotor improvements . in conclusion , these factors are related not only to the injury but specific parameters of step training . these factors have been widely varied in experimental sci and are likely contributing to different conclusions about step training 's efficacy in an incomplete rat model of sci . our results suggest that 30 minutes of manually assisted step training initiated in the subacute stage of recovery can maximize potential locomotor gains . we found that 30 minutes of quadrupedal step training improved overground weight support , coordination , and ankle / hip range of motion . we also found that 30 minutes of daily training did not improve precision paw placement , bladder function , or allodynia . while 60 minutes of daily training does not result in even higher bbb scores or an increase in the percentage of animals that can weight - support , longer daily training sessions result in additional benefits to nonlocomotor functions ( allodynia and bladder function ) , which would substantially influence a patient 's quality of life .

we previously demonstrated that daily , hour - long training sessions significantly improved both locomotor ( limb kinematics , gait , and hindlimb flexor - extensor bursting patterns ) and nonlocomotor ( bladder function and at - level mechanical allodynia ) functions following a moderate contusive spinal cord injury . the amount of training needed to achieve this recovery is unknown . furthermore , whether this recovery is induced primarily by neuronal activity below the lesion or other aspects related to general exercise is unclear . therefore , the current study objectives were to ( 1 ) test the efficacy of 30 minutes of step training for recovery following a clinically relevant contusion injury in male wistar rats and ( 2 ) test the efficacy of training without hindlimb engagement . the results indicate that as little as 30 minutes of step training six days per week enhances overground locomotion in male rats with contusive spinal cord injury but does not alter allodynia or bladder function . thirty minutes of forelimb - only exercise did not alter locomotion , allodynia , or bladder function , and neither training protocol altered the amount of in - cage activity . taken together , locomotor improvements were facilitated by hindlimb step training for 30 minutes , but longer durations of training are required to affect nonlocomotor systems .

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

studies in lower vertebrates have been useful for modeling how the cpg performs in the absence of supraspinal and afferent feedback as well as determining the roles of neurotransmitters and afferent stimuli ( load , speed , and perturbations ) . within the context of spinal cord injury ( sci ) , central pattern generation has become a conceptual basis for locomotor training after injury [ 2 , 3 ] . in rats , cats , and humans , a large amount of spontaneous recovery can occur following sci , and this recovery is closely related to white matter sparing [ 46 ] . specifically , this recovery has mainly been attributed to spared fibers in the ventral and ventral lateral funiculi where the rubrospinal , reticulospinal , and vestibulospinal tracts are located [ 7 , 8 ] . yet , the recovery achieved via training is generally specific to the task practiced , for example , stand , step , or swim training [ 9 , 10 ] . the amount of activity imposed on the limbs is also a crucial component of locomotor rehabilitation . importantly , step training on a treadmill using body weight support should provide a high number of repetitions to facilitate motor learning . recovery potential therefore is a function of ( 1 ) amount of sparing / injury severity , ( 2 ) task specificity , and ( 3 ) amount of activity ( activity dependent plasticity ) . other aspects of step training or general exercise may also contribute to sci recovery , including environmental enrichment , intermittent hypoxia , and general improvements in body strength and psychological well - being . more studies are needed to determine the efficacy and mechanisms of training on functional outcomes ( including locomotion , allodynia , and autonomic functions ) after incomplete contusions in combination with appropriate control groups , kinematics , and overground locomotion ( for review see ) . therefore , a clinically relevant rat contusion model of sci may provide very useful information for the study and translation of locomotor training . research with spinally complete and incomplete rodents has affirmed central pattern generation and identified important aspects of training and sci locomotion [ 11 , 14 , 15 ] . while it is clear that training influences aspects of treadmill locomotion post - sci , basic research studies have yielded conflicting outcomes regarding overground locomotion [ 1619 ] . furthermore , very little is known about the efficacy of step training on nonlocomotor functions . we previously demonstrated that 60 minutes of training 7 days per week significantly improved both locomotor and nonlocomotor functions , such as open field locomotion , hindlimb kinematics , at - level mechanical allodynia , and bladder function in contused rats . in the current study , we ( 1 ) tested the efficacy of a 30-minute body weight supported treadmill step training paradigm for the recovery of overground locomotion using qualitative scoring ( bbb ) and quantitative ( kinematic ) locomotor tests , ( 2 ) determined the contribution of in - cage activity to spontaneous locomotor recovery versus training - induced recovery , and ( 3 ) examined bladder function and allodynia ( pain response to a nonnoxious stimulus ) . all animal procedures were performed according to the nih guidelines , and the protocols were reviewed and approved by the institutional animal use and care committee at the university of louisville , school of medicine . training began at two weeks after sci for thirty minutes per day , six days per week and continued for six weeks . animals were anesthetized with an intraperitoneal dose of ketamine ( 80 mg / kg , ketoset , fort dodge laboratories , fort dodge , ia ) and xylazine ( 10 mg / kg , anased , lloyd laboratories , shenandoah , ia ) mixture . the muscle was closed with suture , the skin closed with michel clips , and topical antibiotic applied . step training was performed on the exer3 treadmill system ( columbus instruments , columbus , oh ) customized with spring scales for body weight support . mission viejo , ca ) and hook - and - loop material and velcro straps . trained animals began quadrupedal step training 2 weeks after sci surgery , 6 days per week , 30 minutes per day , for 6 weeks , at 22 cm / s . trainers adjusted the body weight support as needed using manual assistance at the hip flexor region to facilitate proper plantar placement , for example , complete toe extension , no ankle rotation , and incorporation of forelimb - hindlimb coordination with minimal assistance . for nontrained animals , one trained , one nontrained , and one forelimb trained animal could be harnessed simultaneously . sham animals were not exposed to the treadmill system and were handled four times per week ( minimum ) . rats were not required to complete the 30-minute session during the first week of training if they exhibited signs of stress , that is , porphyrin staining around the eyes or nose , irregular breathing , or excessive diarrhea . an open field locomotor assessment , the basso - beattie - bresnahan ( bbb ) scale , was used to evaluate hindlimb function in the rats . once per week , each animal was placed in an open field and tested for 4 minutes by the same two scorers , who were presented with the trained , forelimb , nontrained , and sham animals in random order ( blinded to group ) . the bbb uses a 21-point scale for locomotion , which rates parameters , such as joint movements ( 08 ) , weight support ( 913 ) , and paw placement ( 1421 ) . after the six - week training period the hindquarters were shaved and the bony landmarks on the lateral side of the left and right hindlimbs were marked with permanent marker : iliac crest , greater trochanter , lateral malleolus , and metatarsophalangeal joint ( ilium , hip , ankle , and toe ) . the iliac crest , hip , ankle ( lateral malleolus ) , toe , and paw were digitized by a blinded observer , and the hip - ankle - toe ( hat ) and iliac crest - hip - ankle ( iha ) angles were marked to quantify the movement of the hindlimbs during overground stepping . four animals ( n = 3 forelimb ; n = 1 nontrain ) could not generate weight bearing steps ( bbb = 8 ; 8 weeks after sci ) ; these animals did not participate in the ri or psi tests . after each crossing , the raters discussed and agreed on the error count . if , at the end of the crossing , the raters did not agree on error count within two errors , the trial was repeated . once per week , testing sessions commenced at approximately the same time of day ( 9 am before the start of training ) . the dorsolateral trunk ( t7t9 dermatomal level ) just above the t9 spinal injury level was tested bilaterally for at - level mechanical hypersensitivity to touch and gentle touch / squeeze . at the start of testing , the top of the cage was removed and the animal was allowed to acclimate to the environment for 2 minutes . while in its cage , each animal was stroked at the dorsolateral trunk five times bilaterally with a number 5 paintbrush ( 1.5 0.5 cm bristles ; average pressure , 15 g ) in an alternating rostral / caudal plane . after each stroking stimulus , the presence / absence of any evoked responses that were indicative of pain was noted : ( 1 ) a freezing response ( stopping of normal activity and staying still in response to the stimulus ) , ( 2 ) escape ( any movement of the animal away from the stimulus probe ) , and ( 3 ) grabbing at or pushing away the stimulus probe with their forepaws . if the animal responded , a lower gram filament was applied to test the animal 's sensitivity . if the animal was not responsive to the initial probing stimulus , the next greatest gram filament ( and repeated if needed ) was used to determine the threshold of the animal 's sensitivity . , evoked pain - like behavioral response to less than 60% of the stimuli less than three of five strokes ) , a gentle squeeze / pressure test was conducted to determine if the animal had increased sensitivity to a stronger mechanical stimulus over a wider surface area ( which also normally does not provoke avoidance behaviors and is thus considered innocuous ) . in this instance , the animal 's skin is gently squeezed with a pair of modified adson tissue forceps ( 2.0 mm wide tips ) , which is equivalent to the 60 g semmes - weinstein monofilament . after the testing session , animals were scored for their degree of at - level allodynia based on a 10-point scale , with 10 being the maximum score an animal could receive . the bladder was emptied , and the tubing was passed through the subcutaneous tissue and exteriorized behind the neck . after a 1.5-hour recovery period , the animal was placed in a darkened box for cystometric recordings . urodynamic data ( voiding and nonvoiding events , voided volumes , and animal movements or spasms ) and experimenter notes were recorded on video for offline playback and analysis with datawave software ( http://www.dwavetech.com/ ) . the in - cage activity of every animal was recorded using an opto - m3 infrared activity monitor ( columbus instruments , columbus , oh ) . , the animal was moving across the cage ) and total movements ( number of beam breaks ) during the active phase ( 6 pm6 am ) . the bladder was blunt dissected away from the prostate , blotted dry , and weighed . the lesion area was quantified as previously described using spot advanced software ( diagnostic instruments , sterling heights , mi ) and the nikon e400 microscope . briefly , white matter was divided into four regions ( dorsal columns , dorsolateral funiculus , ventrolateral funiculus , and the ventral funiculus ) and each area was subdivided into left and right sides . medial edges of the dorsal horn , and the tips of the ventral horn were used as landmarks for the divisions . the percent of white matter sparing ( wms ) was determined by dividing the white matter remaining at the epicenter , .5 mm rostrally , and 1.0 mm rostrally by the average area of white matter present in intact sections . one - way repeated measures analysis of variance ( anova ) ( fixed effects ) was performed for tests of within subject and between subject effects followed by bonferroni post hoc t - tests for the bbb , allodynia , and activity . one - way anova ( fixed effects ) was used for cystometry parameters , bladder weight , gait , and kinematics followed by bonferroni post hoc t - tests , significance level p < .05 . at the initiation of training ( 2 weeks after sci ) all groups functionally displayed plantar paw placement with occasional weight support ( bbb scores : trained : 9.61 1.7 ; forelimb : 9.5 2.2 ; nontrained 9.3 2.0 ) . after 3 weeks of training ( 5 weeks after sci ) , trained animals ' bbb scores were significantly higher compared to pretraining and remained significantly higher through the rest of the study ( figure 1 ) . no differences were found between sham and trained animals after week 3 ( figure 2 ) . however , the trained group was also not significantly different from the forelimb or nontrain groups . the trained animals recovered to bbb scores necessitating the assessment of forelimb - hindlimb coordination . importantly , although the bbb can be used to assess coordination , we also utilized a more objective assessment of coordination , the regularity index ( ri ) . the ri is a score of plantar footfall pattern and limb coordination , and the trained group scored significantly better than the nontrained group ( figure 4 ) . analyses of gait parameters did not detect differences between groups for stride length , stride time , base of support , or toe velocity ( data not shown ) . sham animals were able to cross the horizontal ladder with only one or two errors ( 1.1 0.51 ) . although trained animals displayed improvements in overground locomotion and limb coordination , their ability to control fine placement of the hind paw remained impaired . quantitative measurements of sensitivity to mechanical stimuli were obtained on two separate occasions prior to injury and then once per week for 8 weeks after sci in the trained , nontrained , and forelimb groups . , all rats exhibited a moderate degree of evoked at - level allodynia consisting of either freezing , escaping , or grabbing ( with or without vocalization ) towards a stimulus filament ranging from 15 g to 0.008 g. these pain - related aversion behaviors persisted throughout the course of training for all sci groups ( allodynia score at 8 weeks after sci : train 5.72 1.44 ; forelimb 5.19 1.47 ; nontrain 5.23 2.33 ) ( figure 5 ) . no significant differences were observed between trained , nontrained , and forelimb groups ( one - way anova , p > .05 ) . bladder weights of all sci animals were larger than shams ( bladder weight in milligrams : sham 143.3 25.2 ; train 226.5 52.7 ; forelimb 287.2 78.9 ; nontrain 240.0 65.6 ; one - way anova p = .013 ) [ 38 , 39 ] . home cage activity was monitored during the active phase and revealed that while all groups significantly decreased activity with time , there were no significant differences between any group when measuring ambulatory or total in - cage movements . this finding suggests that home cage activity was not a significant contributor to improved locomotion in the trained sci group . there were no significant differences between any group when analyzing weight gain ( each group gained approximately 100 grams ) , injury parameters , white matter at the epicenter , .5 mm , or 1.0 mm ( table 1 ) . the white matter was further subdivided into ventral and ventral lateral funiculi and gray matter of the ventral horn was also calculated at the epicenter , .5 mm , and 1.0 mm . there were no differences detected between any group when analyzing the ventral portions of the spinal cord . these data suggest that functional differences observed between groups can not be attributed to differences in the amount of spared white or gray matter . the method of training used in this study facilitated additional recovery of the trained group ( beyond the substantial amount of spontaneous recovery that occurred during the first two weeks following sci [ 18 , 26 , 40 ] ) and is the first to show overground improvements using quantitative kinematic and gait analyses in addition to qualitative open field scoring for contused male rats . the differences in locomotor parameters of trained versus nontrained and forelimb controls could not be attributed to differences in home cage activity , similar to a study comparing spontaneous exercise and enriched environment in which the amount of activity did not correlate to locomotor recovery . additionally , like other studies using activity based therapies [ 16 , 18 , 42 ] , we found that training did not increase white matter sparing , even when analyzing subdivisions of the spinal cord , such as the ventrolateral funiculus . after 6 weeks of training , almost all trained sci animals had achieved weight supported stepping and degrees of forelimb - hindlimb coordination . compared to our previous findings , as little as 30 minutes of daily locomotor training can improve weight bearing locomotor ability in sci animals ( 60 minutes of daily training did not further improve bbb scores ) . many studies perform quantitative kinematic measurements on the treadmill ( bipedal and quadrupedal ) where the hindlimb is passively extended during stance , the trunk is supported by a harness , and partial body weight support is provided . here , we used overground kinematic and gait analyses to quantify the training effects on full weight bearing overground locomotion compared to sham and sci controls . our results extend these studies and indicate that training facilitates normal step cycle trajectories during overground locomotion by decreasing ankle extension and excursion , increasing hip excursion , and increasing the plantar stepping index . step training that provides alternate limb loading and rhythmic repeated steps can promote neural activity and improve emg patterns and amplitudes [ 17 , 46 , 47 ] , which could explain how ankle extension ( during stance ) , plantar paw placement , and weight support improved with training . although training promoted the recovery of overground locomotion , training did not improve the ability to cross a horizontal ladder . locomotor training has been reported as having beneficial effects on bladder function in clinical settings [ 5153 ] . indeed , we previously demonstrated that 60 min of training significantly improved bladder function . however , our results according to cystometry and bladder weights in this study and sci - induced polyuria in our previous study indicate that 30 minutes of training did not affect bladder function after sci . indeed , we found that , with 60 minutes of training , both quadrupedal and forelimb training improved the maximum bladder contraction amplitude and reduced sci - induced polyuria . with respect to sensory function , the emergence of neuropathic pain after sci significantly impacts patients ' quality of life and interferes with functional recovery [ 5658 ] . in the clinical setting , we previously demonstrated that 60 min of locomotor training resulted in a significant improvement in at - level allodynia scores compared to the nontrained group . this effect was observed after 3 weeks of training and remained consistent throughout the course of training . other rodent models of sci , employing varying degrees of locomotor training intensity and duration , also report attenuations of allodynic responses following either below - level to the plantar aspects of the paws [ 62 , 63 ] or at - level mechanical threshold testing . in this study , 30 minutes of step training was not sufficient to ameliorate the onset of trunk at - level allodynia after sci . differences across studies may be attributed to the spinal location ( cervical versus thoracic ) and type of injury model ( contusion versus compression ) , gender , magnitude , and intensity of training as well as the region being tested ( trunk versus paw ) . overall , the degree of spared spinal pathways , spontaneous recovery , and the rhythmic weight bearing load during stepping , which may promote activation of cutaneous and proprioceptive afferents , may all be driving factors influencing improvements in tactile sensation . in fact , although not statistically different , the forelimb trained sci animals scored slightly worse on multiple locomotor parameters , including bbb score , ankle flexion and extension , and ladder errors . in contrast , according to our finding that 60 minutes of forelimb training significantly altered some parameters of urinary tract function , other mechanisms must be responsible for these nonlocomotor improvements . in conclusion , these factors are related not only to the injury but specific parameters of step training . these factors have been widely varied in experimental sci and are likely contributing to different conclusions about step training 's efficacy in an incomplete rat model of sci . our results suggest that 30 minutes of manually assisted step training initiated in the subacute stage of recovery can maximize potential locomotor gains . we found that 30 minutes of quadrupedal step training improved overground weight support , coordination , and ankle / hip range of motion . we also found that 30 minutes of daily training did not improve precision paw placement , bladder function , or allodynia . while 60 minutes of daily training does not result in even higher bbb scores or an increase in the percentage of animals that can weight - support , longer daily training sessions result in additional benefits to nonlocomotor functions ( allodynia and bladder function ) , which would substantially influence a patient 's quality of life .

inflammation of the dental pulp ( pulpitis ) can be a progressive and devastating pain experience characterized by spontaneous or provoked pain , hyperalgesia , allodynia and difficulty in achieving adequate local anesthesia . the activation of dental pulp or peridental nociceptors during endodontic inflammation elicits a pain response that contributes to approximately 90% of dental emergency visits in both private dental clinics and in hospitals . the economic implication of these emergency visits is reported to cost almost us$1 billion per year . however , despite the cost and the prevalence of endodontic disease and the great discomfort associated with it , the fundamental molecular aspects of its pathogenesis are still not fully understood . the current literature on pulpal immune response to microbial infection continues to expand but very little is known on the regulatory mechanisms behind pulpal disease . cells comprising the human dental pulp trigger immune responses to a complex array of microorganisms that invade dental tissues . these immunocompetent cells also form mechanical barriers ( i.e. odontoblasts ) , detect and transmit sensations ( nerve fibers ) or differentiate ( i.e. dental pulp stem cells ) to limit infection , signal injury and promote repair , respectively . these cascades of events resulting from dental pulp stimulation by microorganisms result in the release of a plethora of immune mediators that trigger pulpal or odontogenic pain , inflammation or in advanced stages , pulpal necrosis . in addition , several studies have suggested that pulpal events can be reflected in the gingival crevicular fluid ( gcf ) through measurable levels of protein markers that correlated with pulpal symptoms . this shows that the dental pulp is not an isolated environment but rather a vital , reactive tissue that communicates with the outside environment . cytokines , cell surface receptors and other protein markers are shown to be either highly increased or decreased in inflamed dental pulp .. a limited number of studies have examined gene expression in inflamed human pulps . these studies , however , did not explore the differences in gene expression between normal and inflamed dental pulp and the clinical presentation of donor patients ( i.e. pain and swelling ) , which may provide biologic explanation on the variability of clinical signs and symptoms of pulpal inflammation that confound diagnosis . although the clinical value of a molecular diagnostic marker may at first appear limited in scope , the emerging correlation of gene expression with clinical signs and symptoms will enhance our understanding on the development of pulpal inflammation - this will add not only to the knowledge base but it will also provide a biological basis for the varying clinical presentations of pulpitis . prior studies that focused on histological findings have shown a wide variation from poor to strong - in correlating clinical signs and symptoms with histological findings . in this study , data from the full genome scan will be utilized to determine if an association exists between gene expression and clinical presentation ( i.e. pain ) of pulpitis patients . the sam software generated gsea data which showed a significantly higher expression of various gene sets that are associated with immune response activation , maintaining cellular function and cell - to - cell interaction , among others ( figure 1 ) in pulpitis samples . this underscores the utility of both the subjective ( patient - derived history ) and objective ( endodontist - performed testing ) diagnostic techniques that clinically delineate a normal from an inflamed dental pulp . furthermore , the results above re - establish the immunocompetency of the dental pulp that has been shown to carry toll - like receptor ( tlr ) -2/4 + cells . among the patients diagnosed with irreversible pulpitis , eight patients reported experiencing zero to mild pain ( ( 30 mm on vas ) and twelve patient reported moderate to severe pain . the mean vas scores for patients who reported zero to mild pain and moderate to severe pain was 6.63 ( sd = 9.29 ) and 81.93 ( sd = 11.21 ) , respectively ( p mild pain samples showed a significantly higher expression of genes involved in adaptive immune system , cytokine to cytokine interaction and cytokine signaling , among others . however , when looking at specific genes , several of them that have key roles in inflammation are significantly under - expressed , unchanged or over - expressed in asymptomatic or mild pain patients compared to those with moderate to severe pain ( table 1 ) . patients often judge the success of treatment and the efficiency of the dentist based on their pain experience . the decision whether to perform root canal therapy ( rct ) to relieve pain or manage infection relies heavily on clinical diagnostic tests which dichotomize the pulpal diagnosis to reversible and irreversible pulpitis . to arrive at a diagnosis , dentists depend mainly on pain history and responses to sensibility tests ( namely thermal tests and electric pulp testing , ) that have been used for decades ; but whether the responses to these clinical tests correlate well with pulpal histopathology or not remains unclear . this study was aimed to identify the differences in gene expression between normal pulps and pulpitis samples and between mild and moderate to severe pulpitis pain , based on the diagnostic guidelines set by the aae and on the patient - reported pain experience at the time of endodontic treatment , respectively . gene set enrichment analysis ( gsea ) was employed for the analysis of the thousands of genes included in the microarray screening . the broad institute defines gsea as a computational method that determines whether an a priori defined set of genes shows statistically significant , concordant differences between two biological states . first , a biological basis at the molecular level can now be correlated not only with the current diagnostic testing procedures but also with the diagnosis of irreversible pulpitis itself . molecular alterations precede histopathological changes and in the case of the dental pulp , there can be a wide array of histological presentations depending on the timing of pulp extirpation ; thus , there are conflicting reports concerning the clinical presentation and actual histopathology of the pulp . studies investigating inflammation at the molecular level may therefore provide a more accurate representation of the inflammatory changes in the dental pulp . next , the results may be used to cluster genes that are overexpressed or underexpressed in inflamed pulps and to use this information as biological basis for future studies on acute pain and on oral biology , especially those that focus on odontogenic pain and regeneration as these studies require a broad understanding of molecular changes that dictate clinical and biological outcomes . the difference in gene expression between normal and inflamed dental pulps ( figure 1 ) may have been the expected result considering that cytokines , chemokines and their receptors are known to be overexpressed in inflammatory conditions ; however , this study is still an important addition to our current understanding of pulp biology as it provides a broad picture or a snap - shot of the molecular changes occurring in the dental pulp during the very moment that the patient is seeking endodontic treatment on the dental chair . another remarkable finding in the study is the overexpressed genes related to inflammation and immune response in pulps from patients who reported zero to mild pain ( 30 mm on vas ) during their endodontic appointment compared to those who had moderate to severe pain ( figure 2 ) . moreover , looking at individual genes separately from the a priori gene sets in the gsea software revealed that genes involved in key inflammatory response like il8 , tnfa , il1a , ilib and several immunoglobulins ( i.e. ighm , ighv4 - 31 , igkv2d-40 ) , among others ( table 1 ) are expressed at relatively lower levels in pulp samples from asymptomatic to mild pain patient compared to those with moderate to severe pain . il8 has been recently associated with complex regional pain syndrome ( crps ) and il1b has been shown to contribute to the upregulation of nerve growth factor ( ngf ) during inflammation that consequently induces inflammatory hyperalgesia . both il8 and il1b are expressed at relatively lower levelsin zero to mild pain pulpitis patients . several genes that are upstream on the inflammatory cascade of both il1b and il8 ( e.g. tlr2 and tlr44 ) are significantly increased in pulpitis samples ( table 1 ) . furthermore , most genes associated with structural formation and integrity ( e.g. col1 - 4 and mmps ) are expressed at relatively higher levels in pulpitis samples . in contrast however , several genes that are known to be pain mediators or associated with increased pain experience did not have significant discordance between mild and moderate to severe pain ( table 1 ) . for example , prostaglandin - endoperoxide synthase 2 or cyclooxygenase-2 ( pgs2 or cox2 ) , an important inflammatory enzyme that has been shown to be significantly elevated in pulpitis tissues did not have expression difference in mild pain but is significantly elevated in pulpitis compared with normal controls ( table 1 ) . associated molecular pattern receptors , including tlrs , are key molecules in the response to pathological stimuli . tlrs are expressed by both immune and non - immune cells in the pulp including neurons , fibroblasts , endothelial cells , epithelial cells and others . they recognize viral and microbial structures as well as self molecules ( such as single stranded rnas ) that may accumulate in non - physiologic amounts or sites . tlr ligands stimulate the production of pro - pain molecules and , in this way , may contribute to the pathogenesis of pain . in this study , the expression of several tlrs ( 1,2,4,6 , 8 and 9 ) were upregulated and tlr 3 was down regulated in inflamed pulps as compared to normal pulps . our data also show that expression of tlr8 was higher in pulpits patients experiencing severe pain as compared to those who reported no pain or mild pain . this is of particular interest given the recent finding that murine tlr7 ( which is phylogenetically and structurally related to human tlr8 ) interacts with trpa1 and that specific mirnas activate nociceptors via tlr7 and trpa1 . arriving at a reliable diagnosis that matches the histopathology of the dental pulp , especially during a pain episode is arguably the most challenging part of endodontics . the dental pulp is a dynamic tissue that is capable of responding to insults and of healing so that diagnosis may change over time . furthermore , individual variations in immunologic and pain response further complicate not only the clinical but also the biological aspect of endodontic diagnosis . this may be the one of the main reasons for the observational differences among studies correlating clinical and histopathological diagnosis as pointed out in the earlier part of this paper . for example , molecular markers collected from dentinal fluids may be used to determine the pulpal status . the study of tissue molecular profiles to better understand and diagnose disease pathophysiology has been examined in another common oral disease- periodontitis . using biopsies from patients diagnosed with either chronic periodontitis or aggressive periodontitis , kebschull et al . , explored whether molecular profiling can form the basis for a pathobiology based classification that correlates with the phenotypic features of the disease . the clusters had distinct molecular signatures which did not align with the current classifications of periodontitis . however , they did translate into distinct phenotypic differences and can provide a basis for novel classification . this study along with current study supports the use of molecular profiling to classify disease . while microarray analysis is a powerful tool to study the simultaneous expression of several genes it has some inherent limitations . these include technical factors such as limited dynamic range , signal saturations and cross - hybridization . it is important to also remember that the cellular profile of inflamed pulps differs from that of normal pulps . the residents cells in normal pulps include fibroblasts , odontoblasts and some immune cells such as macrophages , dendritic cells and mast cells . inflamed pulps are characterized by an influx of immune cells and as such the results reported here may be in part , due to differences in the cellular makeup as well . the present study shows that there are differences in gene expression between normal and inflamed pulps and ; between zero to mild and moderate to severe pulpitis pain . among the genes reported to have a strong association with pain and inflammation , il8 showed the highest fold change ( 35.9 , q - value = 0 ) in pulpitis samples and it is significantly under - expressed in mild pain compared with moderate to severe pain ( 0.14 , q - value = 0 ) . il8 is increased in gingival crevicular fluid from patients with acute pulpitis and might be a useful measurement for staging patients with acute pulpitis . this study provides a molecular basis for the clinical diagnosis of pulpitis and for the differences or similarities between asymptomatic to mild pulpal pain vis - - vis moderate to severe pain . with an enhanced understanding of pulpal inflammation , future studies on treatment and management of pulpitis and on pain associated with it the sam software generated gsea data which showed a significantly higher expression of various gene sets that are associated with immune response activation , maintaining cellular function and cell - to - cell interaction , among others ( figure 1 ) in pulpitis samples . this underscores the utility of both the subjective ( patient - derived history ) and objective ( endodontist - performed testing ) diagnostic techniques that clinically delineate a normal from an inflamed dental pulp . furthermore , the results above re - establish the immunocompetency of the dental pulp that has been shown to carry toll - like receptor ( tlr ) -2/4 + cells . among the patients diagnosed with irreversible pulpitis , eight patients reported experiencing zero to mild pain ( ( 30 mm on vas ) and twelve patient reported moderate to severe pain . the mean vas scores for patients who reported zero to mild pain and moderate to severe pain was 6.63 ( sd = 9.29 ) and 81.93 ( sd = 11.21 ) , respectively ( p mild pain samples showed a significantly higher expression of genes involved in adaptive immune system , cytokine to cytokine interaction and cytokine signaling , among others . however , when looking at specific genes , several of them that have key roles in inflammation are significantly under - expressed , unchanged or over - expressed in asymptomatic or mild pain patients compared to those with moderate to severe pain ( table 1 ) . patients often judge the success of treatment and the efficiency of the dentist based on their pain experience . the decision whether to perform root canal therapy ( rct ) to relieve pain or manage infection relies heavily on clinical diagnostic tests which dichotomize the pulpal diagnosis to reversible and irreversible pulpitis . to arrive at a diagnosis , dentists depend mainly on pain history and responses to sensibility tests ( namely thermal tests and electric pulp testing , ) that have been used for decades ; but whether the responses to these clinical tests correlate well with pulpal histopathology or not remains unclear . this study was aimed to identify the differences in gene expression between normal pulps and pulpitis samples and between mild and moderate to severe pulpitis pain , based on the diagnostic guidelines set by the aae and on the patient - reported pain experience at the time of endodontic treatment , respectively . gene set enrichment analysis ( gsea ) was employed for the analysis of the thousands of genes included in the microarray screening . the broad institute defines gsea as a computational method that determines whether an a priori defined set of genes shows statistically significant , concordant differences between two biological states . first , a biological basis at the molecular level can now be correlated not only with the current diagnostic testing procedures but also with the diagnosis of irreversible pulpitis itself . molecular alterations precede histopathological changes and in the case of the dental pulp , there can be a wide array of histological presentations depending on the timing of pulp extirpation ; thus , there are conflicting reports concerning the clinical presentation and actual histopathology of the pulp . studies investigating inflammation at the molecular level may therefore provide a more accurate representation of the inflammatory changes in the dental pulp . next , the results may be used to cluster genes that are overexpressed or underexpressed in inflamed pulps and to use this information as biological basis for future studies on acute pain and on oral biology , especially those that focus on odontogenic pain and regeneration as these studies require a broad understanding of molecular changes that dictate clinical and biological outcomes . the difference in gene expression between normal and inflamed dental pulps ( figure 1 ) may have been the expected result considering that cytokines , chemokines and their receptors are known to be overexpressed in inflammatory conditions ; however , this study is still an important addition to our current understanding of pulp biology as it provides a broad picture or a snap - shot of the molecular changes occurring in the dental pulp during the very moment that the patient is seeking endodontic treatment on the dental chair . another remarkable finding in the study is the overexpressed genes related to inflammation and immune response in pulps from patients who reported zero to mild pain ( 30 mm on vas ) during their endodontic appointment compared to those who had moderate to severe pain ( figure 2 ) . moreover , looking at individual genes separately from the a priori gene sets in the gsea software revealed that genes involved in key inflammatory response like il8 , tnfa , il1a , ilib and several immunoglobulins ( i.e. ighm , ighv4 - 31 , igkv2d-40 ) , among others ( table 1 ) are expressed at relatively lower levels in pulp samples from asymptomatic to mild pain patient compared to those with moderate to severe pain . il8 has been recently associated with complex regional pain syndrome ( crps ) and il1b has been shown to contribute to the upregulation of nerve growth factor ( ngf ) during inflammation that consequently induces inflammatory hyperalgesia . both il8 and il1b are expressed at relatively lower levelsin zero to mild pain pulpitis patients . several genes that are upstream on the inflammatory cascade of both il1b and il8 ( e.g. tlr2 and tlr44 ) are significantly increased in pulpitis samples ( table 1 ) . furthermore , most genes associated with structural formation and integrity ( e.g. col1 - 4 and mmps ) are expressed at relatively higher levels in pulpitis samples . in contrast however , several genes that are known to be pain mediators or associated with increased pain experience did not have significant discordance between mild and moderate to severe pain ( table 1 ) . for example , prostaglandin - endoperoxide synthase 2 or cyclooxygenase-2 ( pgs2 or cox2 ) , an important inflammatory enzyme that has been shown to be significantly elevated in pulpitis tissues did not have expression difference in mild pain but is significantly elevated in pulpitis compared with normal controls ( table 1 ) . associated molecular pattern receptors , including tlrs , are key molecules in the response to pathological stimuli . tlrs are expressed by both immune and non - immune cells in the pulp including neurons , fibroblasts , endothelial cells , epithelial cells and others . they recognize viral and microbial structures as well as self molecules ( such as single stranded rnas ) that may accumulate in non - physiologic amounts or sites . tlr ligands stimulate the production of pro - pain molecules and , in this way , may contribute to the pathogenesis of pain . in this study , the expression of several tlrs ( 1,2,4,6 , 8 and 9 ) were upregulated and tlr 3 was down regulated in inflamed pulps as compared to normal pulps . our data also show that expression of tlr8 was higher in pulpits patients experiencing severe pain as compared to those who reported no pain or mild pain . this is of particular interest given the recent finding that murine tlr7 ( which is phylogenetically and structurally related to human tlr8 ) interacts with trpa1 and that specific mirnas activate nociceptors via tlr7 and trpa1 . arriving at a reliable diagnosis that matches the histopathology of the dental pulp , especially during a pain episode is arguably the most challenging part of endodontics . the dental pulp is a dynamic tissue that is capable of responding to insults and of healing so that diagnosis may change over time . furthermore , individual variations in immunologic and pain response further complicate not only the clinical but also the biological aspect of endodontic diagnosis . this may be the one of the main reasons for the observational differences among studies correlating clinical and histopathological diagnosis as pointed out in the earlier part of this paper . for example , molecular markers collected from dentinal fluids may be used to determine the pulpal status . the study of tissue molecular profiles to better understand and diagnose disease pathophysiology has been examined in another common oral disease- periodontitis . using biopsies from patients diagnosed with either chronic periodontitis or aggressive periodontitis , kebschull et al . , explored whether molecular profiling can form the basis for a pathobiology based classification that correlates with the phenotypic features of the disease . the clusters had distinct molecular signatures which did not align with the current classifications of periodontitis . however , they did translate into distinct phenotypic differences and can provide a basis for novel classification . this study along with current study supports the use of molecular profiling to classify disease . while microarray analysis is a powerful tool to study the simultaneous expression of several genes it has some inherent limitations . these include technical factors such as limited dynamic range , signal saturations and cross - hybridization . it is important to also remember that the cellular profile of inflamed pulps differs from that of normal pulps . the residents cells in normal pulps include fibroblasts , odontoblasts and some immune cells such as macrophages , dendritic cells and mast cells . inflamed pulps are characterized by an influx of immune cells and as such the results reported here may be in part , due to differences in the cellular makeup as well . the present study shows that there are differences in gene expression between normal and inflamed pulps and ; between zero to mild and moderate to severe pulpitis pain . among the genes reported to have a strong association with pain and inflammation , il8 showed the highest fold change ( 35.9 , q - value = 0 ) in pulpitis samples and it is significantly under - expressed in mild pain compared with moderate to severe pain ( 0.14 , q - value = 0 ) . il8 is increased in gingival crevicular fluid from patients with acute pulpitis and might be a useful measurement for staging patients with acute pulpitis . this study provides a molecular basis for the clinical diagnosis of pulpitis and for the differences or similarities between asymptomatic to mild pulpal pain vis - - vis moderate to severe pain . with an enhanced understanding of pulpal inflammation , future studies on treatment and management of pulpitis and on pain associated with it eligible patients seeking treatment at the school of dentistry were recruited to participate in the study . demographic details , history of odontogenic pain , vital signs ( blood pressure , pulse rate ) , medical history ( including full details of any medication ) and patient 's smoking and oral habits were recorded . the inclusion criteria were adults ( 18 years or older ) presenting for endodontic treatment with no evidence of periapical pathoses ( i.e radiolucency , swelling , pressure sensitivity ) and no previous pulp therapy ( i.e. , pulp capping , etc ) . exclusion criteria were conditions requiring antibiotic prophylaxis or additional treatment procedures , debilitating disease , chronic pain , diabetes mellitus , hematological disorders or a history of taking centrally acting drugs ( e.g. tricyclic antidepressants ) known to interfere with the release of various pain mediators and/or modify pain experience within the previous 6 months or over - the - counter pain medicine within the last six hours , chronic use of medication known to affect the immune response and patients who were immunocompromised . diagnosis of normal pulp ( np ) and irreversible pulpitis ( ip ) was based on subjective and objective findings and was in accordance with the american association of endodontists ( aae ) guidelines . in brief , np is a clinical diagnostic category in which the pulp is symptom - free and normally responsive to diagnostic testing on the other hand , ip is clinical diagnosis indicating that the vital , inflamed pulp is incapable of healing . the symptoms include lingering and exacerbated pain in response to thermal stimuli , spontaneous pain , referred pain or no clinical symptoms but inflammation is evident after caries excavation or trauma . pain history , cold test ( endo ice refrigerant spray , coltene inc , cuyahoga falls , oh ) and electric pulp testing ( vitality scanner 2006 , sybron endodontics , orange , ca ) were used to determine pulpal diagnosis . percussion , palpation , probing , mobility and radiographs were used to rule out periapical pathosis . if lingering pain is present or significantly evoked by a stimulus compared with a control tooth , the patient was asked to quantify the intensity of pain by placing a mark on a 10-cm vas with anchors of no pain and worst pain imaginable . using a modified version of the vas recommended by jensen et al , a vas mark of 30 mm was classified under mild , 31 - 74 mm under moderate and 75 - 100 under severe pain . a total of 40 pulp samples from 28 patients with ip and from seven patients with np were included in the study . twelve of the ip samples presented with moderate to severe pain at the time of pulp extirpation . the total number of samples was based on a recently published paper by our group on the differences of micro - rna expression between normal and inflamed pulps and on a power analysis . with 20 samples in each group , we had 80% power to detect an effect size of 0.9 using a threshold of p<0.05 for significance . if we required a more stringent threshold of p<0.001 , then we would have 80% power to detect an effect size of 1.4 . given that large effect sizes are common in microarray experiments , we had adequate power to detect the genes that exhibited the largest expression differences between the two groups . moreover , we increased the power as much as needed by increasing the acceptable false discovery rate . given that this initial analysis is intended to be exploratory , a higher false discovery rate is acceptable in this study . thus , this study had adequate power to detect the genes with the largest group differences . the inflamed pulps were extirpated during root canal treatment and the normal pulps were collected right after tooth extraction for orthodontic or restorative purposes or for third molar removal . pulp collection methods were adapted from awawdeh et al with modifications . briefly , local anesthesia ( 2% lidocaine with 1:100,000 epinephrine ) was administered and an access cavity was prepared under rubber dam isolation . pulp tissue was extirpated using a sterile barbed broach or hedstrm - file , placed into a pre - weighed eppendorf tube , immediately frozen in liquid nitrogen and stored at 70c . for the controls and for some patients who chose to have extraction instead of root canal treatment , all extractions were performed under local anesthesia . immediately after extraction , each tooth were split in a vice fitted with a cutting edge and the pulp tissue was removed using a barbed broach or hedstrm - file . the pulp was then placed in an eppendorf tube , immediately frozen in liquid nitrogen and stored at 70c . total rna was isolated using a commercial kit ( qiagen rneasy kit , valencia , ca ) following the manufacturer 's specifications . quantity and purity of the total rna was analyzed using nano - drop ( thermo scientific , wilmington , de ) and bioanalyzer ( agilent technologies , sta . microarray experiments were conducted at the microarray core facility of unc - chapel hill lineberger cancer center . the nugen ovation pico wta system v2 and the nugen encore biotin module were used to prepare rna for hybridization onto affymetrix genechip human gene 2.0 arrays . briefly , 25 ng of total rna was converted into amplified spia cdna using the nugen ovation pico wta system v2 and accompanying user guide . 2.5 ug of spia cdna was fragmented and labeled using the nugen encore biotin module and accompanying user guide . the cocktail for hybridization onto an affymetrix human gene 2.1 st peg plate was prepared using the nugen ovation picosl wt system v2 user guide 's cocktail assembly table . the hybridization cocktails were denatured and hybridized on the human gene 2.1 st peg plate in the affymetrix genetitan mc instrument . basic data analysis of the arrays was carried out using the affymetrix expression console software . changes in gene expressions were analyzed by gene set enrichment analysis ( gsea ) ( broad institute , cambridge , ma ) and its leading edge analysis software that uses the permutation method . the difference in gene expression levels between groups were determined by the significance analysis of microarray ( sam ) program using a false discovery rate ( q - value ) of 5% . since this study analyzed over 53,600 genes , only those that were previously reported to play key roles in pain and inflammation will be discussed in this paper . in addition , only the key genes with a sam fold change of over 1.5 ( overexpressed ) and under 0.5 ( underexpressed ) to signify a potentially relevant clinical effect are reported . the inclusion criteria were adults ( 18 years or older ) presenting for endodontic treatment with no evidence of periapical pathoses ( i.e radiolucency , swelling , pressure sensitivity ) and no previous pulp therapy ( i.e. , pulp capping , etc ) . exclusion criteria were conditions requiring antibiotic prophylaxis or additional treatment procedures , debilitating disease , chronic pain , diabetes mellitus , hematological disorders or a history of taking centrally acting drugs ( e.g. tricyclic antidepressants ) known to interfere with the release of various pain mediators and/or modify pain experience within the previous 6 months or over - the - counter pain medicine within the last six hours , chronic use of medication known to affect the immune response and patients who were immunocompromised . diagnosis of normal pulp ( np ) and irreversible pulpitis ( ip ) was based on subjective and objective findings and was in accordance with the american association of endodontists ( aae ) guidelines . in brief , np is a clinical diagnostic category in which the pulp is symptom - free and normally responsive to diagnostic testing on the other hand , ip is clinical diagnosis indicating that the vital , inflamed pulp is incapable of healing . the symptoms include lingering and exacerbated pain in response to thermal stimuli , spontaneous pain , referred pain or no clinical symptoms but inflammation is evident after caries excavation or trauma . pain history , cold test ( endo ice refrigerant spray , coltene inc , cuyahoga falls , oh ) and electric pulp testing ( vitality scanner 2006 , sybron endodontics , orange , ca ) were used to determine pulpal diagnosis . percussion , palpation , probing , mobility and radiographs were used to rule out periapical pathosis . if lingering pain is present or significantly evoked by a stimulus compared with a control tooth , the patient was asked to quantify the intensity of pain by placing a mark on a 10-cm vas with anchors of no pain and worst pain imaginable . using a modified version of the vas recommended by jensen et al , a vas mark of 30 mm was classified under mild , 31 - 74 mm under moderate and 75 - 100 under severe pain . a total of 40 pulp samples from 28 patients with ip and from seven patients with np were included in the study . twelve of the ip samples presented with moderate to severe pain at the time of pulp extirpation . the total number of samples was based on a recently published paper by our group on the differences of micro - rna expression between normal and inflamed pulps and on a power analysis . with 20 samples in each group , we had 80% power to detect an effect size of 0.9 using a threshold of p<0.05 for significance . if we required a more stringent threshold of p<0.001 , then we would have 80% power to detect an effect size of 1.4 . given that large effect sizes are common in microarray experiments , we had adequate power to detect the genes that exhibited the largest expression differences between the two groups . moreover , we increased the power as much as needed by increasing the acceptable false discovery rate . given that this initial analysis is intended to be exploratory , a higher false discovery rate is acceptable in this study . thus , this study had adequate power to detect the genes with the largest group differences . the inflamed pulps were extirpated during root canal treatment and the normal pulps were collected right after tooth extraction for orthodontic or restorative purposes or for third molar removal . pulp collection methods were adapted from awawdeh et al with modifications . briefly , local anesthesia ( 2% lidocaine with 1:100,000 epinephrine ) was administered and an access cavity was prepared under rubber dam isolation . pulp tissue was extirpated using a sterile barbed broach or hedstrm - file , placed into a pre - weighed eppendorf tube , immediately frozen in liquid nitrogen and stored at 70c . the root canal treatment was then completed . for the controls and for some patients who chose to have extraction instead of root canal treatment , all extractions were performed under local anesthesia . immediately after extraction , each tooth were split in a vice fitted with a cutting edge and the pulp tissue was removed using a barbed broach or hedstrm - file . the pulp was then placed in an eppendorf tube , immediately frozen in liquid nitrogen and stored at 70c . total rna was isolated using a commercial kit ( qiagen rneasy kit , valencia , ca ) following the manufacturer 's specifications . quantity and purity of the total rna was analyzed using nano - drop ( thermo scientific , wilmington , de ) and bioanalyzer ( agilent technologies , sta . microarray experiments were conducted at the microarray core facility of unc - chapel hill lineberger cancer center . the nugen ovation pico wta system v2 and the nugen encore biotin module were used to prepare rna for hybridization onto affymetrix genechip human gene 2.0 arrays . briefly , 25 ng of total rna was converted into amplified spia cdna using the nugen ovation pico wta system v2 and accompanying user guide . 2.5 ug of spia cdna was fragmented and labeled using the nugen encore biotin module and accompanying user guide . the cocktail for hybridization onto an affymetrix human gene 2.1 st peg plate was prepared using the nugen ovation picosl wt system v2 user guide 's cocktail assembly table . the hybridization cocktails were denatured and hybridized on the human gene 2.1 st peg plate in the affymetrix genetitan mc instrument . basic data analysis of the arrays was carried out using the affymetrix expression console software . changes in gene expressions were analyzed by gene set enrichment analysis ( gsea ) ( broad institute , cambridge , ma ) and its leading edge analysis software that uses the permutation method . the difference in gene expression levels between groups were determined by the significance analysis of microarray ( sam ) program using a false discovery rate ( q - value ) of 5% . since this study analyzed over 53,600 genes , only those that were previously reported to play key roles in pain and inflammation will be discussed in this paper . in addition , only the key genes with a sam fold change of over 1.5 ( overexpressed ) and under 0.5 ( underexpressed ) to signify a potentially relevant clinical effect are reported .

the cost , prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis . this study was aimed to identify the genetic contributors to pulpal pain and inflammation . inflamed pulps were collected from patients diagnosed with irreversible pulpitis ( n=20 ) . normal pulps from teeth extracted for various reasons served as controls ( n=20 ) . pain level was assessed using a visual analog scale ( vas ) . genome - wide microarray analysis was performed using affymetrix genetitan multichannel instrument . the difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate ( q - value ) of 5% . genes involved in immune response , cytokine - cytokine receptor interaction and signaling , integrin cell surface interactions , and others were expressed at relatively higher levels in the in the pulpitis group . moreover , several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients ( 30 mm on vas ) compared to those with moderate to severe pain . this exploratory study provides a molecular basis for the clinical diagnosis of pulpitis . with an enhanced understanding of pulpal inflammation , future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology .

Introduction Results and Discussion Normal and pulpitis samples exhibited differentially expressed genes Differences in gene expression between mild and moderate to severe pain Materials and Methods Patient selection and pulpal diagnosis Sample collection RNA Extraction and Microarray analysis

however , despite the cost and the prevalence of endodontic disease and the great discomfort associated with it , the fundamental molecular aspects of its pathogenesis are still not fully understood . in addition , several studies have suggested that pulpal events can be reflected in the gingival crevicular fluid ( gcf ) through measurable levels of protein markers that correlated with pulpal symptoms . cytokines , cell surface receptors and other protein markers are shown to be either highly increased or decreased in inflamed dental pulp .. a limited number of studies have examined gene expression in inflamed human pulps . these studies , however , did not explore the differences in gene expression between normal and inflamed dental pulp and the clinical presentation of donor patients ( i.e. pain and swelling ) , which may provide biologic explanation on the variability of clinical signs and symptoms of pulpal inflammation that confound diagnosis . although the clinical value of a molecular diagnostic marker may at first appear limited in scope , the emerging correlation of gene expression with clinical signs and symptoms will enhance our understanding on the development of pulpal inflammation - this will add not only to the knowledge base but it will also provide a biological basis for the varying clinical presentations of pulpitis . in this study , data from the full genome scan will be utilized to determine if an association exists between gene expression and clinical presentation ( i.e. pain ) of pulpitis patients . the sam software generated gsea data which showed a significantly higher expression of various gene sets that are associated with immune response activation , maintaining cellular function and cell - to - cell interaction , among others ( figure 1 ) in pulpitis samples . among the patients diagnosed with irreversible pulpitis , eight patients reported experiencing zero to mild pain ( ( 30 mm on vas ) and twelve patient reported moderate to severe pain . the mean vas scores for patients who reported zero to mild pain and moderate to severe pain was 6.63 ( sd = 9.29 ) and 81.93 ( sd = 11.21 ) , respectively ( p mild pain samples showed a significantly higher expression of genes involved in adaptive immune system , cytokine to cytokine interaction and cytokine signaling , among others . however , when looking at specific genes , several of them that have key roles in inflammation are significantly under - expressed , unchanged or over - expressed in asymptomatic or mild pain patients compared to those with moderate to severe pain ( table 1 ) . patients often judge the success of treatment and the efficiency of the dentist based on their pain experience . to arrive at a diagnosis , dentists depend mainly on pain history and responses to sensibility tests ( namely thermal tests and electric pulp testing , ) that have been used for decades ; but whether the responses to these clinical tests correlate well with pulpal histopathology or not remains unclear . this study was aimed to identify the differences in gene expression between normal pulps and pulpitis samples and between mild and moderate to severe pulpitis pain , based on the diagnostic guidelines set by the aae and on the patient - reported pain experience at the time of endodontic treatment , respectively . gene set enrichment analysis ( gsea ) was employed for the analysis of the thousands of genes included in the microarray screening . first , a biological basis at the molecular level can now be correlated not only with the current diagnostic testing procedures but also with the diagnosis of irreversible pulpitis itself . molecular alterations precede histopathological changes and in the case of the dental pulp , there can be a wide array of histological presentations depending on the timing of pulp extirpation ; thus , there are conflicting reports concerning the clinical presentation and actual histopathology of the pulp . studies investigating inflammation at the molecular level may therefore provide a more accurate representation of the inflammatory changes in the dental pulp . next , the results may be used to cluster genes that are overexpressed or underexpressed in inflamed pulps and to use this information as biological basis for future studies on acute pain and on oral biology , especially those that focus on odontogenic pain and regeneration as these studies require a broad understanding of molecular changes that dictate clinical and biological outcomes . the difference in gene expression between normal and inflamed dental pulps ( figure 1 ) may have been the expected result considering that cytokines , chemokines and their receptors are known to be overexpressed in inflammatory conditions ; however , this study is still an important addition to our current understanding of pulp biology as it provides a broad picture or a snap - shot of the molecular changes occurring in the dental pulp during the very moment that the patient is seeking endodontic treatment on the dental chair . another remarkable finding in the study is the overexpressed genes related to inflammation and immune response in pulps from patients who reported zero to mild pain ( 30 mm on vas ) during their endodontic appointment compared to those who had moderate to severe pain ( figure 2 ) . moreover , looking at individual genes separately from the a priori gene sets in the gsea software revealed that genes involved in key inflammatory response like il8 , tnfa , il1a , ilib and several immunoglobulins ( i.e. ighm , ighv4 - 31 , igkv2d-40 ) , among others ( table 1 ) are expressed at relatively lower levels in pulp samples from asymptomatic to mild pain patient compared to those with moderate to severe pain . both il8 and il1b are expressed at relatively lower levelsin zero to mild pain pulpitis patients . col1 - 4 and mmps ) are expressed at relatively higher levels in pulpitis samples . in contrast however , several genes that are known to be pain mediators or associated with increased pain experience did not have significant discordance between mild and moderate to severe pain ( table 1 ) . for example , prostaglandin - endoperoxide synthase 2 or cyclooxygenase-2 ( pgs2 or cox2 ) , an important inflammatory enzyme that has been shown to be significantly elevated in pulpitis tissues did not have expression difference in mild pain but is significantly elevated in pulpitis compared with normal controls ( table 1 ) . tlrs are expressed by both immune and non - immune cells in the pulp including neurons , fibroblasts , endothelial cells , epithelial cells and others . in this study , the expression of several tlrs ( 1,2,4,6 , 8 and 9 ) were upregulated and tlr 3 was down regulated in inflamed pulps as compared to normal pulps . our data also show that expression of tlr8 was higher in pulpits patients experiencing severe pain as compared to those who reported no pain or mild pain . furthermore , individual variations in immunologic and pain response further complicate not only the clinical but also the biological aspect of endodontic diagnosis . this may be the one of the main reasons for the observational differences among studies correlating clinical and histopathological diagnosis as pointed out in the earlier part of this paper . using biopsies from patients diagnosed with either chronic periodontitis or aggressive periodontitis , kebschull et al . , explored whether molecular profiling can form the basis for a pathobiology based classification that correlates with the phenotypic features of the disease . while microarray analysis is a powerful tool to study the simultaneous expression of several genes it has some inherent limitations . it is important to also remember that the cellular profile of inflamed pulps differs from that of normal pulps . inflamed pulps are characterized by an influx of immune cells and as such the results reported here may be in part , due to differences in the cellular makeup as well . the present study shows that there are differences in gene expression between normal and inflamed pulps and ; between zero to mild and moderate to severe pulpitis pain . among the genes reported to have a strong association with pain and inflammation , il8 showed the highest fold change ( 35.9 , q - value = 0 ) in pulpitis samples and it is significantly under - expressed in mild pain compared with moderate to severe pain ( 0.14 , q - value = 0 ) . il8 is increased in gingival crevicular fluid from patients with acute pulpitis and might be a useful measurement for staging patients with acute pulpitis . this study provides a molecular basis for the clinical diagnosis of pulpitis and for the differences or similarities between asymptomatic to mild pulpal pain vis - - vis moderate to severe pain . with an enhanced understanding of pulpal inflammation , future studies on treatment and management of pulpitis and on pain associated with it the sam software generated gsea data which showed a significantly higher expression of various gene sets that are associated with immune response activation , maintaining cellular function and cell - to - cell interaction , among others ( figure 1 ) in pulpitis samples . among the patients diagnosed with irreversible pulpitis , eight patients reported experiencing zero to mild pain ( ( 30 mm on vas ) and twelve patient reported moderate to severe pain . the mean vas scores for patients who reported zero to mild pain and moderate to severe pain was 6.63 ( sd = 9.29 ) and 81.93 ( sd = 11.21 ) , respectively ( p mild pain samples showed a significantly higher expression of genes involved in adaptive immune system , cytokine to cytokine interaction and cytokine signaling , among others . however , when looking at specific genes , several of them that have key roles in inflammation are significantly under - expressed , unchanged or over - expressed in asymptomatic or mild pain patients compared to those with moderate to severe pain ( table 1 ) . patients often judge the success of treatment and the efficiency of the dentist based on their pain experience . to arrive at a diagnosis , dentists depend mainly on pain history and responses to sensibility tests ( namely thermal tests and electric pulp testing , ) that have been used for decades ; but whether the responses to these clinical tests correlate well with pulpal histopathology or not remains unclear . this study was aimed to identify the differences in gene expression between normal pulps and pulpitis samples and between mild and moderate to severe pulpitis pain , based on the diagnostic guidelines set by the aae and on the patient - reported pain experience at the time of endodontic treatment , respectively . gene set enrichment analysis ( gsea ) was employed for the analysis of the thousands of genes included in the microarray screening . first , a biological basis at the molecular level can now be correlated not only with the current diagnostic testing procedures but also with the diagnosis of irreversible pulpitis itself . molecular alterations precede histopathological changes and in the case of the dental pulp , there can be a wide array of histological presentations depending on the timing of pulp extirpation ; thus , there are conflicting reports concerning the clinical presentation and actual histopathology of the pulp . studies investigating inflammation at the molecular level may therefore provide a more accurate representation of the inflammatory changes in the dental pulp . next , the results may be used to cluster genes that are overexpressed or underexpressed in inflamed pulps and to use this information as biological basis for future studies on acute pain and on oral biology , especially those that focus on odontogenic pain and regeneration as these studies require a broad understanding of molecular changes that dictate clinical and biological outcomes . the difference in gene expression between normal and inflamed dental pulps ( figure 1 ) may have been the expected result considering that cytokines , chemokines and their receptors are known to be overexpressed in inflammatory conditions ; however , this study is still an important addition to our current understanding of pulp biology as it provides a broad picture or a snap - shot of the molecular changes occurring in the dental pulp during the very moment that the patient is seeking endodontic treatment on the dental chair . another remarkable finding in the study is the overexpressed genes related to inflammation and immune response in pulps from patients who reported zero to mild pain ( 30 mm on vas ) during their endodontic appointment compared to those who had moderate to severe pain ( figure 2 ) . moreover , looking at individual genes separately from the a priori gene sets in the gsea software revealed that genes involved in key inflammatory response like il8 , tnfa , il1a , ilib and several immunoglobulins ( i.e. ighm , ighv4 - 31 , igkv2d-40 ) , among others ( table 1 ) are expressed at relatively lower levels in pulp samples from asymptomatic to mild pain patient compared to those with moderate to severe pain . both il8 and il1b are expressed at relatively lower levelsin zero to mild pain pulpitis patients . col1 - 4 and mmps ) are expressed at relatively higher levels in pulpitis samples . in contrast however , several genes that are known to be pain mediators or associated with increased pain experience did not have significant discordance between mild and moderate to severe pain ( table 1 ) . for example , prostaglandin - endoperoxide synthase 2 or cyclooxygenase-2 ( pgs2 or cox2 ) , an important inflammatory enzyme that has been shown to be significantly elevated in pulpitis tissues did not have expression difference in mild pain but is significantly elevated in pulpitis compared with normal controls ( table 1 ) . tlrs are expressed by both immune and non - immune cells in the pulp including neurons , fibroblasts , endothelial cells , epithelial cells and others . in this study , the expression of several tlrs ( 1,2,4,6 , 8 and 9 ) were upregulated and tlr 3 was down regulated in inflamed pulps as compared to normal pulps . our data also show that expression of tlr8 was higher in pulpits patients experiencing severe pain as compared to those who reported no pain or mild pain . furthermore , individual variations in immunologic and pain response further complicate not only the clinical but also the biological aspect of endodontic diagnosis . this may be the one of the main reasons for the observational differences among studies correlating clinical and histopathological diagnosis as pointed out in the earlier part of this paper . using biopsies from patients diagnosed with either chronic periodontitis or aggressive periodontitis , kebschull et al . , explored whether molecular profiling can form the basis for a pathobiology based classification that correlates with the phenotypic features of the disease . while microarray analysis is a powerful tool to study the simultaneous expression of several genes it has some inherent limitations . it is important to also remember that the cellular profile of inflamed pulps differs from that of normal pulps . inflamed pulps are characterized by an influx of immune cells and as such the results reported here may be in part , due to differences in the cellular makeup as well . the present study shows that there are differences in gene expression between normal and inflamed pulps and ; between zero to mild and moderate to severe pulpitis pain . among the genes reported to have a strong association with pain and inflammation , il8 showed the highest fold change ( 35.9 , q - value = 0 ) in pulpitis samples and it is significantly under - expressed in mild pain compared with moderate to severe pain ( 0.14 , q - value = 0 ) . il8 is increased in gingival crevicular fluid from patients with acute pulpitis and might be a useful measurement for staging patients with acute pulpitis . this study provides a molecular basis for the clinical diagnosis of pulpitis and for the differences or similarities between asymptomatic to mild pulpal pain vis - - vis moderate to severe pain . with an enhanced understanding of pulpal inflammation , future studies on treatment and management of pulpitis and on pain associated with it eligible patients seeking treatment at the school of dentistry were recruited to participate in the study . tricyclic antidepressants ) known to interfere with the release of various pain mediators and/or modify pain experience within the previous 6 months or over - the - counter pain medicine within the last six hours , chronic use of medication known to affect the immune response and patients who were immunocompromised . diagnosis of normal pulp ( np ) and irreversible pulpitis ( ip ) was based on subjective and objective findings and was in accordance with the american association of endodontists ( aae ) guidelines . if lingering pain is present or significantly evoked by a stimulus compared with a control tooth , the patient was asked to quantify the intensity of pain by placing a mark on a 10-cm vas with anchors of no pain and worst pain imaginable . using a modified version of the vas recommended by jensen et al , a vas mark of 30 mm was classified under mild , 31 - 74 mm under moderate and 75 - 100 under severe pain . twelve of the ip samples presented with moderate to severe pain at the time of pulp extirpation . the total number of samples was based on a recently published paper by our group on the differences of micro - rna expression between normal and inflamed pulps and on a power analysis . moreover , we increased the power as much as needed by increasing the acceptable false discovery rate . given that this initial analysis is intended to be exploratory , a higher false discovery rate is acceptable in this study . the inflamed pulps were extirpated during root canal treatment and the normal pulps were collected right after tooth extraction for orthodontic or restorative purposes or for third molar removal . the hybridization cocktails were denatured and hybridized on the human gene 2.1 st peg plate in the affymetrix genetitan mc instrument . basic data analysis of the arrays was carried out using the affymetrix expression console software . the difference in gene expression levels between groups were determined by the significance analysis of microarray ( sam ) program using a false discovery rate ( q - value ) of 5% . since this study analyzed over 53,600 genes , only those that were previously reported to play key roles in pain and inflammation will be discussed in this paper . tricyclic antidepressants ) known to interfere with the release of various pain mediators and/or modify pain experience within the previous 6 months or over - the - counter pain medicine within the last six hours , chronic use of medication known to affect the immune response and patients who were immunocompromised . diagnosis of normal pulp ( np ) and irreversible pulpitis ( ip ) was based on subjective and objective findings and was in accordance with the american association of endodontists ( aae ) guidelines . using a modified version of the vas recommended by jensen et al , a vas mark of 30 mm was classified under mild , 31 - 74 mm under moderate and 75 - 100 under severe pain . twelve of the ip samples presented with moderate to severe pain at the time of pulp extirpation . the total number of samples was based on a recently published paper by our group on the differences of micro - rna expression between normal and inflamed pulps and on a power analysis . moreover , we increased the power as much as needed by increasing the acceptable false discovery rate . given that this initial analysis is intended to be exploratory , a higher false discovery rate is acceptable in this study . the inflamed pulps were extirpated during root canal treatment and the normal pulps were collected right after tooth extraction for orthodontic or restorative purposes or for third molar removal . the hybridization cocktails were denatured and hybridized on the human gene 2.1 st peg plate in the affymetrix genetitan mc instrument . basic data analysis of the arrays was carried out using the affymetrix expression console software . the difference in gene expression levels between groups were determined by the significance analysis of microarray ( sam ) program using a false discovery rate ( q - value ) of 5% . since this study analyzed over 53,600 genes , only those that were previously reported to play key roles in pain and inflammation will be discussed in this paper .

virus - like particle ( vlp ) has been extensively studied for its vaccine capacity in the last 20 years . more than a dozen of vlp - based vaccines have been tested in clinical trials . two of them against hepatitis b and human papilloma viruses are commercially available as prophylactics . the merits of vlp - based vaccine rest on its viral mimicry and devoid of genetic materials , hence alleviating the safety concerns . the repetitive and ordered structures of vlps are conducive to opsonization , resulting in enhanced phagocytosis [ 3 , 4 ] , and are capable to activate b cells in a t cell - independent manner [ 5 , 6 ] . owing to the virus - like dimension and particulate nature , vlps are easily taken up by antigen - presenting cells ( apcs ) [ 7 , 8 ] and more efficient in cross - presentation of antigens to mhc i molecules compared to the soluble antigen and hence the possibility of activating antigen - specific cytotoxic t lymphocytes ( ctls ) . although the vlp - based vaccines have the potential to induce ctl response , most of them favor the production of humoral immunity but poorly evoke ctl response if additional stimuli for innate immune system and , in particular , apc were neglected . it is somewhat surprising that vlps derived from baculovirus system incorporating viral structural proteins of influenza virus were found to induce both cell - mediated and humoral immunity to confer full protection from viral challenge [ 1012 ] . this distinct feature drew our attention on the interaction between vlps and cells since most of the vlp - based strategies for vaccine development do not involve antigens with functions of ligand binding and membrane fusion in contrast with the hemagglutinin of influenza vlp . influenza a virus is an enveloped rna virus with three transmembrane proteins : hemagglutinin ( ha ) , neuraminidase ( na ) , and ion channel protein ( m2 ) along with matrix protein 1 ( m1 ) underlying the viral envelope . the major tasks of these proteins include ligand binding and membrane fusion for ha [ 13 , 14 ] , spreading of viral progeny and facilitating entry of the virus for na [ 1517 ] , proton transfer for viral uncoating as well as assembly and budding of the virus for m2 [ 1820 ] , recruitment of viral components to the assembly site , and a major driving force for viral budding for m1 [ 21 , 22 ] . several laboratories have produced insect cell - derived vlps encompassing the above - mentioned influenza proteins for vaccine development . however , there has been no report on the characterization of the interaction between vlps and cells . to afford a better understanding of these interactions , we used fluorescent images and dye dequenching in this study in the hope of shedding some light on the mechanism of antigen presentation , and virus - mediated membrane fusion and its inhibition . ha ( a / thailand/1(kan-1)/2004/h5n1 ) , na ( a / viet nam/1203/2004/h5n1 ) , m1 ( a / wsn/33/h1n1 ) and m2 ( a / wsn/33/h1n1 ) , influenza cdna sequences were amplified by pcr ( primer sequences were listed in the table 1 ) . the ha pcr products were cloned into bamhi / noti site under the control of the polyhedron promoter , the m1 pcr products into xhoi / kpni site under p10 promoter of the pfastbac dual baculovirus transfer vector ( invitrogen ) , the m2 pcr products into ecori /hindiii site under p10 promoter of the pfastbac dual baculovirus transfer vector ( invitrogen ) , and the na - egfp pcr products into xhoi / kpni site under p10 promoter of the pfastbac dual baculovirus transfer vector ( invitrogen ) . all the inserted sequences were confirmed by dna sequence analysis ( mission biotech inc . , production of recombinant baculoviruses followed the manufacturer 's manual of bac - to - bac baculovirus system ( invitrogen ) . briefly , the pfastbac dual plasmids were transformed into e.coli strain dh10bac ( invitrogen ) and selected on lb plate containing kanamycin ( invitrogen ) , gentamicin ( invitrogen ) , tetracycline ( invitrogen ) , bluo - gal ( invitrogen ) , and iptg ( biorad ) . the recombinant bacmids were confirmed by sequencing and then transfected into spodoptera frugiperda ( sf9 ) cells for recombinant baculovirus packaging via the aid of cellfectin reagent ( invitrogen ) . after 4 days , the recombinant baculovirus in the supernatant was collected as p1 viral stock and further amplified as p2 viral stock for vlp purification . the virus titer of viral stock was determined by the end - point dilution in sf9 cells . 11496 - 015 , invitrogen ) were maintained as suspension cultures in sf900 ii sfm ( invitrogen ) at 27c . for the production of ha vlp , 300 ml of sf9 cells at 2 10cells / ml were coinfected with dual ha , m1 and dual na , m2 recombinant baculovirus at moi of 3 and 1 , respectively , whereas for the production of ha vlp , the dual ha , m1 recombinant baculovirus were replaced with m1 only recombinant baculovirus . to produce na - egfp vlp , the same infection procedure was carried out except for the replacement of dual na , m2 with dual na - egfp , m2 recombinant baculovirus . at 72 h post - infection , the vlps were precipitated at 33000 rpm and 4c for 2 h ( rps40st rotor , hitachi ) . the particles were resuspended in pbs buffer , loaded on a 060% ( w / v ) discontinuous sucrose gradient , and centrifuged for 4 h at 33000 rpm and 4c ( rps40st rotor , hitachi ) . after ultracentrifugation , fractions were collected from the top of the gradient and analyzed by western blotting . fractions containing ha , na , m1 , and m2 proteins were collected , and the protein concentration was determined by bca protein assay kit ( cat . protein samples were resolved by 12.5% sds polyacrylamide gel and then transferred to pvdf membranes . the membrane blots were blocked with 5% nonfat milk in tris - buffered saline containing 0.1% tween-20 and probed with primary antibodies against ha ( abcam ab21297 ) , na ( abcam ab70759 ) , m1 ( abcam ab25918 ) , m2 ( novus nb100 - 2073 ) , separately . the presence of each protein was detected by hrp - conjugated secondary antibody and enhanced chemiluminescence ( ecl ) plus western blot detection system ( amersham bioscience ) . the blot images were captured by the las-3000 imaging system ( fujifilm , tokyo , japan ) . vlps suspended in pbs were mixed with equal volume of r18- or dio - containing pbs to final concentration of 10 m of r18 or dio ( molecular probes , invitrogen ) . the mixture was incubated in dark for 1 h at room temperature for r18 labeling while incubated for 20 min at 37c for dio incorporation . the r18-labeled vlps were washed three times with pbs , and the unincorporated dyes were filtered away by a microcon devise with mw cutoff of 50 kda . the fluorecent - labeled vlps were mounted on a glass support and visualized by inverted fluorescence microscope ( aviovert , carl zeiss inc . ) with oil immersion objective ( plan - apo 100x , n.a . the images were processed by metamorph software ( carl zeiss meditec , gottingen germany ) . , santa barbara , ca ) equipped with a microcuvette and a 30 mw , 685 nm gaas laser light source . one milliliter of pbs was filtrated through a 0.02 m inorganic membrane filter ( anodisc , whatman international ltd . , maidstone , england ) as the dls background . for the hydrodynamic radius ( rh ) measurement , one microliter of vlp solution was directly mixed with the pbs and transferred in the microcuvette . data collection and analysis were performed on the astra v software ( wyatt technology corp . the morphology of the influenza vlp was observed by transmission electron microscope jem 2010 ( jeol ltd . , tokyo japan ) . 3 l of diluted influenza vlps were loaded onto formvar - carbon - coated 300-mesh copper grids ( polysciences inc . ) and allowed to absorb for 3 minutes . the grids were wicked dry with filter paper , rinsed once with pbs , and negatively stained with 1% ammonium phosphotungstate ph 7 . grids were examined by tem at 200 kv accelerating voltage for the presence of influenza virus . 20 l of na - egfp vlp ( total protein amount 15.6 g ) was diluted in 50 l of pbs with varying ph by titration with 25 mm of citric acid . the excitation wavelength was 470 nm while emission spectra spanned 490 to 560 nm . excitation and emission slit widths were set at 2.5 and 10 nm , respectively . the fluorescent - labeled vlps were diluted in ice - cold dmem medium containing 50 mm nh4cl and added to cover slips with a monolayer of grown a549 cells . the maximum amount of nh4cl was tested for 24 h for minimal cellular morphological change . the cover slips were stood on ice for 1 h to prevent vlp engulfed by pinocytosis . to show that the endocytotic process is mediated by the binding between viral ha and cellular sialylated receptors , a549 cells were treated with neuraminidase from vibrio cholerae ( sigma ) at 4 10 unit / ml and 37c for 2 h prior to the addition of fluorescent - labeled vlps . after 1 h incubation , the unbound vlps were washed away with ice - cold dmem medium , and the cells were cultured in dmem medium containing 50 mm nh4cl at 37c for 2 h , which was replaced with a medium containing 5 m of did dye for 15 min before the cells were washed extensively with pbs and fixed with 4% paraformaldehyde in pbs at room temperature for 10 min and mounted with 50% glycerol in pbs . the r18- and dio - labeled vlps taken up by a549 cells were observed by zeiss lsm510 meta laser scanning confocal microscope using oil immersion objective ( plan - apo 100x , n. a. 1.4 ) in multitrack channel mode . excitation wavelengths and emission filters were 488 nm / band - pass 500530 nm for egfp and dio , 561 nm / band - pass 575630 nm for r18 , and 561 nm / band -pass 651704 nm for did . 5 l of r18-labeled vlps ( protein estimation 2.4 g ) were mixed with variant concentration of fetuin in final volume of 200 l and incubated at r.t . for 5 min . the mixtures were then added into equal volume of jurkat cells ( 1 10 cells ) and incubated at 37c for 10 min . the cell - bound vlps were collected by centrifugation at 200 g for 5 min and lysed with octaethylene glycol dodecyl ether ( c12e8 , calbiochem ) in a final concentration of 3 mm . the r18 signal was detected by fluorescence spectrophotometer f4500 ( hitachi ) with excitation at 540 nm and emission at 578 nm . excitation and emission slit widths were set at 2.5 and 10 nm , respectively . experiments of surface plasmon resonance ( spr ) were conducted to evaluate the binding affinity of ha2-specific mab iif4 to ha and havlps . in brief , the vlp was immobilized on an activated cm5 sensor chip , which was placed in the chamber of a biacore 3000 biosensor system ( biacore ab , uppsala , sweden ) . iif4 used as the analyte was injected over the cm5 sensor chip at 10 l / min flow rate at ph 7.4 or 5.0 in sodium phosphate buffer to examine the ph - dependent conformational change as observed for the native influenza virus . the sensor surface was regenerated with solution of 0.1 m nacl and 0.02 n naoh . 20 l of vlp was diluted in 200 l of pbs , and then added onto poly - l - lysine - treated coverslip on ice for 2 h. after pbs washing , the ph 5 treated vlp was incubated with low ph buffer ( 10 mm hepes , 10 mm mes in pbs , ph 5.0 ) for 2 min , rinsed with pbs , and fixed with 4% paraformaldehyde and subsequently blocked with 10% fbs in pbs at rt for 1 h. the vlp was probed with iif4 mab ( kindly provided by dr . vareckova , 100x diluted ) and subsequently hybridized with fitc conjugated antimouse igg ( 500x diluted ) at 37c for 1 h. the coverslips were mounted on a microscope slide and visualized with fluorescence microscope ( carl zeiss , germany ) . the images were captured and processed with metamorph software ( molecular devices , ca , usa ) . the fusion process was monitored by fluorescence dequenching of r18 due to the lateral diffusion and dilution from viral to cellular membrane as a result of membrane fusion [ 29 , 30 ] . 6 10 of jurkat cells pretreated with 25 mm of nh4cl at 37c for 15 min were suspended in 500 l of pbs containing 25 mm of nh4cl and added 10 l of r18-labeled vlps ( protein estimation 4.7 g ) and then incubated at 37c for 10 min . the cells were sedimented by centrifugation at 4c for 5 min at 200 g and rapidly resuspended in 1 ml of pbs . fusion of vlps with plasma membrane was triggered by lowering the ph to 5 with 0.25 m of citric acid . the maximum fluorescence was obtained by lysing the viral and cellular membrane with c12e8 at final concentration of 3 mm after each experiment . the fluorescent increase was detected by fluorescence spectrophotometer f4500 ( hitachi ) with excitation at 540 nm and emission at 578 nm . excitation and emission slit widths were set at 2.5 and 10 nm , respectively . the fluorescent images were captured by inverted fluorescence microscope ( aviovert , carl zeiss inc . ) with oil immersion objective ( plan - apo 40x , n.a . 1.3 ) and processed by metamorph software ( carl zeiss meditec , gottingen , germany ) . figure 1 illustrates the construction of pfastbac dual plasmids containing influenza m1/ha , na / m2 , /ha , and na - egfp / m2 gene pairs for the production of recombinant baculoviruses . using these recombinant baculoviruses , havlps , havlps , and na - egfp vlp figure 2 displays the western blot results ( panel a ) of the expressed proteins incorporated in ha and havlps . the incorporated proteins were expressed at similar level except for ha protein devoid in havlps as expected . it is noted that the fraction of cleavage for hemagglutinin in the present expression system is estimated to be 33.7% . the morphology of the expressed vlp as visualized by transmission electron microscopy shown in figure 2(b ) is nearly spherical with a diameter of ~100 nm , close to the virus in the native context [ 31 , 32 ] . the radius of vlp measured by light scattering was 63.3 0.13 nm with a convergent size distribution ( figure 2(c ) ) . binding of hemagglutinins to cellular sialylated receptors initiates the host endocytotic process for influenza virus entry [ 3335 ] . equal amount of havlp or havlp , with regard to each protein concentration , was added to a549 cells . compared to havlp , ha - incorporated vlps were significantly endocytosed by a549 cells . to confirm that the endocytic process is mediated by ha binding to the sialylated receptor , after 2 h of treatment , most of the sialic acids exposed on the cell surface were removed as confirmed by sialic acid - specific lectin staining ( see figure s1 in supplementary material available online at doi:10.11/2010/506363 ) . as shown in figure 3 , removal of sialic acids starkly decreased havlp engulfed by a549 cells . also , attachment of vlp to another suspended cell line jurkat cell could be hampered by fetuin ( figure 3(f ) ) , which is a sialylated glycoprotein [ 36 , 37 ] and used as a model inhibitor for influenza virus infection [ 3841 ] , in a dose - dependent manner . taken together , the havlp was endocytosed by a549 cells through the binding of viral ha and cellular sialylated receptor . iif4 mab has been reported to be specific to the epitope within ha2 aa 125175 of the h3 subtype of influenza a virus and is well cross - reacting with h4 and h5 viruses [ 4244 ] . previous studies demonstrated that iif4 epitopes on ha2 became accessible after acid treatment on ha [ 42 , 45 ] . the binding affinity of ha2-specific mab iif4 to ha vlps at acidic ph was 0.18 0.05 nm , 32-fold higher than binding affinity at neutral ph ( figures 4(a ) and 4(c ) and table 2 ) . this implied that the ha on vlp membrane could expose ha2 subunit leading to fusion of the viral and cellular membranes as a result of lowered ph . to further support the spr result that the havlp has much higher affinity to iif4 mab at ph 5 than at ph 7.4 , acidic buffer - treated ha vlp was recognizable by ha2 iif4 mab whereas at natural ph ha vlp and havlp were not , in agreement with the spr measurement and indicating that the ha2 portion on the vlp is exposed at acidic ph . fluorescent dequenching of r18 was employed to monitor the fusion process of influenza virus with culture cells [ 29 , 30 ] . thus r18-labeled vlps were mixed with jurkat cells and collected by centrifugation of the cells . as shown in figure 5(a ) , ha vlp could adhere to the cells surface and form heterogeneous fluorescent dots while ha vlp could be barely detected . after lowering the external ph of the cells , the cell - bound vlps underwent fusion with the cells , as manifested by more homogenous dispersion of fluorescent signals around the cell . the fusion activity of ha vlp was reaffirmed by the fluorescence dequenching experiment displayed in figure 5(b ) showing that only ha vlp induced dramatic fluorescence increase in response to acidic ph . since the na activity has been inferred unnecessary for influenza vlp release from sf9 insect cells [ 4649 ] , the extracellular domain of na was replaced with the egfp protein to demonstrate the possibility of accommodating other protein antigens on the vlp membrane to extend the application of influenza vlp as an antigen carrier . to construct the membrane - anchored chimera , the amino terminal 61 amino acids of na protein were retained and fused with egfp . the na - egfp vlps were stained with lipophilic dye r18 to characterize the virus - like particles incorporated with egfp . colocalization of na - egfp and r18 confirms that the virus - like particle was loaded with na - egfp . to investigate the uptake of na - egfp - incorporated vlp by cells , the vlps were labeled with r18 and then admixed with a549 cells at 4c to prevent pinocytosis . as shown in figures 6(d)6(f ) , na - egfp vlp can be taken up by a549 cells , and 20.6 3.7% of the endocytotic vesicles contain the egfp fluorescence . using pfastbac dual baculovirus transfer vector , we produced insect cell derived vlps containing ha , m1 , na , and m2 from influenza virus as well as egfp - incorporated vlps . despite the low na content or deprived activity in the egfp - fused na , it was reported that the proteins expressed in sf9 insect cells were devoid of sialylation as a result of the absence of detectable sialyltransferase activities [ 46 , 47 ] and cmp - sialic acids [ 48 , 49 ] and were n - glycosylated in high mannose type . it implies that vlp production in insect cells is viable without the aid of na activity for viral progeny release if ha is incorporated . also , n - glycans in high mannose type could possibly enhance the ha binding to its sialylated receptors and facilitate the uptake of vlps by apcs . from a previous report , ha expression in insect cells was retarded for posttranslational proteolysis as noted in our western blot result . however , the proteolytic process does not impair the binding capacity of ha but does for membrane fusion , as demonstrated in our fusion test ( figure 5 ) . it is noted that some vlps did not fuse with the plasma membrane of the cells when ph was lowered to 5 and clumped on the cell surface . it raises an interesting question : since insect cell - derived vlp can induce cell - mediated immune response [ 1012 ] , how are the ha proteins anchored on the late endosomal membrane as a result of ha - induced membrane fusion or accumulated in the endosome lumen involved in cross presentation to mhc i molecules , leading to ha - specific ctl induction ? to address this question , mammalian cell - derived influenza vlp with or without proteolytic site in ha protein could be tested for ha - specific ctl induction , protein processing as well as mhc i loading . the size of expressed vlp is estimated to be ~100 nm while the average size deduced from dynamic light scattering is ~120 nm . the discrepancy between the two data can be at least partly accounted for by the fact that the latter reports the hydrodynamic diameter of the particle , namely , including the hydrated water molecules surrounding the vlp , thus the larger size . low ph - triggered conformational change can be detected by mab iif4 with the epitope within ha2 125 - 175 , which was exposed upon lowering ph to 5.0 as deduced from 33-fold increase in binding affinity to iif4 when switched to acidic ph . again , this reaffirms that the constructed vlp can be used as a valid mimic to the native virus for the functional and structural study of hemagglutinin . further investigation of the biological activity for the synthesized vlp was performed on fusion to the jurkat cell membrane induced by ha ( figure 5 ) as probed by dequenching of the embedded r18 label due to its dilution arising from membrane merger . the result established the function exerted by ha as havlp did not exhibit fusion activity . by measuring the fluorescence intensity of r18 , ic50 of fetuin on binding inhibition of havlp to jurkat cell was estimated to be 147 m ( 7.1 mg / ml ) , compared to a previous result of 6.4 mg / ml for inhibition of viral infection by focus - forming assays . based on viral binding and fusion both experiments can be used as screening platforms for inhibitors targeting viral entry . however , it reinforced that the application na chimera for antigen incorporation in vlp was feasible . the egfp - vlp could be expanded for its utility as a reporter tag , particularly as a ph sensor inside the ha - targeted cells . further study on the time variation of egfp in the endosome can be conducted to monitor the time course of the fusion process and trafficking of vlp within the cell , which may provide insight into the antigen presentation of the virus and the dynamics and the route of virus invasion in the cell . the extensive biophysical characterization of influenza vlp containing m1 , ha , na , and m2 viral structural proteins established a reliable protocol of synthesizing a large quantity of the virus mimetic for use in vaccine study , in particular for the ha as the major antigen . the incorporation of a fluorescent label demonstrates the feasible use of vlp as an antigen carrier as well as a possible tool for microscopic observation of the virus dynamics and interaction with the cellular components within the cell .

we have constructed virus - like particles ( vlps ) harboring hemagglutinin ( ha ) , neuraminidase ( na ) , matrix protein 1 ( m1 ) , and proton channel protein ( m2 ) using baculovirus as a vector in the sf9 insect cell . the size of the expressed vlp was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy . recognition of ha on the vlp surface by the ha2-specific monoclonal antibody iif4 at acidic ph , as probed by surface plasmon resonance , indicated the ph - induced structural rearrangement of ha . uptake of the particle by a549 mediated by ha - sialylose receptor interaction was visualized by the fluorescent - labeled vlp . the ha - promoted cell - virus fusion activity was illustrated by fluorescence imaging on the jurkat cells incubated with rhodamine - loaded vlp performed at fusogenic ph . furthermore , the green fluorescence protein ( gfp ) was fused to na to produce vlp with a ph - sensitive probe , expanding the use of vlp as an antigen carrier and a tool for viral tracking .

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

virus - like particle ( vlp ) has been extensively studied for its vaccine capacity in the last 20 years . the repetitive and ordered structures of vlps are conducive to opsonization , resulting in enhanced phagocytosis [ 3 , 4 ] , and are capable to activate b cells in a t cell - independent manner [ 5 , 6 ] . owing to the virus - like dimension and particulate nature , vlps are easily taken up by antigen - presenting cells ( apcs ) [ 7 , 8 ] and more efficient in cross - presentation of antigens to mhc i molecules compared to the soluble antigen and hence the possibility of activating antigen - specific cytotoxic t lymphocytes ( ctls ) . it is somewhat surprising that vlps derived from baculovirus system incorporating viral structural proteins of influenza virus were found to induce both cell - mediated and humoral immunity to confer full protection from viral challenge [ 1012 ] . this distinct feature drew our attention on the interaction between vlps and cells since most of the vlp - based strategies for vaccine development do not involve antigens with functions of ligand binding and membrane fusion in contrast with the hemagglutinin of influenza vlp . influenza a virus is an enveloped rna virus with three transmembrane proteins : hemagglutinin ( ha ) , neuraminidase ( na ) , and ion channel protein ( m2 ) along with matrix protein 1 ( m1 ) underlying the viral envelope . the major tasks of these proteins include ligand binding and membrane fusion for ha [ 13 , 14 ] , spreading of viral progeny and facilitating entry of the virus for na [ 1517 ] , proton transfer for viral uncoating as well as assembly and budding of the virus for m2 [ 1820 ] , recruitment of viral components to the assembly site , and a major driving force for viral budding for m1 [ 21 , 22 ] . several laboratories have produced insect cell - derived vlps encompassing the above - mentioned influenza proteins for vaccine development . however , there has been no report on the characterization of the interaction between vlps and cells . to afford a better understanding of these interactions , we used fluorescent images and dye dequenching in this study in the hope of shedding some light on the mechanism of antigen presentation , and virus - mediated membrane fusion and its inhibition . ha ( a / thailand/1(kan-1)/2004/h5n1 ) , na ( a / viet nam/1203/2004/h5n1 ) , m1 ( a / wsn/33/h1n1 ) and m2 ( a / wsn/33/h1n1 ) , influenza cdna sequences were amplified by pcr ( primer sequences were listed in the table 1 ) . the ha pcr products were cloned into bamhi / noti site under the control of the polyhedron promoter , the m1 pcr products into xhoi / kpni site under p10 promoter of the pfastbac dual baculovirus transfer vector ( invitrogen ) , the m2 pcr products into ecori /hindiii site under p10 promoter of the pfastbac dual baculovirus transfer vector ( invitrogen ) , and the na - egfp pcr products into xhoi / kpni site under p10 promoter of the pfastbac dual baculovirus transfer vector ( invitrogen ) . briefly , the pfastbac dual plasmids were transformed into e.coli strain dh10bac ( invitrogen ) and selected on lb plate containing kanamycin ( invitrogen ) , gentamicin ( invitrogen ) , tetracycline ( invitrogen ) , bluo - gal ( invitrogen ) , and iptg ( biorad ) . after 4 days , the recombinant baculovirus in the supernatant was collected as p1 viral stock and further amplified as p2 viral stock for vlp purification . the virus titer of viral stock was determined by the end - point dilution in sf9 cells . for the production of ha vlp , 300 ml of sf9 cells at 2 10cells / ml were coinfected with dual ha , m1 and dual na , m2 recombinant baculovirus at moi of 3 and 1 , respectively , whereas for the production of ha vlp , the dual ha , m1 recombinant baculovirus were replaced with m1 only recombinant baculovirus . to produce na - egfp vlp , the same infection procedure was carried out except for the replacement of dual na , m2 with dual na - egfp , m2 recombinant baculovirus . fractions containing ha , na , m1 , and m2 proteins were collected , and the protein concentration was determined by bca protein assay kit ( cat . the membrane blots were blocked with 5% nonfat milk in tris - buffered saline containing 0.1% tween-20 and probed with primary antibodies against ha ( abcam ab21297 ) , na ( abcam ab70759 ) , m1 ( abcam ab25918 ) , m2 ( novus nb100 - 2073 ) , separately . the blot images were captured by the las-3000 imaging system ( fujifilm , tokyo , japan ) . the r18-labeled vlps were washed three times with pbs , and the unincorporated dyes were filtered away by a microcon devise with mw cutoff of 50 kda . the fluorecent - labeled vlps were mounted on a glass support and visualized by inverted fluorescence microscope ( aviovert , carl zeiss inc . ) , santa barbara , ca ) equipped with a microcuvette and a 30 mw , 685 nm gaas laser light source . for the hydrodynamic radius ( rh ) measurement , one microliter of vlp solution was directly mixed with the pbs and transferred in the microcuvette . the morphology of the influenza vlp was observed by transmission electron microscope jem 2010 ( jeol ltd . the grids were wicked dry with filter paper , rinsed once with pbs , and negatively stained with 1% ammonium phosphotungstate ph 7 . 20 l of na - egfp vlp ( total protein amount 15.6 g ) was diluted in 50 l of pbs with varying ph by titration with 25 mm of citric acid . the fluorescent - labeled vlps were diluted in ice - cold dmem medium containing 50 mm nh4cl and added to cover slips with a monolayer of grown a549 cells . to show that the endocytotic process is mediated by the binding between viral ha and cellular sialylated receptors , a549 cells were treated with neuraminidase from vibrio cholerae ( sigma ) at 4 10 unit / ml and 37c for 2 h prior to the addition of fluorescent - labeled vlps . after 1 h incubation , the unbound vlps were washed away with ice - cold dmem medium , and the cells were cultured in dmem medium containing 50 mm nh4cl at 37c for 2 h , which was replaced with a medium containing 5 m of did dye for 15 min before the cells were washed extensively with pbs and fixed with 4% paraformaldehyde in pbs at room temperature for 10 min and mounted with 50% glycerol in pbs . the r18- and dio - labeled vlps taken up by a549 cells were observed by zeiss lsm510 meta laser scanning confocal microscope using oil immersion objective ( plan - apo 100x , n. a. excitation wavelengths and emission filters were 488 nm / band - pass 500530 nm for egfp and dio , 561 nm / band - pass 575630 nm for r18 , and 561 nm / band -pass 651704 nm for did . the cell - bound vlps were collected by centrifugation at 200 g for 5 min and lysed with octaethylene glycol dodecyl ether ( c12e8 , calbiochem ) in a final concentration of 3 mm . experiments of surface plasmon resonance ( spr ) were conducted to evaluate the binding affinity of ha2-specific mab iif4 to ha and havlps . in brief , the vlp was immobilized on an activated cm5 sensor chip , which was placed in the chamber of a biacore 3000 biosensor system ( biacore ab , uppsala , sweden ) . iif4 used as the analyte was injected over the cm5 sensor chip at 10 l / min flow rate at ph 7.4 or 5.0 in sodium phosphate buffer to examine the ph - dependent conformational change as observed for the native influenza virus . 20 l of vlp was diluted in 200 l of pbs , and then added onto poly - l - lysine - treated coverslip on ice for 2 h. after pbs washing , the ph 5 treated vlp was incubated with low ph buffer ( 10 mm hepes , 10 mm mes in pbs , ph 5.0 ) for 2 min , rinsed with pbs , and fixed with 4% paraformaldehyde and subsequently blocked with 10% fbs in pbs at rt for 1 h. the vlp was probed with iif4 mab ( kindly provided by dr . the fusion process was monitored by fluorescence dequenching of r18 due to the lateral diffusion and dilution from viral to cellular membrane as a result of membrane fusion [ 29 , 30 ] . fusion of vlps with plasma membrane was triggered by lowering the ph to 5 with 0.25 m of citric acid . the fluorescent increase was detected by fluorescence spectrophotometer f4500 ( hitachi ) with excitation at 540 nm and emission at 578 nm . figure 1 illustrates the construction of pfastbac dual plasmids containing influenza m1/ha , na / m2 , /ha , and na - egfp / m2 gene pairs for the production of recombinant baculoviruses . using these recombinant baculoviruses , havlps , havlps , and na - egfp vlp figure 2 displays the western blot results ( panel a ) of the expressed proteins incorporated in ha and havlps . it is noted that the fraction of cleavage for hemagglutinin in the present expression system is estimated to be 33.7% . the morphology of the expressed vlp as visualized by transmission electron microscopy shown in figure 2(b ) is nearly spherical with a diameter of ~100 nm , close to the virus in the native context [ 31 , 32 ] . the radius of vlp measured by light scattering was 63.3 0.13 nm with a convergent size distribution ( figure 2(c ) ) . compared to havlp , ha - incorporated vlps were significantly endocytosed by a549 cells . to confirm that the endocytic process is mediated by ha binding to the sialylated receptor , after 2 h of treatment , most of the sialic acids exposed on the cell surface were removed as confirmed by sialic acid - specific lectin staining ( see figure s1 in supplementary material available online at doi:10.11/2010/506363 ) . as shown in figure 3 , removal of sialic acids starkly decreased havlp engulfed by a549 cells . also , attachment of vlp to another suspended cell line jurkat cell could be hampered by fetuin ( figure 3(f ) ) , which is a sialylated glycoprotein [ 36 , 37 ] and used as a model inhibitor for influenza virus infection [ 3841 ] , in a dose - dependent manner . taken together , the havlp was endocytosed by a549 cells through the binding of viral ha and cellular sialylated receptor . iif4 mab has been reported to be specific to the epitope within ha2 aa 125175 of the h3 subtype of influenza a virus and is well cross - reacting with h4 and h5 viruses [ 4244 ] . the binding affinity of ha2-specific mab iif4 to ha vlps at acidic ph was 0.18 0.05 nm , 32-fold higher than binding affinity at neutral ph ( figures 4(a ) and 4(c ) and table 2 ) . this implied that the ha on vlp membrane could expose ha2 subunit leading to fusion of the viral and cellular membranes as a result of lowered ph . to further support the spr result that the havlp has much higher affinity to iif4 mab at ph 5 than at ph 7.4 , acidic buffer - treated ha vlp was recognizable by ha2 iif4 mab whereas at natural ph ha vlp and havlp were not , in agreement with the spr measurement and indicating that the ha2 portion on the vlp is exposed at acidic ph . thus r18-labeled vlps were mixed with jurkat cells and collected by centrifugation of the cells . after lowering the external ph of the cells , the cell - bound vlps underwent fusion with the cells , as manifested by more homogenous dispersion of fluorescent signals around the cell . the fusion activity of ha vlp was reaffirmed by the fluorescence dequenching experiment displayed in figure 5(b ) showing that only ha vlp induced dramatic fluorescence increase in response to acidic ph . since the na activity has been inferred unnecessary for influenza vlp release from sf9 insect cells [ 4649 ] , the extracellular domain of na was replaced with the egfp protein to demonstrate the possibility of accommodating other protein antigens on the vlp membrane to extend the application of influenza vlp as an antigen carrier . the na - egfp vlps were stained with lipophilic dye r18 to characterize the virus - like particles incorporated with egfp . colocalization of na - egfp and r18 confirms that the virus - like particle was loaded with na - egfp . to investigate the uptake of na - egfp - incorporated vlp by cells , the vlps were labeled with r18 and then admixed with a549 cells at 4c to prevent pinocytosis . as shown in figures 6(d)6(f ) , na - egfp vlp can be taken up by a549 cells , and 20.6 3.7% of the endocytotic vesicles contain the egfp fluorescence . using pfastbac dual baculovirus transfer vector , we produced insect cell derived vlps containing ha , m1 , na , and m2 from influenza virus as well as egfp - incorporated vlps . despite the low na content or deprived activity in the egfp - fused na , it was reported that the proteins expressed in sf9 insect cells were devoid of sialylation as a result of the absence of detectable sialyltransferase activities [ 46 , 47 ] and cmp - sialic acids [ 48 , 49 ] and were n - glycosylated in high mannose type . it implies that vlp production in insect cells is viable without the aid of na activity for viral progeny release if ha is incorporated . also , n - glycans in high mannose type could possibly enhance the ha binding to its sialylated receptors and facilitate the uptake of vlps by apcs . however , the proteolytic process does not impair the binding capacity of ha but does for membrane fusion , as demonstrated in our fusion test ( figure 5 ) . it is noted that some vlps did not fuse with the plasma membrane of the cells when ph was lowered to 5 and clumped on the cell surface . it raises an interesting question : since insect cell - derived vlp can induce cell - mediated immune response [ 1012 ] , how are the ha proteins anchored on the late endosomal membrane as a result of ha - induced membrane fusion or accumulated in the endosome lumen involved in cross presentation to mhc i molecules , leading to ha - specific ctl induction ? to address this question , mammalian cell - derived influenza vlp with or without proteolytic site in ha protein could be tested for ha - specific ctl induction , protein processing as well as mhc i loading . the size of expressed vlp is estimated to be ~100 nm while the average size deduced from dynamic light scattering is ~120 nm . the discrepancy between the two data can be at least partly accounted for by the fact that the latter reports the hydrodynamic diameter of the particle , namely , including the hydrated water molecules surrounding the vlp , thus the larger size . low ph - triggered conformational change can be detected by mab iif4 with the epitope within ha2 125 - 175 , which was exposed upon lowering ph to 5.0 as deduced from 33-fold increase in binding affinity to iif4 when switched to acidic ph . again , this reaffirms that the constructed vlp can be used as a valid mimic to the native virus for the functional and structural study of hemagglutinin . further investigation of the biological activity for the synthesized vlp was performed on fusion to the jurkat cell membrane induced by ha ( figure 5 ) as probed by dequenching of the embedded r18 label due to its dilution arising from membrane merger . the result established the function exerted by ha as havlp did not exhibit fusion activity . by measuring the fluorescence intensity of r18 , ic50 of fetuin on binding inhibition of havlp to jurkat cell was estimated to be 147 m ( 7.1 mg / ml ) , compared to a previous result of 6.4 mg / ml for inhibition of viral infection by focus - forming assays . however , it reinforced that the application na chimera for antigen incorporation in vlp was feasible . the egfp - vlp could be expanded for its utility as a reporter tag , particularly as a ph sensor inside the ha - targeted cells . further study on the time variation of egfp in the endosome can be conducted to monitor the time course of the fusion process and trafficking of vlp within the cell , which may provide insight into the antigen presentation of the virus and the dynamics and the route of virus invasion in the cell . the extensive biophysical characterization of influenza vlp containing m1 , ha , na , and m2 viral structural proteins established a reliable protocol of synthesizing a large quantity of the virus mimetic for use in vaccine study , in particular for the ha as the major antigen . the incorporation of a fluorescent label demonstrates the feasible use of vlp as an antigen carrier as well as a possible tool for microscopic observation of the virus dynamics and interaction with the cellular components within the cell .

brain metastases are the most common form of brain cancer and exceed the number of primary brain tumors by at least fourfold , with an estimated 98,000170,000 new cases of brain metastases per year in north america [ 1 , 2 ] . these figures will further rise as a result of an ageing population , increasing use of improved diagnostic imaging and advancements in systemic and local cancer therapies . the primary tumors most likely to metastasize to the brain are located in the lung ( 50% ) , breast ( 1525% ) , skin ( melanoma ) ( 520% ) , colon - rectum , and kidney [ 3 , 4 ] . in addition , most other malignant tumors can metastasize to the brain . in up to 15% of patients , the primary site is unknown . the diagnosis of brain metastases ( bm ) is devastating for both patients and caregivers as evidenced by the heavy burden of disease and significant impact on quality of life ( qol ) . clinical manifestations can include focal neurological deficits , seizures , raised intracranial pressure , and alteration in cognition , personality , and ultimately functional status . this is primarily due to the heterogeneity in cancer type and extent , patient demographics and clinical status , and treatment history and options . to better understand the factors influencing survival , a recursive partitioning analysis ( rpa ) of data from three radiation therapy oncology group ( rtog ) prospective brain metastases trials was performed identifying 3 classes of patients based on age , performance status , systemic disease control , and presence of extracranial metastases ( table 1 ) . these classes predict median survival ( 7.1 , 4.2 , and 2.3 months for classes i , ii , and iii patients , resp . ) and suggest that many rpa iii patients will benefit little from aggressive treatment . more aggressive strategies are recommended in younger patients with controlled systemic cancer , minimal extra - cranial metastases , and high - performance status . based on a review of more than four thousand bm patients , the disease - specific graded prognostic assessment ( ds - gpa ) can produce survival estimates better tailored to the primary malignancy . in some cancers , additional factors will be of prognostic importance ( hormone receptor status for breast cancer and number of brain metastases in lung cancer , melanoma , and renal cell carcinoma ) while in others performance status will dominate prognosis ( gastrointestinal malignancies ) . the ongoing expansion in the number and complexity of therapeutic options seen over the past three decades has added further intricacy to the management of bm patients . treatment decisions must therefore be tailored to each individual , based on a complex array of patient- and tumour - specific characteristics , as no single algorithm is appropriate for every patient . close collaboration is required between general medical and radiation oncologists , neuro - oncologists , and neurosurgeons in a multidisciplinary setting where multiple treatment modalities can be offered . finally , there is a lack of consensus amongst practitioners on the optimum treatment strategy and it is often the experience of the local treating team that governs decision making . this trend is underpinned by persistent controversies and limitations in the data supporting management guidelines . whilst accepted medical practice , this review aims to discuss the existing literature , some of the key on - going controversies , and emerging trends surrounding local control ( lc ) of bm . the last 30 years have seen important changes in the management of brain metastases . below is a timeline displaying the major landmarks and some of the notable related publications ( figure 1 ) . the palliative use of corticosteroids in patients harbouring cerebral metastases is necessary for symptom control as it aids in alleviating peritumoral edema [ 9 , 10 ] . however , the dose and duration of corticosteroid use is variable . although recommended for short - term symptom control , patients often remain on prolonged regimens with related complications . these complications are well known and can negatively impact quality of life due to the associated adverse effects such as myopathy , hyperglycemia , weight gain , immune suppression , insomnia , emotional lability , and occasional severe psychiatric disturbances . although only studied in a 48-patient trial from the pre - ct scan era , whole - brain radiation therapy ( wbrt ) has been the mainstay of metastatic brain tumour therapy for decades . following wbrt , overall survival is typically 3 - 4 months . the goals of wbrt include relief of symptoms from existing bm and prevention of future bm . common north american regimens use parallel - opposed megavoltage beams to deliver 2037.5 gy in 515 daily fractions over 13 weeks . acute complications include encephalopathy , cerebral edema , nausea and vomiting , alopecia , dermatitis , mucositis , otitis externa , and fatigue . late complications can include optic and otic toxicities , endocrinopathies , and decline in neurocognitive function . although potentially debilitating , the real incidence of severe radiation encephalopathy is poorly documented . in a study published in 1989 , of particular note , four of these patients received a high dose per fraction ( 5 or 6 gy ) while the other patient received a concurrent radiation sensitizer . the 15 patients who received less than 3 gy fractions did not develop dementia . various authors now agree that the delayed late - radiation injury to the brain may be overstated and less relevant with respect to outcome . however , as patients survive longer with their primary cancer long - term adverse outcomes of wbrt are of significant concern and a number of trials have and continue to address these issues . without such prospective investigations it is difficult to discern the contributing effect that multiple systemic therapies can have on cognitive function . wbrt is often used alone in rpa class iii patients whose main alternative treatment option is best supportive care . in this setting , both overall response rate and neurologic improvement range from 50% to 60% [ 16 , 17 ] . wbrt can also be used in conjunction with local treatment ( surgery or stereotactic radiosurgery ) in rpa class i / ii patients whose alternative is local treatment alone or local treatment combined with systemic treatment . surgical excision of cerebral metastases is often used in patients with rpa class i / ii , harbouring single lesions , and minimal or controlled systemic disease . the goals of surgery include establishment of a diagnosis , lc in noneloquent locations , and rapid relief of symptoms ( e.g. , mass effect , hemorrhage , hydrocephalus ) . currently , surgery involves intraoperative image guidance , microsurgical techniques , perioperative neurologic monitoring , and awake craniotomy with cortical mapping . these and other refinements [ 19 , 20 ] have resulted in significant reduction in postoperative complications which can include infection , neurologic deficits ( including potential impairments of cognition , speech , movement , sensation , vision , hearing , and coordination ) , cerebral hemorrhage , and/or infarction , the incidence of which have been reduced to under 5% [ 21 , 22 ] . unlike infiltrating primary brain tumours , bms are usually well demarcated from surrounding brain parenchyma . this increases the ability to achieve a gross total resection with a minimum of morbidity , especially new neurological deficits . a classical and much quoted randomized controlled trial ( rct ) by patchell et al . investigated the value of adding surgery to wbrt . they demonstrated that resection of a single bm leads to improved overall survival ( os ; driven by increased lc ) , qol , and lc compared to wbrt alone . similar results were demonstrated in another rct . however , only a quarter to a third of bm patients have a single lesion [ 26 , 27 ] and of these nearly half are not suitable for surgery ( inaccessibility of the tumor , extensive systemic disease , unfit for surgery , etc . ) . furthermore , surgical resection does not eradicate microscopic disease in the operative bed nor does it address disease elsewhere in the brain . a follow - up rct led by patchell showed that the addition of adjuvant wbrt following surgical resection reduced recurrence at the surgical site compared to surgery alone . moreover , adjuvant wbrt following surgery prevented subsequent development of distant bms and reduced neurological death when compared to surgery alone . os and qol were reported as not affected raising doubt as to the value of wbrt in preserving qol and functional independence . stereotactic radiosurgery ( srs ) is a technique whereby multiple convergent beams deliver high - energy x - rays , gamma rays , or protons to a discrete radiographically defined target . due to the rapid fall - off of dose outside the target volume , the radiation dose delivered to the normal brain tissue distant from the tumour is clinically insignificant . the advantages of srs are the relative ease of tolerability for patients and the ability to treat deep - seated , surgically inaccessible lesions as well as those traditionally considered radioresistant such as melanoma and renal cell carcinoma . a number of srs devices are in clinical use , including the gamma knife ( gk , elekta ab , stockholm ) dedicated linear accelerators such as the cyberknife ( accuray , sunnyvale , ca ; mounted on a robotic arm ) or the novalis tx ( brainlab / varian medical systems , palo alto , ca / munich , germany ; multi - leaf collimator providing beam shaping ) as well are various modified conventional linear accelerators . radiosurgery has revolutionized the treatment of brain metastases since its first use in north america in the late 1980s and , after more than 30 years of experience , has established itself as a valid therapeutic option providing a high degree of lc for small ( 3-up to 4 cm ) metastases . the ability of upfront srs to improve survival has been demonstrated prospectively relatively recently [ 3538 ] . a literature review by the american society for therapeutic radiology and oncology ( astro ) summarized the available data according to the level of evidence . based on level i iii evidence , for selected patients with single , small ( up to 4 cm ) brain metastases ( up to 3 lesions and 4 in one randomized trial ) , the addition of srs boost to wbrt improves brain control as compared with wbrt alone . there are two prospective randomized studies that have been published in extenso on the subject [ 36 , 38 ] . the strongest evidence comes from the radiation therapy oncology group ( rtog ) 9508 trial , which randomized 164 patients to wbrt and srs boost versus 167 patients to wbrt alone . the brain metastases could be a maximum diameter of 4 cm for the largest lesion and the additional lesions could not exceed 3 cm . for these three randomized trials , local brain control at 1 year ranged from 82% to 92% in the srs boost arm versus 0%71% in the whole - brain alone arm . treatment with radiosurgery alone appears to result in the same overall survival as the combination of srs and wbrt . however , local and distant brain control is significantly poorer with omission of upfront wbrt ( level i iii evidence ) [ 36 , 39 ] . despite this , there is a recent trend toward withholding adjuvant wbrt from initial treatment and deferring it until other treatments have failed . the concerns are long - term neurotoxicity ( especially in longer surviving patients ) , the availability of effective salvage treatments , and the fact that adjuvant wbrt does not translate into a prolonged os or have an impact on preservation of performance status or functional independence [ 14 , 40 ] . it seems that in patients with a limited number of brain metastases ( one to three metastases ) , who are initially treated with either radiosurgery or surgery , wbrt can be withheld if serial imaging for followup is performed . there remains a paradox that patients without active systemic disease and good performance status may be both more likely to benefit from wbrt and more likely to be harmed by its late toxicities . patients with a poor performance status or active systemic disease may either die before developing new metastases or reseed their brain from uncontrolled extra - cranial tumors . some of the nihilism that surrounded the management of oligometastases in the past still persists with multiple metastases patients . this is likely due to the perceived poor outcomes despite available treatments and the high incidence of concurrent , active systemic disease . there is a subset of patients , however , who have treated or controlled systemic disease and have maintained good neurologic function primarily due to small tumor size . traditionally , fractionated wbrt to a dose of approximately 30 gy has been administered . surgical resection has been offered rarely to these patients , because the morbidity of resection in multiple brain locations was believed to be excessive , and the risk for developing additional tumors perceived to be high . radiosurgery can be performed in most brain locations , irrespective of regional brain function . accordingly , radiosurgery can be used to treat multiple metastases in one setting [ 4143 ] . in a retrospective study of 323 patients , chang et al . analyzed the efficacy of radiosurgery in treating patients with various numbers of brain metastases . when assessing patient survival and progression - free survival times as a function of the number of bms , they reported no statistical difference between survival times after radiosurgery . although remote disease progression was more frequent in patients with > 15 bms , there was no statistical difference in local control rates . these findings identified srs as a treatment option for local control of metastatic lesions and raised the notion that srs might offer improved survival in patients with multiple metastatic brain lesions . in a separate retrospective study , bhatnagar et al . devised a recursive partitioning analysis of 205 patients with four or more bm who were treated with srs in one setting . srs was used alone or in conjunction with wbrt , or after failure of wbrt . with a median marginal radiosurgery dose of 16 gy and median total treatment volume of 6.8 cc , they identified two distinct cohorts of patients . patients with a total treatment volume of < 7 cc and < 7 brain metastases ( 46 ) were found to have extended survival following srs . those patients with > 7 cc and > 7 bms had a significantly poorer survival following srs . these studies confirmed a role for srs in the treatment of patients with multiple bms , and identified a subgroup of patients with improved survival following srs . like conventional surgery , however , srs is a focal treatment and its role remains limited by the risk of development of further tumors outside the initial irradiation volumes . with the complex needs of patients with brain metastases , therapeutic options evolve based on clinical judgement and case series . the authors believe that two emerging treatment paradigms will soon integrate the mainstream and thus warrant further clinical studies . the postoperative delivery of wbrt for patients with bm aims to sterilize residual disease in the tumor bed as well as other sites of occult disease in the brain . , in the absence of wbrt , 46% of patients with an mri confirmed complete resection had a recurrence in the original site at a median of 27 weeks . the addition of high - dose wbrt ( 50.4 gy ) decreased this recurrence to 10% with a time to recurrence of 52 weeks . this series , as most others , did not censure patients for lc at the time of last imaging leading to failure rate underestimations . in addition , few physicians use such high doses of wbrt in clinical practice either because of resource limitations or for fear of neurocognitive effects . finally , complete resection is not always confirmed by immediate post - operative mr imaging . with these caveats , the actual 1 - 2 year local recurrence rate is likely often higher in clinical practice . stereotactic radiosurgery to the surgical bed in addition to or without wbrt is an emerging trend in the treatment of brain metastases . a retrospective review carried out at the mcgill university health center , montreal , canada ( muhc ) of patients treated with surgery followed by wbrt revealed an actuarial rate of local recurrence of 67% at 2 years . a retrospective series from rades et al . [ 45 , 46 ] compared 2 groups of patients with rpa class i to ii disease with 1 - 2 resectable brain metastases : resection plus wbrt and same treatment plus a fractionated boost to the tumor bed ( 5 fractions of 3 gy or 5 fractions of 2 gy each ) . in this series , the boost improved both lc ( 2-year lc after complete resection 88% versus 32% , p < 0.001 ) and overall survival ( 1-year os 66% versus 41% , p < 0.001 ) . in a 5-year experience at the muhc using wbrt plus a tumor bed srs boost of 10 gy in 44 patients , the actuarial 2-year lc was 91% . the median survival was 17 months , and with a median followup of 10 months , only 11% developed new metastases [ 4749 ] . without the srs boost , the 1- and 2-year actuarial lc rates were 52% and 33% , respectively , statistically worse than with tumor bed srs ( p < 0.001 ) and in keeping with the data of rades et al . three recent retrospective studies have described the use of srs or fractionated stereotactic radiotherapy following surgery without wbrt . soltys et al . reported a 1-year actuarial lc rate of 79% with no deaths secondary to neurologic causes in a series of 72 patients treated at stanford university . in a series of 40 patients , mathieu et al . do et al . demonstrated that an actuarial 1-year lc rate was 82% in 30 patients , 19 ( 63% ) of which developed recurrences in new intracranial sites . the concern with tumor bed srs alone is that srs is limited by the size of the tumor cavity and prone to geographic miss . some groups have advocated the inclusion of a 2 mm margin around the tumor cavity to improve lc . as previously discussed , wbrt has a proven effect in reducing the occurrence of new brain metastases . in patchell 's study , aoyama et al . demonstrated a reduction from 64 to 42% with no difference in toxicity . in addition , the above studies looking at srs wbrt demonstrated greater local control rates with intact brain metastases . it seems reasonable to expect that in a similar fashion , the combination of srs and wbrt would show improved local control at the surgical bed . moreover , wbrt eliminates any risk of complete marginal miss from difficulties in defining a proper post - operative target . the use of a reduced dose of srs opens this option to patients with large tumors and lesions approximating critical structures . post - operative intracranial failure most commonly occurs in the tumor bed , despite the use of adjuvant wbrt . although evidence suggests that postoperative srs provides increased lc , a prospective trial comparing this strategy to wbrt alone has not been carried out and is the subject of a current national cancer institute trial ( nct00003320 ) . if the number of published series can be used as an indicator , tumor bed srs alone is being used more and more commonly . if this strategy can provide equivalent lc to wbrt with less neuro - cognitive toxicity , its adoption will grow rapidly . advances in chemotherapeutic agents for systemic cancer have improved patient survival and overall prognosis . as the number of patients with prolonged survival rises , and the use of upfront wbrt decreases , an increase in a subgroup of patients with bm who have failed initial treatment and developed recurrence is likely . although previously neglected , this growing subgroup merits specific studies to determine the optimum treatment strategy to extend survival and minimize morbidity . at our institution , over a third of the eligible patient population undergo upfront srs alone . the therapeutic strategies offered to these patients continue to evolve but finding level 1 evidence to guide physicians remains challenging , as studies in this area are very limited . brain relapses comprise not only lesions that have progressed locally following initial treatment but also new lesions that have developed at a distance or a combination of the two . in this context , surgical resection is often not appropriate and not indicated as more than two - thirds of patients have multiple brain metastases and large tumors are scarce as closer mri followup allows early detection of new metastases . current chemotherapy regimens or small molecule inhibitors used for targeted therapy of systemic cancer are typically not effective for bm as the blood - brain barrier limits bioavailability in the tumour tissue . nonsurgical salvage treatment options include wbrt for wbrt - nave patients , repeat wbrt , salvage srs , or a combination of radiation modalities . although salvage wbrt alone is a widely accepted traditional approach at most institutions , there are no studies that evaluate its use in patients whose initial management did not include wbrt , in other words salvage wbrt after failure of initial treatment with srs . conversely , the use of repeat wbrt in patients who have failed previous treatment with wbrt has been reported but its use remains controversial and doubts persist as to the therapeutic ratio [ 5457 ] . largely retrospective data show improvement in neurologic function in 3142% of patients , median duration of response of 2.5 months , and median survival of 4 - 5 months following reirradiation with 2025 gy over multiple fractions . the strongest predictors of a favourable outcome following re - irradiation were a good performance status and a good response to initial treatment . late complications are largely unreported but proponents argue that these need to be balanced by the likely occurrence of neurological deterioration if lesions are left untreated . salvage radiosurgery as defined by the use of srs to treat brain relapses is being offered on an individual basis and is in many cases accepted as sole treatment . indeed , this indication was at the very origin of the use of srs for bm . although its role at the time of progressive brain metastases previously treated with either whole - brain radiotherapy or srs alone has not been fully elucidated , there is evidence to suggest that it provides good lc thus extending bdfs . however , there are theoretical concerns of higher rates of radiation necrosis associated with repeat srs at local failure with the consequent increased risk of neurological deficits . a number of retrospective case series exist analyzing salvage srs in patients whose initial management was wbrt along [ 5962 ] . . showed 100% lc in 21 lesions . in one of the larger series of the subsequent decade , nol et al . reported 1- and 2-year lc rates of 91 and 84% with a median survival of 7.8 months in 54 patients whose median interval between the end of wbrt and salvage srs was 9 months . one- and 2-year new brain event - free survival rates were 65% and 57% , respectively . although 24% of patients developed new metastases , none of the patients died from cerebral cause , and only 5% of the treated lesions recurred with 28% of patients being still alive at 2 years . larger more recent study , chao et al . showed an overall survival of 9.9 months following salvage srs in 111 patients . lc was 68 and 59% at 1 and 2 years , respectively , with 25 and 31% of patients developing local and distant recurrence , respectively . only 3.6 % of patients developed complications ( radiation necrosis in 2 patients , seizures in 1 , and severe fatigue in 1 ) . one prospective study showed a median survival of 6 months from salvage srs with lc of 19 treated lesions . although predictive factors were not consistent in the literature , the interval between wbrt and srs seem to predict overall survival . a few studies examine the use of salvage srs after initial treatment with srs [ 6366 ] . two of these studies present survival data from the date of salvage srs [ 63 , 64 ] . kwon et al . presented a series of 43 patients who underwent salvage srs in which median survival from the time of srs to recurrent / progressive disease was 32 weeks and the lc rate of retreated lesions at 6 months was 91% . multivariate analysis revealed that rpa class was the only predictor for overall survival . in the case series by chen et al . , of 45 patients , median survival from the time of srs for recurrent brain metastases was 28 weeks . both authors noted that repeated srs may extend survival at least as long as the first srs intervention had . unfortunately , these studies are retrospective and the reported results are subject to selection bias . no comparison is made with other competing strategies , making the evidence class ii and iii . nevertheless , they provide compelling support for repeat or salvage srs being effective in treating locally progressive or new lesions after initial srs . arguably , this strategy should be considered in favourable rpa class patients in order to minimize morbidity , maximize patient quality of life and perception of disease burden , and to reduce cost [ 6871 ] . prospective trials are needed to determine the clinical value and compare srs and wbrt salvage modalities at the time of local or distant failure . the goal of brain metastases management is to minimize morbidity and mortality , improve patient quality of life , and reduce associated treatment costs . in an era of healthcare budget constraints , it is imperative that clinicians adopt fiscally responsible standards to guide their practice . several reports have examined the treatment outcomes and cost - effectiveness of current management paradigms . in a retrospective study , metha and colleagues assessed the survival and quality of life outcome data for patients with solitary brain metastasis , who were randomized to receive wbrt , surgery + wbrt , or radiosurgery alone . when comparing the relative cost ratios of surgery and radiosurgery , and analyzing the cost - effectiveness ( cost per year of median survival ) of each modality , radiosurgery yielded greater survival and functional independence versus surgery or wbrt alone . more recently , lee et al . examined the outcomes and cost - effectiveness of treating 156 patients with multiple brain metastases , randomized to receive radiosurgery or wbrt . the mortality rate for radiosurgery - treated patients , with multiple brain metastases ( 25 lesions ) and a good initial kps score , was found to be significantly better . radiosurgery also resulted in better posttreatment kps scores , improved quality of life , and higher cost - effectiveness when compared to wbrt . these findings highlight the need for prospective clinical trials to comprehensively study the clinical and economic efficacy of various treatment modalities , especially in a time of increasing healthcare costs and significant budget constraints . over the last three decades a major shift in the philosophy guiding the treatment of patients with brain metastases has occurred . the traditional nihilistic expectation of rapid neurological decline and inevitable neurological demise is no longer acceptable , and the focus has moved from palliation to achieving sustained brain disease control , systemic disease control , and preserved neurological function . thus , with a variety of aggressive treatment options available to them , patients can now benefit not only from longer overall survival but also extended brain disease - free survival and improved quality of life . these parameters and others including the prolongation of functional independent status , reduction in burden of focal neurological deficits , neurocognitive preservation , and freedom from seizures have now become established treatment goals and have taken over overall survival as primary endpoints in major brain metastases trials . with current management strategies , patients with limited brain metastases are often more likely to succumb from their systemic disease than from their brain tumour(s ) . continual progress in systemic cancer treatments has led to a steady increase in patient survival and overall prognosis . paradoxically , as control of systemic disease improves , the occurrence of brain metastases and its associated neurological morbidity and mortality will increase . the management of brain metastases has seen a paradigm shift from palliation toward aggressive intervention to achieve control of brain disease . following surgical resection , however , more recent data has highlighted the potential benefit of more aggressive local control measures involving surgical resection and srs in addition to wbrt . radiosurgery alone , surgical resection with adjuvant srs , and advanced chemotherapeutic agents have been increasingly used amidst concerns of the potential neurotoxicity of wbrt . following surgery , intracranial failure most commonly occurs in the tumor bed , despite wbrt . postoperative srs boost to the tumor cavity may provide acceptable local control without many of the toxicities of wbrt . the efficacy of this strategy remains to be tested in a comparative prospective trial . optimum management strategies at the time of failure remain unclear and include salvage wbrt , salvage srss or a combination of the two . in summary , although much progress has been made in the last 3 decades , numerous challenges lay ahead in establishing evidence - based guidelines in this challenging group of patients . the field of neuro - oncology is witnessing an exciting evolution in management of brain metastases . the future holds great promise and opportunity to carry out focused clinical investigations to demonstrate efficacy of our treatment paradigms . proposed management goals will not only aim to increase overall survival and brain disease - free survival , but also to improve quality of life and prolong functional independent status whilst minimizing neurological deficit .

the last 30 years have seen major changes in attitude toward patients with cerebral metastases . this paper aims to outline the major landmarks in this transition and the therapeutic strategies currently used . the controversies surrounding control of brain disease are discussed , and two emerging management trends are reviewed : tumor bed radiosurgery and salvage radiation .

1. Introduction 2. Therapeutic Strategies and Supporting Evidence 3. Emerging Trends in Treatment of BM 4. Conclusions

brain metastases are the most common form of brain cancer and exceed the number of primary brain tumors by at least fourfold , with an estimated 98,000170,000 new cases of brain metastases per year in north america [ 1 , 2 ] . the primary tumors most likely to metastasize to the brain are located in the lung ( 50% ) , breast ( 1525% ) , skin ( melanoma ) ( 520% ) , colon - rectum , and kidney [ 3 , 4 ] . in addition , most other malignant tumors can metastasize to the brain . the diagnosis of brain metastases ( bm ) is devastating for both patients and caregivers as evidenced by the heavy burden of disease and significant impact on quality of life ( qol ) . clinical manifestations can include focal neurological deficits , seizures , raised intracranial pressure , and alteration in cognition , personality , and ultimately functional status . this is primarily due to the heterogeneity in cancer type and extent , patient demographics and clinical status , and treatment history and options . to better understand the factors influencing survival , a recursive partitioning analysis ( rpa ) of data from three radiation therapy oncology group ( rtog ) prospective brain metastases trials was performed identifying 3 classes of patients based on age , performance status , systemic disease control , and presence of extracranial metastases ( table 1 ) . these classes predict median survival ( 7.1 , 4.2 , and 2.3 months for classes i , ii , and iii patients , resp . ) more aggressive strategies are recommended in younger patients with controlled systemic cancer , minimal extra - cranial metastases , and high - performance status . in some cancers , additional factors will be of prognostic importance ( hormone receptor status for breast cancer and number of brain metastases in lung cancer , melanoma , and renal cell carcinoma ) while in others performance status will dominate prognosis ( gastrointestinal malignancies ) . the ongoing expansion in the number and complexity of therapeutic options seen over the past three decades has added further intricacy to the management of bm patients . close collaboration is required between general medical and radiation oncologists , neuro - oncologists , and neurosurgeons in a multidisciplinary setting where multiple treatment modalities can be offered . this trend is underpinned by persistent controversies and limitations in the data supporting management guidelines . whilst accepted medical practice , this review aims to discuss the existing literature , some of the key on - going controversies , and emerging trends surrounding local control ( lc ) of bm . the last 30 years have seen important changes in the management of brain metastases . below is a timeline displaying the major landmarks and some of the notable related publications ( figure 1 ) . the palliative use of corticosteroids in patients harbouring cerebral metastases is necessary for symptom control as it aids in alleviating peritumoral edema [ 9 , 10 ] . although recommended for short - term symptom control , patients often remain on prolonged regimens with related complications . these complications are well known and can negatively impact quality of life due to the associated adverse effects such as myopathy , hyperglycemia , weight gain , immune suppression , insomnia , emotional lability , and occasional severe psychiatric disturbances . acute complications include encephalopathy , cerebral edema , nausea and vomiting , alopecia , dermatitis , mucositis , otitis externa , and fatigue . late complications can include optic and otic toxicities , endocrinopathies , and decline in neurocognitive function . in a study published in 1989 , of particular note , four of these patients received a high dose per fraction ( 5 or 6 gy ) while the other patient received a concurrent radiation sensitizer . wbrt is often used alone in rpa class iii patients whose main alternative treatment option is best supportive care . in this setting , both overall response rate and neurologic improvement range from 50% to 60% [ 16 , 17 ] . surgical excision of cerebral metastases is often used in patients with rpa class i / ii , harbouring single lesions , and minimal or controlled systemic disease . the goals of surgery include establishment of a diagnosis , lc in noneloquent locations , and rapid relief of symptoms ( e.g. , mass effect , hemorrhage , hydrocephalus ) . currently , surgery involves intraoperative image guidance , microsurgical techniques , perioperative neurologic monitoring , and awake craniotomy with cortical mapping . these and other refinements [ 19 , 20 ] have resulted in significant reduction in postoperative complications which can include infection , neurologic deficits ( including potential impairments of cognition , speech , movement , sensation , vision , hearing , and coordination ) , cerebral hemorrhage , and/or infarction , the incidence of which have been reduced to under 5% [ 21 , 22 ] . this increases the ability to achieve a gross total resection with a minimum of morbidity , especially new neurological deficits . they demonstrated that resection of a single bm leads to improved overall survival ( os ; driven by increased lc ) , qol , and lc compared to wbrt alone . however , only a quarter to a third of bm patients have a single lesion [ 26 , 27 ] and of these nearly half are not suitable for surgery ( inaccessibility of the tumor , extensive systemic disease , unfit for surgery , etc . ) moreover , adjuvant wbrt following surgery prevented subsequent development of distant bms and reduced neurological death when compared to surgery alone . os and qol were reported as not affected raising doubt as to the value of wbrt in preserving qol and functional independence . stereotactic radiosurgery ( srs ) is a technique whereby multiple convergent beams deliver high - energy x - rays , gamma rays , or protons to a discrete radiographically defined target . due to the rapid fall - off of dose outside the target volume , the radiation dose delivered to the normal brain tissue distant from the tumour is clinically insignificant . the advantages of srs are the relative ease of tolerability for patients and the ability to treat deep - seated , surgically inaccessible lesions as well as those traditionally considered radioresistant such as melanoma and renal cell carcinoma . a number of srs devices are in clinical use , including the gamma knife ( gk , elekta ab , stockholm ) dedicated linear accelerators such as the cyberknife ( accuray , sunnyvale , ca ; mounted on a robotic arm ) or the novalis tx ( brainlab / varian medical systems , palo alto , ca / munich , germany ; multi - leaf collimator providing beam shaping ) as well are various modified conventional linear accelerators . radiosurgery has revolutionized the treatment of brain metastases since its first use in north america in the late 1980s and , after more than 30 years of experience , has established itself as a valid therapeutic option providing a high degree of lc for small ( 3-up to 4 cm ) metastases . based on level i iii evidence , for selected patients with single , small ( up to 4 cm ) brain metastases ( up to 3 lesions and 4 in one randomized trial ) , the addition of srs boost to wbrt improves brain control as compared with wbrt alone . the brain metastases could be a maximum diameter of 4 cm for the largest lesion and the additional lesions could not exceed 3 cm . treatment with radiosurgery alone appears to result in the same overall survival as the combination of srs and wbrt . despite this , there is a recent trend toward withholding adjuvant wbrt from initial treatment and deferring it until other treatments have failed . the concerns are long - term neurotoxicity ( especially in longer surviving patients ) , the availability of effective salvage treatments , and the fact that adjuvant wbrt does not translate into a prolonged os or have an impact on preservation of performance status or functional independence [ 14 , 40 ] . it seems that in patients with a limited number of brain metastases ( one to three metastases ) , who are initially treated with either radiosurgery or surgery , wbrt can be withheld if serial imaging for followup is performed . patients with a poor performance status or active systemic disease may either die before developing new metastases or reseed their brain from uncontrolled extra - cranial tumors . this is likely due to the perceived poor outcomes despite available treatments and the high incidence of concurrent , active systemic disease . traditionally , fractionated wbrt to a dose of approximately 30 gy has been administered . surgical resection has been offered rarely to these patients , because the morbidity of resection in multiple brain locations was believed to be excessive , and the risk for developing additional tumors perceived to be high . in a retrospective study of 323 patients , chang et al . analyzed the efficacy of radiosurgery in treating patients with various numbers of brain metastases . although remote disease progression was more frequent in patients with > 15 bms , there was no statistical difference in local control rates . these findings identified srs as a treatment option for local control of metastatic lesions and raised the notion that srs might offer improved survival in patients with multiple metastatic brain lesions . devised a recursive partitioning analysis of 205 patients with four or more bm who were treated with srs in one setting . with a median marginal radiosurgery dose of 16 gy and median total treatment volume of 6.8 cc , they identified two distinct cohorts of patients . patients with a total treatment volume of < 7 cc and < 7 brain metastases ( 46 ) were found to have extended survival following srs . those patients with > 7 cc and > 7 bms had a significantly poorer survival following srs . these studies confirmed a role for srs in the treatment of patients with multiple bms , and identified a subgroup of patients with improved survival following srs . like conventional surgery , however , srs is a focal treatment and its role remains limited by the risk of development of further tumors outside the initial irradiation volumes . with the complex needs of patients with brain metastases , therapeutic options evolve based on clinical judgement and case series . the authors believe that two emerging treatment paradigms will soon integrate the mainstream and thus warrant further clinical studies . the postoperative delivery of wbrt for patients with bm aims to sterilize residual disease in the tumor bed as well as other sites of occult disease in the brain . , in the absence of wbrt , 46% of patients with an mri confirmed complete resection had a recurrence in the original site at a median of 27 weeks . finally , complete resection is not always confirmed by immediate post - operative mr imaging . stereotactic radiosurgery to the surgical bed in addition to or without wbrt is an emerging trend in the treatment of brain metastases . a retrospective review carried out at the mcgill university health center , montreal , canada ( muhc ) of patients treated with surgery followed by wbrt revealed an actuarial rate of local recurrence of 67% at 2 years . [ 45 , 46 ] compared 2 groups of patients with rpa class i to ii disease with 1 - 2 resectable brain metastases : resection plus wbrt and same treatment plus a fractionated boost to the tumor bed ( 5 fractions of 3 gy or 5 fractions of 2 gy each ) . in this series , the boost improved both lc ( 2-year lc after complete resection 88% versus 32% , p < 0.001 ) and overall survival ( 1-year os 66% versus 41% , p < 0.001 ) . in a 5-year experience at the muhc using wbrt plus a tumor bed srs boost of 10 gy in 44 patients , the actuarial 2-year lc was 91% . the median survival was 17 months , and with a median followup of 10 months , only 11% developed new metastases [ 4749 ] . without the srs boost , the 1- and 2-year actuarial lc rates were 52% and 33% , respectively , statistically worse than with tumor bed srs ( p < 0.001 ) and in keeping with the data of rades et al . demonstrated that an actuarial 1-year lc rate was 82% in 30 patients , 19 ( 63% ) of which developed recurrences in new intracranial sites . the concern with tumor bed srs alone is that srs is limited by the size of the tumor cavity and prone to geographic miss . some groups have advocated the inclusion of a 2 mm margin around the tumor cavity to improve lc . as previously discussed , wbrt has a proven effect in reducing the occurrence of new brain metastases . moreover , wbrt eliminates any risk of complete marginal miss from difficulties in defining a proper post - operative target . the use of a reduced dose of srs opens this option to patients with large tumors and lesions approximating critical structures . post - operative intracranial failure most commonly occurs in the tumor bed , despite the use of adjuvant wbrt . although evidence suggests that postoperative srs provides increased lc , a prospective trial comparing this strategy to wbrt alone has not been carried out and is the subject of a current national cancer institute trial ( nct00003320 ) . if the number of published series can be used as an indicator , tumor bed srs alone is being used more and more commonly . as the number of patients with prolonged survival rises , and the use of upfront wbrt decreases , an increase in a subgroup of patients with bm who have failed initial treatment and developed recurrence is likely . at our institution , over a third of the eligible patient population undergo upfront srs alone . the therapeutic strategies offered to these patients continue to evolve but finding level 1 evidence to guide physicians remains challenging , as studies in this area are very limited . brain relapses comprise not only lesions that have progressed locally following initial treatment but also new lesions that have developed at a distance or a combination of the two . in this context , surgical resection is often not appropriate and not indicated as more than two - thirds of patients have multiple brain metastases and large tumors are scarce as closer mri followup allows early detection of new metastases . conversely , the use of repeat wbrt in patients who have failed previous treatment with wbrt has been reported but its use remains controversial and doubts persist as to the therapeutic ratio [ 5457 ] . largely retrospective data show improvement in neurologic function in 3142% of patients , median duration of response of 2.5 months , and median survival of 4 - 5 months following reirradiation with 2025 gy over multiple fractions . the strongest predictors of a favourable outcome following re - irradiation were a good performance status and a good response to initial treatment . a number of retrospective case series exist analyzing salvage srs in patients whose initial management was wbrt along [ 5962 ] . . in one of the larger series of the subsequent decade , nol et al . reported 1- and 2-year lc rates of 91 and 84% with a median survival of 7.8 months in 54 patients whose median interval between the end of wbrt and salvage srs was 9 months . although 24% of patients developed new metastases , none of the patients died from cerebral cause , and only 5% of the treated lesions recurred with 28% of patients being still alive at 2 years . lc was 68 and 59% at 1 and 2 years , respectively , with 25 and 31% of patients developing local and distant recurrence , respectively . only 3.6 % of patients developed complications ( radiation necrosis in 2 patients , seizures in 1 , and severe fatigue in 1 ) . a few studies examine the use of salvage srs after initial treatment with srs [ 6366 ] . presented a series of 43 patients who underwent salvage srs in which median survival from the time of srs to recurrent / progressive disease was 32 weeks and the lc rate of retreated lesions at 6 months was 91% . both authors noted that repeated srs may extend survival at least as long as the first srs intervention had . unfortunately , these studies are retrospective and the reported results are subject to selection bias . arguably , this strategy should be considered in favourable rpa class patients in order to minimize morbidity , maximize patient quality of life and perception of disease burden , and to reduce cost [ 6871 ] . the goal of brain metastases management is to minimize morbidity and mortality , improve patient quality of life , and reduce associated treatment costs . several reports have examined the treatment outcomes and cost - effectiveness of current management paradigms . in a retrospective study , metha and colleagues assessed the survival and quality of life outcome data for patients with solitary brain metastasis , who were randomized to receive wbrt , surgery + wbrt , or radiosurgery alone . when comparing the relative cost ratios of surgery and radiosurgery , and analyzing the cost - effectiveness ( cost per year of median survival ) of each modality , radiosurgery yielded greater survival and functional independence versus surgery or wbrt alone . more recently , lee et al . examined the outcomes and cost - effectiveness of treating 156 patients with multiple brain metastases , randomized to receive radiosurgery or wbrt . radiosurgery also resulted in better posttreatment kps scores , improved quality of life , and higher cost - effectiveness when compared to wbrt . over the last three decades a major shift in the philosophy guiding the treatment of patients with brain metastases has occurred . the traditional nihilistic expectation of rapid neurological decline and inevitable neurological demise is no longer acceptable , and the focus has moved from palliation to achieving sustained brain disease control , systemic disease control , and preserved neurological function . thus , with a variety of aggressive treatment options available to them , patients can now benefit not only from longer overall survival but also extended brain disease - free survival and improved quality of life . these parameters and others including the prolongation of functional independent status , reduction in burden of focal neurological deficits , neurocognitive preservation , and freedom from seizures have now become established treatment goals and have taken over overall survival as primary endpoints in major brain metastases trials . with current management strategies , patients with limited brain metastases are often more likely to succumb from their systemic disease than from their brain tumour(s ) . paradoxically , as control of systemic disease improves , the occurrence of brain metastases and its associated neurological morbidity and mortality will increase . the management of brain metastases has seen a paradigm shift from palliation toward aggressive intervention to achieve control of brain disease . radiosurgery alone , surgical resection with adjuvant srs , and advanced chemotherapeutic agents have been increasingly used amidst concerns of the potential neurotoxicity of wbrt . following surgery , intracranial failure most commonly occurs in the tumor bed , despite wbrt . the efficacy of this strategy remains to be tested in a comparative prospective trial . in summary , although much progress has been made in the last 3 decades , numerous challenges lay ahead in establishing evidence - based guidelines in this challenging group of patients . the field of neuro - oncology is witnessing an exciting evolution in management of brain metastases . proposed management goals will not only aim to increase overall survival and brain disease - free survival , but also to improve quality of life and prolong functional independent status whilst minimizing neurological deficit .

the wnt/-catenin pathway has been implicated in the development and progression of melanoma and a wide range of cancer types , including colorectal cancer , breast cancer , esophageal carcinoma , and liver cancer [ 13 ] . under normal conditions , increases in expression and binding of certain wnt ligands to frizzled receptor or mutations in specific components of the -catenin degradation assembly deactivate this regulatory mechanism . nuclear -catenin stimulates transcription of a large number of tcf/-catenin responsive genes that include cyclin d1 , c - myc [ 5 , 6 ] , and the melanocyte - specific gene , microphthalmia - associated transcription factor mitf - m . thus , accumulation of nuclear -catenin as observed in several cancer types is considered a marker of canonical wnt/-catenin pathway deregulation and unfavorable prognosis [ 3 , 8 ] . previous studies have reported an association between nuclear -catenin accumulation and melanoma progression and suggested nuclear -catenin to be a marker of poor prognosis [ 1 , 7 ] . however , recent studies have shown that contrary to breast and colon cancer , metastatic progression of melanoma is associated with decreases in nuclear and cytoplasmic -catenin expression [ 9 , 10 ] . moreover , clinical , genetic , and histological studies suggest that nuclear and cytoplasmic -catenin may be used as biomarkers of good prognosis in melanoma [ 1114 ] . recently , hhr6 , a human homologue of the yeast rad6 gene and a principal component of the postreplication dna repair pathway , has been identified as an important regulator of canonical wnt/-catenin signaling [ 15 , 16 ] . hhr6 , referred hereafter as rad6 , stabilizes -catenin by polyubiquitin modifications that render -catenin resistant to 26s proteasomal degradation . furthermore , rad6 is a transcriptional target of -catenin , thus revealing a positive feedback loop between -catenin - mediated activation of rad6 gene expression and rad6-induced -catenin stabilization . rad6 expression is low in normal breast tissues ; however , increases in rad6 protein expression are detected in hyperplastic , ductal carcinoma in situ ( dcis ) and invasive breast carcinomas . we have previously demonstrated a role for rad6 in breast cancer progression through its regulatory effect on the canonical wnt/-catenin pathway . since the decrease / loss of nuclear -catenin [ 9 , 10 ] , rather than increases as in breast cancer , is linked to melanoma progression , it is not known whether rad6 and -catenin work in concert to promote melanoma pathogenesis . furthermore , rad6 expression in the skin has not been investigated , and there are no data on the role of rad6 in the pathogenesis of benign ( nevi ) and malignant ( melanoma ) melanocytic lesions . it is important to address this gap in knowledge because of the unmet medical need for new effective antimelanoma therapies and because rad6 and -catenin have been identified as therapeutic targets [ 19 , 20 ] . in this study , we examined rad6 and -catenin expressions in serial sections of nevi , primary , and metastatic melanomas to determine their potential roles in melanoma development and metastatic progression . our data suggest that membranous relocation of -catenin and upregulation of rad6 are independent markers of melanoma development and progression . we also offer a hypothesis that explains the role of membranous -catenin relocation and decreasing cytoplasmic -catenin in melanoma development , a phenomenon that has been linked to unfavorable prognosis [ 9 , 21 , 22 ] . cases were retrieved from the files of the pinkus dermatopathology laboratory ( pdl ) , a private dermatopathology laboratory located in monroe , mi . preserved paraffin - embedded tissue specimens collected for each case were assigned an accession code that excluded patient identifier information . nevus and primary melanoma cases were selected for study using random numbers generated by a uniform random number generator ( stata / mp 13.1 ) . the study groups consisted of 30 cases of melanocytic nevi , 29 cases of primary cutaneous melanoma , and 29 cases of metastatic cutaneous melanoma . the study includes all metastatic cutaneous melanoma samples that were archived between 2010 and 2012 . the number of cases for each nevus and primary melanoma subtype was determined to reflect the lesion 's relative representation in cases obtained at the pdl during the above period . atypical nevi were diagnosed using criteria originally proposed by clark and lesion architecture as reviewed by roth et al . . primary antibodies used in the study are as follows : ( i ) anti--catenin ( is702 ) was purchased from dako ( glostrup , denmark ) and used in an undiluted form ; and ( ii ) anti - rad6 ( ab31917 ) was purchased from abcam ( cambridge , ma ) and used at a 1 : 500 dilution . in humans , the yeast homologous rad6 gene is duplicated and the proteins encoded by the two genes hhr6a ( or rad6a ) and hhr6b ( rad6b ) from chromosomes xq24-q25 and 5q23-q31 , respectively , share 95% identical amino acid residues . neither ab31917 , our own rad6 antibody , nor any other commercially available anti - rad6 antibody is currently able to distinguish between rad6a and rad6b proteins . therefore , rather than referring as rad6a or rad6b , we refer to the protein detected by the antibody as rad6 . briefly , five - micrometer sections were deparaffinized in xylene and rehydrated in graded ethanol . for antigen retrieval , sections were microwaved in citrate buffer ph 6.0 ( biogenex , san ramon , ca , usa ) for 12 min at 95c and cooled for 30 min prior to immunostaining . sections were incubated with 3% hydrogen peroxide for 15 min , followed by incubation with primary antibody for 60 min . an automated immunostainer ( i6000 ; biogenex ) was utilized for subsequent incubation steps : sections were incubated in multilink biotinylated anti - igg for 20 min , horseradish peroxidase conjugated secondary antibody for 20 min , followed by development with 3-amino-9-ethyl - carbazole for 10 min ( biogenex ) . all incubation steps were performed at room temperature , and sections were washed with tris - buffered saline between incubations . lung and colon cancer tissues were included as positive controls for immunostaining with anti--catenin antibody , and breast cancer tissues were included as positive controls for staining with anti - rad6 antibody . stained sections were independently enumerated by two coauthors ( d. r. mehregan and m. campbell ) , who were blinded to patient medical records for each case . blinded enumeration was performed under light microscopy at 400x magnification , and an ocular grid consisting of a simple square lattice of 100 test points was utilized to count the number of positively and negatively stained melanomas or nevus cells for each section . per section , the total number of positively and negatively stained cells was counted for each of three sequential horizontal fields . the mean value of the three fields was used to estimate the relative density of cells in a specimen . to increase assessment accuracy , all positively and negatively stained melanomas or nevus cells in a visual field when independent readings for positively stained cells differed by 20% for a given section , both evaluators reviewed the section together to establish a consensus reading . a specimen was considered negative if less than 4% of the cells were immunostained for rad6 or -catenin . a tumor was considered to be stained with high intensity if > 50% of the cells in a specimen expressed rad6 or -catenin , similar to the criteria used by mineta et al . . kruskal - wallis tests were used to compare groups on the basis of continuous variables such as age and percent positive cells . chi - square tests for differences in proportions were used to compare groups on the basis of categorical variables such as gender and -catenin localization . spearman 's rank correlation was used to assess the pairwise association between age and percent of rad6 positive and -catenin positive cells . multinomial logistic regression was used to assess the simultaneous association of rad6 and age with diagnostic group . adjustments were made for multiple comparisons using wilcoxon rank sum tests with bonferroni correction for pairwise comparisons . our analysis included 30 individuals diagnosed with nevi , 29 with primary melanoma , and 29 with metastatic melanoma ( table 1 ) . these groups differed marginally with respect to gender ( p = 0.08 ) and significantly in age ( p = 0.0001 ) . significant age differences were observed between individuals with nevi and those with either primary melanomas ( p = 0.02 ) or metastatic melanomas ( p = 0.0001 ) . in contrast , no age difference is observed between individuals with primary and metastatic melanomas ( p = 0.27 ) . significant differences in age were also observed between groups defined by -catenin localization ( p = 0.007 ) . individuals with -catenin localized in the cytoplasm were significantly younger than individuals with -catenin localization at the cell membrane ( p = 0.02 ) and marginally younger than individuals with -catenin localization at both the cytoplasm and the cell membrane ( p = 0.05 ) . when age was categorized as < 50 , 5060 , and > 60 years , there were statistically significant differences in rad6 expression between the groups ( p = 0.0008 ) , although there is substantial variability ( table 2 ) . median rad6 is greater in the group of people older than 60 years compared to the 5060 years old group ( p = 0.04 ) and < 50 years old group ( p = 0.001 ) . a 20% discrepancy of positively stained cells between the two evaluators was observed in fewer than 5% of the cases , and these cases were evaluated together to establish a consensus reading . melanoma development and progression were not associated with significant changes in percentage of specimens expressing -catenin . -catenin staining was observed in 97% of nevi and in all primary and metastatic melanomas . also , the percentages of nevi ( 93% ) , primary melanoma ( 97% ) , and metastatic melanoma ( 93% ) that expressed -catenin in more than 50% of the cells did not differ significantly ( figures 1 , 2 , and 3 ) . however , significant differences were observed between the percentages of nevi ( 59% ) , primary melanoma ( 90% ) , and metastatic melanoma ( 56% ) that expressed -catenin in more than 90% of the cells ( p = 0.02 ; figure 2 ) . these differences were greatly impacted by the percentage of primary melanomas ( 48% ) that expressed -catenin in 100% of the cells , which was approximately twofold higher than the percentages of nevi ( 21% ) or metastatic melanoma ( 26% ) ( data not shown ) . none of the nevi or melanomas expressed -catenin in the nucleus ( figure 1 ) . the percentage of tumors that expressed membranous -catenin increased dramatically from nevi ( 10% ) to primary and metastatic melanomas ( 83% and 93% , resp . ; concurrently with this increase , the percentage of tumors that expressed cytoplasmic -catenin decreased from nevi ( 90% ) to primary and metastatic melanomas ( 45% and 38% , resp . ; in contrast , no significant differences were observed between the percentages of primary and metastatic melanomas that expressed -catenin at either the membrane ( 83% and 93% , resp . ; p = 0.289 ) or the cytoplasm ( 45% and 38% , resp . ; p = 0.633 ; figure 4 ) . while all four nevi types ( junctional , intradermal , compound , and atypical ) expressed -catenin in the cytoplasm , only junctional and atypical nevi expressed -catenin at the plasma membrane . as opposed to nevi types , the three primary melanoma types examined ( superficial spreading , nodular , and lentigo maligna ) did not differ in -catenin localization as -catenin was localized at the plasma membrane , in the cytoplasm , or in both ( figure 4 ) . conversely , all primary melanomas ( 100% ) and the majority of metastatic melanomas ( 96% ) exhibited greater than 50% rad6 expression . the increase in tumor populations expressing rad6 from 37% of nevi to 100% of primary and metastatic melanomas was significant ( p = 0.0001 ; figures 1 , 2 , and 3 ) . melanoma progression from primary to metastatic disease was not associated with changes in the ( i ) percentage of melanomas expressing rad6 ( 100% of primary and metastatic melanomas ) , or ( ii ) percentage of melanomas expressing rad6 in more than 50% of the tumor cells ( 100% and 96% of primary and metastatic melanomas , resp . ) . the increase in proportion of tumor populations , expressing rad6 in more than 50% of the cells in primary melanoma ( 67% ) versus metastatic melanoma ( 79% ) was not significant ( p = 0.37 ; figure 2 ) . this study was not designed to test whether the distribution of the tumor cells positive for rad6 is the same between the subtypes of nevi . however , the percentages of benign tumors that lacked rad6 were similar between atypical nevi ( 62% ) and the group of other three nevi types ( 59% ) . interestingly , only one of the 30 nevi ( atypical nevus , 3% ) expressed rad6 in > 80% of the cells , and none of the primary and metastatic melanomas expressed rad6 in < 40% of the cells ( figure 2 ) . these results prompted us to examine whether rad6 expression can serve as a marker for histological diagnosis of melanoma . using a multiple logistic regression model , we found that the strength of rad6 expression is a strong predictor of melanoma ( p < 0.001 ) even when age group ( p = 0.65 ) and gender ( p = 0.24 ) are included in the model . the model predicts that every 1% increase in rad6 expression results in a 9% increase in the probability that a lesion is melanoma . if we assume that a predicted probability of > 0.5 indicates melanoma , the model with only rad6 has sensitivity of 93% and specificity of 80% . these results are very encouraging ; however , they need to be validated in a larger study . the expression profiles of -catenin and rad6 differed considerably in nevi . approximately 93% of nevi expressed -catenin in more than 50% of the cells , whereas only 27% of the same population of nevi expressed rad6 ( figure 2 ) . -catenin and rad6 expressions in nevi were not significantly correlated ( r = 0.06 ; p = 0.77 ) . there is a 2.7-fold difference in the percentage of primary melanomas ( 100% ) expressing rad6 compared to nevi ( 37% ) and virtually no difference in -catenin expression between primary and metastatic melanomas ( 100% ) . accordingly , rad6 and -catenin expressions in primary melanoma were not correlated ( r < 0.001 , p > 0.99 ) . a significant correlation between rad6 and -catenin positive cells however , this association diminished ( r = 0.40 , p = 0.05 ) following the exclusion of two observations which are disproportionally influential ( one with < 50% positive rad6 and one with < 50% positive -catenin ) . this is the first study to characterize rad6 expression in cutaneous benign and malignant melanocytic tumors . in this study , we examined the association between rad6 and -catenin expressions in benign and malignant melanocytic tumors to determine whether rad6 works in concert with -catenin to influence melanoma development and progression . rad6 and -catenin positively regulate each other in breast cancer [ 15 , 18 ] . however , while -catenin has been implicated in the pathogenesis of melanoma and other cancer types , data about the role of rad6 in cancer pathogenesis are mostly limited to breast cancer . therefore , we hypothesized that comparison of rad6 and -catenin expressions in the same nevi and melanoma tumors would help determine whether these two signals collaborate to promote melanoma development and progression as they do in breast cancer [ 15 , 28 ] . accumulation of nuclear and cytoplasmic -catenin has been implicated in driving the development and progression of several cancer types ( e.g. , colon and ovarian cancers ) [ 2931 ] . however , our results show that the expression levels of -catenin do not contribute to melanoma initiation and progression since no difference in -catenin levels was found between nevi , primary melanoma , and metastatic melanoma ( 93%97% of all samples expressed -catenin in > 50% of the tumor cells ) . the high expression levels of -catenin are in line with the crucial role of -catenin in differentiation and proliferation of both normal melanocytes and metastatic melanoma cells . also , our findings are in agreement with previous reports of positive -catenin staining in nevi ( 100% ) and primary melanoma ( 95% , 94% ) but are higher than reported in metastatic melanoma ( 75% , 68% ) [ 9 , 21 ] . the variation in expression of -catenin levels in metastatic melanomas between the studies while we studied only melanoma metastases to the skin , other studies either obtained 58% of their specimens from lymph nodes , tonsil , and liver or did not identify the anatomical site of their metastases [ 9 , 21 ] . furthermore , different anatomical sites may regulate dissimilar antigen expressions in metastases that originate from the same primary tumor in the same patient [ 33 , 34 ] . previous studies have shown higher percentages of nuclear -catenin in nevi than in melanoma ( 84% versus 33% , and 44% versus 15% ) [ 9 , 22 ] . those observations provided the basis for the currently held concept that loss of nuclear and cytoplasmic -catenin suggest poor prognosis and decreased overall survival of melanoma patients [ 12 , 22 ] . in light of these data , the absence of nuclear -catenin in all the nevi and melanomas analyzed in our study was surprising . usage of different anti--catenin antibodies may explain in part the discrepancy in nuclear -catenin expression observed between the studies . however , our results are consistent with the lack of nuclear -catenin reported in four studies which comprised 57 nevi , more than 55 primary melanomas , and 20 metastatic melanomas [ 21 , 3537 ] . moreover , nuclear -catenin was not found in either the nevus portion or the melanoma portion of 15 cutaneous lesions and was absent in additional 42 primary melanomas . in another study of 70 primary melanomas , nuclear -catenin was reported in only 6.4% of the melanomas . finally , in a study of 230 primary and metastatic melanomas , nuclear -catenin was reported in only 13 cases ( 5.6% ) and therefore those cases were excluded from analysis . taken together , the absence or negligible amount of nuclear -catenin detected in the aforementioned studies as well as ours suggests possible extranuclear roles for -catenin in nevi and melanoma . this notion is supported by a role for cytoplasmic -catenin to execute functions that do not require nuclear translocation ( e.g. , activation of map kinase p38 and nf - kb ) [ 37 , 40 ] . a major finding of this study is the association between melanoma development and intracellular redistribution of -catenin . the percentage of cases that expressed -catenin on the cell membrane increased dramatically from 10% in nevi to 83% and 93% in primary and metastatic melanomas , respectively . concurrently , the percentage of cases that expressed cytoplasmic -catenin decreased from 90% in nevi to 45% and 39% in primary and metastatic melanomas , respectively ( figure 4 ) . we hypothesize that the relocation of -catenin from the cytoplasm to the cell membrane may serve as a deactivating mechanism of canonical wnt/-catenin signaling and that the resulting reduction in cytoplasmic -catenin level may contribute to the malignant transformation of melanocytic nevi . the proposed hypothesis is supported by the following observations : ( i ) as in our study , bachmann et al . also reported an association between nevus to melanoma development and relocation of -catenin to the cell membrane . nevertheless , the authors of that study did not offer an explanation for their observation ; ( ii ) our analysis of the data of kagashita et al . showed -catenin decrease in the cytoplasm and increase at the cell membrane and that these changes in -catenin distribution corresponded with the malignant transition of nevi ; ( iii ) wnt4 signal has been identified as a mechanism that can drive -catenin relocation from cytoplasm to cell membrane ; and ( iv ) -catenin relocation from cytoplasm to cell membrane has been reported to block -catenin signaling in a human embryonic kidney ( hek293 ) cell line . of note , this hypothesis can explain how despite the abundant -catenin expression in melanoma [ 1 , 7 ] , cytoplasmic -catenin is selectively decreased , a phenomenon that has been associated with unfavorable melanoma prognosis [ 9 , 21 , 22 ] . our current efforts are directed towards determining if the increases in membranous -catenin observed in primary and metastatic melanomas result from relocation of existing molecules in the cytoplasm or deposition of newly generated -catenin at the membranous site . rad6 has been implicated in early breast cancer development since an increase in rad6 levels is observed in adenosis and benign hyperplasias as compared to normal tissue . in contrast , our findings do not support rad6 to play a similar role in nevus formation as in benign breast neoplasia , since 63% of the nevi were negative for rad6 . rad6 has also been implicated in breast cancer progression because rad6 levels increase with progression from ductal carcinoma in situ to invasive primary carcinoma and metastatic cancer [ 15 , 28 ] . in accordance with the upregulation of rad6 in early stages of breast cancer development as compared to benign hyperplasia [ 15 , 17 ] , we observed a striking increase in rad6 expression in primary melanoma when compared to nevi . while all primary melanomas displayed strong rad6 staining ( > 50% of the tumor cells ) , rad6 was negative in 63% of the nevi . these findings suggest that rad6 may play a role in malignant transformation of nevi as in breast cancer . progression of melanoma from primary to metastatic disease was not significantly associated with further changes in the percentage of tumors expressing rad6 or rad expression intensity as > 50% of tumor cells stained positively in 100% and 96% of primary melanomas and metastatic melanomas , respectively . these findings suggest that rad6 may play a sustained role in melanoma metastasis as it does in melanoma development . in benign and malignant breast tumors , rad6 stabilizes -catenin , and , in turn , -catenin positively upregulates rad6 transcription [ 1517 ] . however , this direct positive correlation between -catenin and rad6 expression does not appear to be conserved in melanoma as the expression profiles of -catenin and rad6 differed considerably in nevi . approximately 93% of nevi expressed -catenin compared to only 27% of nevi that expressed rad6 in more than 50% of their cells ( figure 2 ) . these observations suggest that the high -catenin expression in nevi is likely driven by regulators other than rad6 . at first glance , it would appear that -catenin and rad6 expressions are correlated in primary melanoma because these proteins were coexpressed in approximately all primary melanomas . also , these findings correspond to the 80% correlation between rad6 and -catenin expressions in primary breast cancer . however , it is unlikely that the high rad6 expression in primary melanoma is driven by the concurrent high -catenin expression , because rad6 expression is low in nevi despite the presence of high cytoplasmic -catenin expression that is comparable to primary melanoma . this notion is confirmed by lack of statistical correlation between rad6 and -catenin expressions in primary melanoma . -catenin is not the only activator of rad6 ; for instance , rad6 is activated by nerve growth factor in nervous tissue . therefore , it is conceivable that , in primary melanoma , rad6 expression is regulated by yet unidentified activators . we also demonstrated that further progression of melanoma from primary to metastatic disease is not associated with a correlation between the -catenin and rad6 expressions . taken together , our study does not support a direct positive interaction between -catenin and rad6 in either benign or malignant melanocytic tumors . we characterized for the first time rad6 expression in cutaneous benign and malignant melanocytic tumors . we are showing a striking upregulation of rad6 from a negative expression in most benign melanocytic tumors to 100% of primary and metastatic melanomas . these findings strongly suggest a role for rad6 in the development of primary melanoma and metastatic disease . we show that in contrast to rad6 , -catenin is expressed in more than 50% of the tumor cells in almost all nevi and melanoma tumors . taken together , in contrast to the rad6 -catenin positive relationship in breast cancer [ 1517 ] , our study does not support a similar positive interaction between -catenin and rad6 in benign or malignant melanocytic tumors . finally , our findings suggest a role for the cytoplasmic to membrane translocation of -catenin in the development of primary melanoma . future studies will determine whether newly generated -catenin at the membranous site coincide with -catenin translocation from the cytoplasm .

we have previously demonstrated that rad6 and -catenin enhance each other 's expression through a positive feedback loop to promote breast cancer development / progression . while -catenin has been implicated in melanoma pathogenesis , rad6 function has not been investigated . here , we examined the relationship between rad6 and -catenin in melanoma development and progression . eighty - eight cutaneous tumors , 30 nevi , 29 primary melanoma , and 29 metastatic melanomas , were immunostained with anti--catenin and anti - rad6 antibodies . strong expression of rad6 was observed in only 27% of nevi as compared to 100% of primary and 96% of metastatic melanomas . -catenin was strongly expressed in 97% of primary and 93% of metastatic melanomas , and unlike rad6 , in 93% of nevi . none of the tumors expressed nuclear -catenin . -catenin was exclusively localized on the cell membrane of 55% of primary , 62% of metastatic melanomas , and only 10% of nevi . cytoplasmic -catenin was detected in 90% of nevi , 17% of primary , and 8% of metastatic melanoma , whereas 28% of primary and 30% of metastatic melanomas exhibited -catenin at both locations . these data suggest that melanoma development and progression are associated with rad6 upregulation and membranous redistribution of -catenin and that -catenin and rad6 play independent roles in melanoma development .

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

the wnt/-catenin pathway has been implicated in the development and progression of melanoma and a wide range of cancer types , including colorectal cancer , breast cancer , esophageal carcinoma , and liver cancer [ 13 ] . nuclear -catenin stimulates transcription of a large number of tcf/-catenin responsive genes that include cyclin d1 , c - myc [ 5 , 6 ] , and the melanocyte - specific gene , microphthalmia - associated transcription factor mitf - m . thus , accumulation of nuclear -catenin as observed in several cancer types is considered a marker of canonical wnt/-catenin pathway deregulation and unfavorable prognosis [ 3 , 8 ] . however , recent studies have shown that contrary to breast and colon cancer , metastatic progression of melanoma is associated with decreases in nuclear and cytoplasmic -catenin expression [ 9 , 10 ] . moreover , clinical , genetic , and histological studies suggest that nuclear and cytoplasmic -catenin may be used as biomarkers of good prognosis in melanoma [ 1114 ] . recently , hhr6 , a human homologue of the yeast rad6 gene and a principal component of the postreplication dna repair pathway , has been identified as an important regulator of canonical wnt/-catenin signaling [ 15 , 16 ] . furthermore , rad6 is a transcriptional target of -catenin , thus revealing a positive feedback loop between -catenin - mediated activation of rad6 gene expression and rad6-induced -catenin stabilization . we have previously demonstrated a role for rad6 in breast cancer progression through its regulatory effect on the canonical wnt/-catenin pathway . since the decrease / loss of nuclear -catenin [ 9 , 10 ] , rather than increases as in breast cancer , is linked to melanoma progression , it is not known whether rad6 and -catenin work in concert to promote melanoma pathogenesis . furthermore , rad6 expression in the skin has not been investigated , and there are no data on the role of rad6 in the pathogenesis of benign ( nevi ) and malignant ( melanoma ) melanocytic lesions . it is important to address this gap in knowledge because of the unmet medical need for new effective antimelanoma therapies and because rad6 and -catenin have been identified as therapeutic targets [ 19 , 20 ] . in this study , we examined rad6 and -catenin expressions in serial sections of nevi , primary , and metastatic melanomas to determine their potential roles in melanoma development and metastatic progression . our data suggest that membranous relocation of -catenin and upregulation of rad6 are independent markers of melanoma development and progression . we also offer a hypothesis that explains the role of membranous -catenin relocation and decreasing cytoplasmic -catenin in melanoma development , a phenomenon that has been linked to unfavorable prognosis [ 9 , 21 , 22 ] . the study groups consisted of 30 cases of melanocytic nevi , 29 cases of primary cutaneous melanoma , and 29 cases of metastatic cutaneous melanoma . primary antibodies used in the study are as follows : ( i ) anti--catenin ( is702 ) was purchased from dako ( glostrup , denmark ) and used in an undiluted form ; and ( ii ) anti - rad6 ( ab31917 ) was purchased from abcam ( cambridge , ma ) and used at a 1 : 500 dilution . neither ab31917 , our own rad6 antibody , nor any other commercially available anti - rad6 antibody is currently able to distinguish between rad6a and rad6b proteins . lung and colon cancer tissues were included as positive controls for immunostaining with anti--catenin antibody , and breast cancer tissues were included as positive controls for staining with anti - rad6 antibody . a specimen was considered negative if less than 4% of the cells were immunostained for rad6 or -catenin . a tumor was considered to be stained with high intensity if > 50% of the cells in a specimen expressed rad6 or -catenin , similar to the criteria used by mineta et al . chi - square tests for differences in proportions were used to compare groups on the basis of categorical variables such as gender and -catenin localization . spearman 's rank correlation was used to assess the pairwise association between age and percent of rad6 positive and -catenin positive cells . multinomial logistic regression was used to assess the simultaneous association of rad6 and age with diagnostic group . our analysis included 30 individuals diagnosed with nevi , 29 with primary melanoma , and 29 with metastatic melanoma ( table 1 ) . in contrast , no age difference is observed between individuals with primary and metastatic melanomas ( p = 0.27 ) . individuals with -catenin localized in the cytoplasm were significantly younger than individuals with -catenin localization at the cell membrane ( p = 0.02 ) and marginally younger than individuals with -catenin localization at both the cytoplasm and the cell membrane ( p = 0.05 ) . a 20% discrepancy of positively stained cells between the two evaluators was observed in fewer than 5% of the cases , and these cases were evaluated together to establish a consensus reading . melanoma development and progression were not associated with significant changes in percentage of specimens expressing -catenin . -catenin staining was observed in 97% of nevi and in all primary and metastatic melanomas . also , the percentages of nevi ( 93% ) , primary melanoma ( 97% ) , and metastatic melanoma ( 93% ) that expressed -catenin in more than 50% of the cells did not differ significantly ( figures 1 , 2 , and 3 ) . however , significant differences were observed between the percentages of nevi ( 59% ) , primary melanoma ( 90% ) , and metastatic melanoma ( 56% ) that expressed -catenin in more than 90% of the cells ( p = 0.02 ; figure 2 ) . these differences were greatly impacted by the percentage of primary melanomas ( 48% ) that expressed -catenin in 100% of the cells , which was approximately twofold higher than the percentages of nevi ( 21% ) or metastatic melanoma ( 26% ) ( data not shown ) . none of the nevi or melanomas expressed -catenin in the nucleus ( figure 1 ) . the percentage of tumors that expressed membranous -catenin increased dramatically from nevi ( 10% ) to primary and metastatic melanomas ( 83% and 93% , resp . ; concurrently with this increase , the percentage of tumors that expressed cytoplasmic -catenin decreased from nevi ( 90% ) to primary and metastatic melanomas ( 45% and 38% , resp . ; in contrast , no significant differences were observed between the percentages of primary and metastatic melanomas that expressed -catenin at either the membrane ( 83% and 93% , resp . while all four nevi types ( junctional , intradermal , compound , and atypical ) expressed -catenin in the cytoplasm , only junctional and atypical nevi expressed -catenin at the plasma membrane . as opposed to nevi types , the three primary melanoma types examined ( superficial spreading , nodular , and lentigo maligna ) did not differ in -catenin localization as -catenin was localized at the plasma membrane , in the cytoplasm , or in both ( figure 4 ) . conversely , all primary melanomas ( 100% ) and the majority of metastatic melanomas ( 96% ) exhibited greater than 50% rad6 expression . the increase in tumor populations expressing rad6 from 37% of nevi to 100% of primary and metastatic melanomas was significant ( p = 0.0001 ; figures 1 , 2 , and 3 ) . melanoma progression from primary to metastatic disease was not associated with changes in the ( i ) percentage of melanomas expressing rad6 ( 100% of primary and metastatic melanomas ) , or ( ii ) percentage of melanomas expressing rad6 in more than 50% of the tumor cells ( 100% and 96% of primary and metastatic melanomas , resp . ) the increase in proportion of tumor populations , expressing rad6 in more than 50% of the cells in primary melanoma ( 67% ) versus metastatic melanoma ( 79% ) was not significant ( p = 0.37 ; figure 2 ) . this study was not designed to test whether the distribution of the tumor cells positive for rad6 is the same between the subtypes of nevi . interestingly , only one of the 30 nevi ( atypical nevus , 3% ) expressed rad6 in > 80% of the cells , and none of the primary and metastatic melanomas expressed rad6 in < 40% of the cells ( figure 2 ) . using a multiple logistic regression model , we found that the strength of rad6 expression is a strong predictor of melanoma ( p < 0.001 ) even when age group ( p = 0.65 ) and gender ( p = 0.24 ) are included in the model . the expression profiles of -catenin and rad6 differed considerably in nevi . approximately 93% of nevi expressed -catenin in more than 50% of the cells , whereas only 27% of the same population of nevi expressed rad6 ( figure 2 ) . -catenin and rad6 expressions in nevi were not significantly correlated ( r = 0.06 ; p = 0.77 ) . there is a 2.7-fold difference in the percentage of primary melanomas ( 100% ) expressing rad6 compared to nevi ( 37% ) and virtually no difference in -catenin expression between primary and metastatic melanomas ( 100% ) . accordingly , rad6 and -catenin expressions in primary melanoma were not correlated ( r < 0.001 , p > 0.99 ) . a significant correlation between rad6 and -catenin positive cells however , this association diminished ( r = 0.40 , p = 0.05 ) following the exclusion of two observations which are disproportionally influential ( one with < 50% positive rad6 and one with < 50% positive -catenin ) . in this study , we examined the association between rad6 and -catenin expressions in benign and malignant melanocytic tumors to determine whether rad6 works in concert with -catenin to influence melanoma development and progression . rad6 and -catenin positively regulate each other in breast cancer [ 15 , 18 ] . however , while -catenin has been implicated in the pathogenesis of melanoma and other cancer types , data about the role of rad6 in cancer pathogenesis are mostly limited to breast cancer . therefore , we hypothesized that comparison of rad6 and -catenin expressions in the same nevi and melanoma tumors would help determine whether these two signals collaborate to promote melanoma development and progression as they do in breast cancer [ 15 , 28 ] . accumulation of nuclear and cytoplasmic -catenin has been implicated in driving the development and progression of several cancer types ( e.g. however , our results show that the expression levels of -catenin do not contribute to melanoma initiation and progression since no difference in -catenin levels was found between nevi , primary melanoma , and metastatic melanoma ( 93%97% of all samples expressed -catenin in > 50% of the tumor cells ) . the high expression levels of -catenin are in line with the crucial role of -catenin in differentiation and proliferation of both normal melanocytes and metastatic melanoma cells . also , our findings are in agreement with previous reports of positive -catenin staining in nevi ( 100% ) and primary melanoma ( 95% , 94% ) but are higher than reported in metastatic melanoma ( 75% , 68% ) [ 9 , 21 ] . the variation in expression of -catenin levels in metastatic melanomas between the studies while we studied only melanoma metastases to the skin , other studies either obtained 58% of their specimens from lymph nodes , tonsil , and liver or did not identify the anatomical site of their metastases [ 9 , 21 ] . previous studies have shown higher percentages of nuclear -catenin in nevi than in melanoma ( 84% versus 33% , and 44% versus 15% ) [ 9 , 22 ] . in light of these data , the absence of nuclear -catenin in all the nevi and melanomas analyzed in our study was surprising . however , our results are consistent with the lack of nuclear -catenin reported in four studies which comprised 57 nevi , more than 55 primary melanomas , and 20 metastatic melanomas [ 21 , 3537 ] . moreover , nuclear -catenin was not found in either the nevus portion or the melanoma portion of 15 cutaneous lesions and was absent in additional 42 primary melanomas . in another study of 70 primary melanomas , nuclear -catenin was reported in only 6.4% of the melanomas . finally , in a study of 230 primary and metastatic melanomas , nuclear -catenin was reported in only 13 cases ( 5.6% ) and therefore those cases were excluded from analysis . taken together , the absence or negligible amount of nuclear -catenin detected in the aforementioned studies as well as ours suggests possible extranuclear roles for -catenin in nevi and melanoma . a major finding of this study is the association between melanoma development and intracellular redistribution of -catenin . the percentage of cases that expressed -catenin on the cell membrane increased dramatically from 10% in nevi to 83% and 93% in primary and metastatic melanomas , respectively . concurrently , the percentage of cases that expressed cytoplasmic -catenin decreased from 90% in nevi to 45% and 39% in primary and metastatic melanomas , respectively ( figure 4 ) . we hypothesize that the relocation of -catenin from the cytoplasm to the cell membrane may serve as a deactivating mechanism of canonical wnt/-catenin signaling and that the resulting reduction in cytoplasmic -catenin level may contribute to the malignant transformation of melanocytic nevi . also reported an association between nevus to melanoma development and relocation of -catenin to the cell membrane . showed -catenin decrease in the cytoplasm and increase at the cell membrane and that these changes in -catenin distribution corresponded with the malignant transition of nevi ; ( iii ) wnt4 signal has been identified as a mechanism that can drive -catenin relocation from cytoplasm to cell membrane ; and ( iv ) -catenin relocation from cytoplasm to cell membrane has been reported to block -catenin signaling in a human embryonic kidney ( hek293 ) cell line . of note , this hypothesis can explain how despite the abundant -catenin expression in melanoma [ 1 , 7 ] , cytoplasmic -catenin is selectively decreased , a phenomenon that has been associated with unfavorable melanoma prognosis [ 9 , 21 , 22 ] . our current efforts are directed towards determining if the increases in membranous -catenin observed in primary and metastatic melanomas result from relocation of existing molecules in the cytoplasm or deposition of newly generated -catenin at the membranous site . rad6 has been implicated in early breast cancer development since an increase in rad6 levels is observed in adenosis and benign hyperplasias as compared to normal tissue . rad6 has also been implicated in breast cancer progression because rad6 levels increase with progression from ductal carcinoma in situ to invasive primary carcinoma and metastatic cancer [ 15 , 28 ] . in accordance with the upregulation of rad6 in early stages of breast cancer development as compared to benign hyperplasia [ 15 , 17 ] , we observed a striking increase in rad6 expression in primary melanoma when compared to nevi . while all primary melanomas displayed strong rad6 staining ( > 50% of the tumor cells ) , rad6 was negative in 63% of the nevi . these findings suggest that rad6 may play a role in malignant transformation of nevi as in breast cancer . progression of melanoma from primary to metastatic disease was not significantly associated with further changes in the percentage of tumors expressing rad6 or rad expression intensity as > 50% of tumor cells stained positively in 100% and 96% of primary melanomas and metastatic melanomas , respectively . these findings suggest that rad6 may play a sustained role in melanoma metastasis as it does in melanoma development . in benign and malignant breast tumors , rad6 stabilizes -catenin , and , in turn , -catenin positively upregulates rad6 transcription [ 1517 ] . however , this direct positive correlation between -catenin and rad6 expression does not appear to be conserved in melanoma as the expression profiles of -catenin and rad6 differed considerably in nevi . approximately 93% of nevi expressed -catenin compared to only 27% of nevi that expressed rad6 in more than 50% of their cells ( figure 2 ) . at first glance , it would appear that -catenin and rad6 expressions are correlated in primary melanoma because these proteins were coexpressed in approximately all primary melanomas . also , these findings correspond to the 80% correlation between rad6 and -catenin expressions in primary breast cancer . however , it is unlikely that the high rad6 expression in primary melanoma is driven by the concurrent high -catenin expression , because rad6 expression is low in nevi despite the presence of high cytoplasmic -catenin expression that is comparable to primary melanoma . this notion is confirmed by lack of statistical correlation between rad6 and -catenin expressions in primary melanoma . -catenin is not the only activator of rad6 ; for instance , rad6 is activated by nerve growth factor in nervous tissue . therefore , it is conceivable that , in primary melanoma , rad6 expression is regulated by yet unidentified activators . we also demonstrated that further progression of melanoma from primary to metastatic disease is not associated with a correlation between the -catenin and rad6 expressions . taken together , our study does not support a direct positive interaction between -catenin and rad6 in either benign or malignant melanocytic tumors . we are showing a striking upregulation of rad6 from a negative expression in most benign melanocytic tumors to 100% of primary and metastatic melanomas . these findings strongly suggest a role for rad6 in the development of primary melanoma and metastatic disease . we show that in contrast to rad6 , -catenin is expressed in more than 50% of the tumor cells in almost all nevi and melanoma tumors . taken together , in contrast to the rad6 -catenin positive relationship in breast cancer [ 1517 ] , our study does not support a similar positive interaction between -catenin and rad6 in benign or malignant melanocytic tumors . finally , our findings suggest a role for the cytoplasmic to membrane translocation of -catenin in the development of primary melanoma .

identification of new therapeutic compound classes and validation of new therapeutic targets remain major hurdles in drug discovery . in the past decade , human sirtuins ( homologues of yeast silent information regulator two or sir-2 ) have emerged as targets for cancer chemotherapy as well as for neurodegenerative and aging - related disorders such as huntington s disease , alzheimer s disease , and diabetes . although strong evidence exists for sirtuins having a central role in these debilitating diseases , their validation as targets for therapeutic intervention using small molecule modulators has been controversial . the most publicized efforts at modulation of sirtuin activity have been with the plant polyphenol resveratrol . this purported sirtuin activator was shown to have highly beneficial effects in animal models of metabolic disorders ( e.g. , diabetes ) and lifespan extension using experimental models that have since been largely shown to be flawed . ex-527 , a potent and selective sirt1 inhibitor ( sirt1 : human sirtuin isoform 1 ) , was found to be devoid of chemotherapeutic effect ; however , cambinol , tenovin-1 , tenovin-6 , and salermide , nonselective sirt1/sirt2 inhibitors , were found to have significant antitumor activity . combined use of a nonselective sirtuin inhibitor niacinamide ( nicotinamide ) and a pan - type i / ii hdac ( i.e. , zinc - dependent histone deacetylases ) inhibitor vorinostat yielded encouraging results in a recent diffuse large b - cell lymphoma phase i clinical trial further validating sirtuins as antilymphoma drug targets . additionally , srt1720 , a potent direct sirt1 activator that was originally developed for its potential in lifespan extension or antiaging activity , was later found to be beneficial in a rat diabetes model employing a mechanism which may involve indirect activation of sirt1 . the recent functional characterization of other sirtuin isoforms such as sirt3 , sirt5 , sirt6 , and sirt7 has further complicated the field as it is becoming increasingly clear that in addition to sirt1 and 2 , these isoforms may also play major roles in aging ( sirt3 , sirt6 ) as well as in cell - proliferation disorders ( sirt7 ) . additional controversies regarding artifacts of popular in vitro assays to identify novel small molecule modulators of sirtuin activity have also hampered the validation of these enzymes for pharmacological intervention . previously , in an attempt to identify isoform selective sirtuin inhibitors , we carried out a phenotypic screen using an nci chemical library that resulted in discovery of cambinol ( 5-[(2-hydroxy-1-naphthyl)methyl]-6-phenyl-2-thioxo-2,3-dihydro-4(1h)-pyrimidinone ) 1 . titration experiments using sirt2 and an acetyl - peptide substrate showed that cambinol was competitive with the peptide substrate but noncompetitive with nad suggesting that its binding site partially overlaps with that of the substrate . cambinol inhibits sirt1 and sirt2 with mid - micromolar ic50 and induces hyperacetylation of p53 , a known sirt1 target , and -tubulin , a sirt2 target , in cell - based deacetylation assays . in addition , cambinol was found to be an effective chemosensitizer for the dna damaging agent etoposide both in p53-proficient and p53-deficient cancer cell lines . as a single agent , cambinol was found to be selectively toxic to bcl6 expressing b - cell lymphoma cells suggesting that it could be used as a lead compound for antilymphoma drug development . cambinol however suffers from major drawbacks including ( 1 ) moderate potency , as it did not induce tumor regression , but a decrease in tumor growth rate , and ( 2 ) poor solubility . since the discovery of cambinol , we have focused on optimization of cambinol s potency and validation of specific sirtuin isoforms ( e.g. , sirt1 and sirt2 ) as targets for b - cell lymphoma chemotherapy . the -naphthol nucleus is absolutely essential for activity , and the aryl ring is also important as replacing it with five - membered or a heterocyclic ring results in significant loss of potency . the pyrimidinedione ( thiouracil ) heteroaryl ring requires a balance of hydrogen bond donor and acceptor groups to maintain potency . cambinol analogues . ( a ) cambinol ; ( b ) schematic ; ( c ) pyrazolone analogues ( e.g. , 8 and 17 ) ; and ( d ) isoxazolone analogues ( e.g. , 24 ) . to further develop the structure activity relationship ( sar ) , we investigated substituting the pyrimidinedione ring ( heteroaryl group , figure 1b ) with five - membered heterocycles ( pyrazolone 1c and isoxazolone 1d ) as well as altering the functional groups on the naphthyl and aryl rings . to guide us with the specific positioning of the functional group to observe increased potency , we conducted saturation transfer difference ( std ) nmr experiments using 20 ( figure 2 ) . the data obtained from std nmr experiments identified portions of the ligand that interact with the receptor and suggest sites available for additional elaboration . cell - based toxicity assays show a significant correlation between sirt2 inhibition and cytotoxicity in the namalwa burkitt s lymphoma cell line . h nmr spectrum and std spectrum of ( 20 ) and sirt1 . the chemical structure of ( 20 ) using naphthalene numbering for clarity . syntheses on new cambinol analogues closely parallel that of the parent compound , cambinol ( scheme 1 ) . target compounds are prepared by a three - step sequence from 2-hydroxy-1-naphthaldehyde starting materials . for cambinol , base - catalyzed knoevenagel condensation with ethyl - benzoylacetate with 2-hydroxy-1-naphthaldehyde affords the ,-unsaturated -ketoester ( scheme 1 ) . sodium borohydride reduction of the ,-unsaturated olefin followed by base - promoted 2-thiouracil formation with thiourea yields cambinol . for naphthyl and quinolyl derivatives of cambinol , hydroxy - naphthaldehyde and hydroxy - quinaldehyde starting materials were prepared by titanium - catalyzed reiche formylation reaction ( 1-carbaldehyde-2-hydroxynaphthalene derivatives ) from the corresponding phenols , and the duff reaction of hexamethylenetetramine ( hmta ) with 6-hydroxy-2-quinolones in the presence of trifluoroacetic acid ( tfa ) afforded the corresponding 5-carbaldehyde-6-hydroxy-2-quinolones , respectively ( schemes 1 and 2 ) . the pyrazolone analogues were prepared by reacting the appropriate -ketoester with hydrazine , while syntheses of the isoxazolone analogues used corresponding hydroxylamine compounds as nucleophiles ( scheme 1 ) . reagents and conditions : ( a ) cl2chome , ticl4 , ch2cl2 , 020 c , 24 h ; ( b ) aryloyl ethyl acetoacetate , piperidine , etoh , reflux , 2 h ; ( c ) nabh4 , pyridine , 20 c , 2 h ; ( d ) thiourea , naoet , etoh , reflux , 18 h ; ( e ) hydrazine , dmf , 20 c , 1.5 h ; ( f ) hydroxylamine , dmf , 60 c , 18 h ; reagents and conditions : ( a ) hmta , tfa , 100 c , 2 h ; ( b ) pox3 , dmf , temp , time x = cl , br ; ( c ) aryloyl ethyl acetoacetate , piperidine , etoh , reflux , 2 h ; ( d ) nabh4 , pyridine , 20 c , 24 h ; ( e ) hydrazine , dmf , 20 c , 1.5 h. sirt1 , sirt2 , and sirt3 enzyme inhibition studies , carried out using the sirt - glo assay ( promega corp . , madison , wi ) at a single inhibitor concentration ( 50 m ) , revealed a range of activities against each target ( tables 1 , 2 , and 3 ) . cambinol , the parent of this class of agents , was found to be an nonselective sirt1 , sirt2 inhibitor with 54% sirt1 inhibition , 46% sirt2 and 16% sirt3 inhibition at a single 50 m concentration ( table 1 ) , and dose response titration against these enzymes with cambinol gave ic50 values very similar to published values ( sirt1 ic50 = 56 m , sirt2 ic50 = 51 m ) and > 200 m sirt3 ic50 . we carried out an std nmr experiment using recombinant sirt1 and compound 20 ( figure 2 ) . the relative saturation transfer enhancement at positions we were able to evaluate is shown in figure 2 . the results show that c-7 proton ( naphthalene numbering is used for clarity since most of the compounds discussed in this section are naphthalene derivatives ) gives the strongest std signal ( 100% relative intensity ) and forms a close contact with the protein . the aryl c-4 methyl protons ( 66% signal intensity ) and c-8 proton ( 74% signal intensity ) form contacts with the protein , but these may be suboptimal . consistent with this , compounds 1 ( cambinol ) , 2 , 6 , 13 , and 22 that contain a small substituent at c-4 ( i.e. , hydrogen or fluorine ) are relatively poor and nonselective sirtuin inhibitors . the most selective compounds 17 and 24 have methyl groups at c-4 suggesting that sirt1 and sirt2 residues that interact with this group are well conserved . likewise , compounds that contain larger than hydrogen ( i.e. , methyl or isopropyl ) substituents at the c-4 position of the aryl moiety tend to favor sirt1 inhibition with the bulkiest substituent ( i.e. , isopropyl , compound 23 ) having the highest selectivity ( > 3-fold , table 4 ) . the std values for the -methylene protons and the nhs of the pyrazolone moiety could not be determined because of the overlap with the water signal and the fast exchange with solvent deuterons , respectively . std values for the c-3 hydrogen and for protons in the phenyl ring ( c-2 , c-3 , c-5 , c-6 ) could not be accurately determined due to overlapping signals . the most significant determinant of the selectivity between sirt1 and sirt2 in the present compound series is the h - bond donor or acceptor at the 2 position of the heteroaryl unit ( i.e. , n h or oxygen ) in 17 and 24 . the reversal of hydrogen bond directionality results in a reversal of selectivity with the hydrogen - bond donor compounds 17 displaying up to a 7.8-fold sirt1 selectivity and the hydrogen bond acceptor 24 a 15.4-fold sirt2 selectivity with approximately similar overall inhibitory activity against the preferred isoform : 26 m ic50 for 17 against sirt1 and 13 m ic50 for 24 against sirt2 . substitution of a hydrophobic aromatic group at c-6 of the naphthalene group gave the most potent sirt3 inhibitor ( compound 8 , sirt3 ic50 6 m ) . this compound however was relatively nonselective with sirt1 and sirt2 ic50 of 41 and 32 m , respectively . the potency of c-6 phenyl substituted naphthalene across the sirt isoforms suggests a conserved hydrophobic pocket present in all three proteins . concentration giving 50% inhibition of sirtuin activity . our previous studies with cambinol demonstrated that sirt1 inhibition could be quantified in cells by determining the level of p53 acetylation following induction of dna damage by the cytotoxic topoisomerase ii poison etoposide . we treated nci - h460 cells with 1 m etoposide in the presence or absence of cambinol 1 or 14 , a sirt1-selective inhibitor ( tables 1 and 4 ) . as expected , the more potent sirt1 inhibitor 14 shows a significantly higher level of p53 acetylation at a given concentration ( figure 3 ) . the sirt2-selective inhibitor 24 induced a rapid and robust dose - dependent increase in -tubulin acetylation in nci - h460 cells ( figure 4 ) . determination of p53 acetylation in nci - h460 cells by western blot following 24 h drug treatment . determination of -tubulin acetylation in nci - h460 cells by western blot following 4 h treatment with trichostatin a ( tsa ) and ( 24 ) . part of the motivation behind these studies was to determine whether sirt1 , sirt2 or sirt3 inhibition , or possibly a subset of these enzymes , was responsible for cambinol s antilymphoma activity . cell - based sirtuin activity assays were carried out by determining the acetylation status of well established sirt1 ( p53 ) and sirt2 ( -tubulin ) in the presence of isoform selective inhibitors . cytotoxicity assays were carried out with the human burkitt s lymphoma cell line namalwa grown under standard conditions . cells were plated in 96-well plates , treated with the test compound or dmso control for 72 h , and viable cells were quantified by luminescence using celltiterglo ( promega corp . , cell viability assays showed that both sirt1- and sirt2-selective series exhibit antiproliferative , cytotoxic activity against the namalwa burkitt s lymphoma cell line , while the sirt3-selective compound was less toxic . in the namalwa cell line , cell death occurs by induction of apoptosis as evidenced by dose - dependent appearance of annexin v - positive cells as indication of early stage apoptosis ( figure 5 ) . in addition , while there is a strong correlation between sirt2 inhibition and namalwa cytotoxicity ( r = 0.56 , p = 0.0014 ) ( figure 6 ) , neither sirt1 ( r = 0.11 ) nor sirt3 ( r = 0.21 ) ( data not shown ) inhibition correlates with namalwa cytotoxicity . three compounds , the sirt1-selective 17 , sirt2-selective 24 and sirt3-selective 8 , were tested against an expanded panel of burkitt s lymphoma ( dakiki , daudi , mutu , oku , ramos and namalwa ) , diffuse large b - cell lymphoma ( su - dhl4 and oci - ly8-lam53 ) , nontransformed epstein barr virus ( ebv ) immortalized b - cell lines ( b1 and b2 ) , and epithelial cancer cell lines ( hct116-colon , mcf7-breast , nci - h460-nonsmall cell lung cancer and ovcar3-ovarian ) ( table 5 ) . the sirt2-selective inhibitor 24 exhibited potent cytotoxicity in both lymphoma and epithelial cancer cell lines with ic50 ranging from 3 to 7 m relative to the nontransformed b - cell lines ( ic50 2228 m ) . facs analysis of namalwa cells treated with dmso ( left ) , 10 m ( 24 ) ( center ) and 25 m ( 24 ) ( right ) for 16 h. cells were stained with annexin v - pe ( y - axis ) and dapi ( x - axis ) . cells entering an early phase of apoptosis are present in the upper left panel . sirt1 ( red circles ) and sirt2 ( blue triangles ) inhibition ( y - axis ) versus namalwa growth inhibition ( x - axis ) . concentration giving 50% growth inhibition following 72 h drug treatment . dakiki , daudi , mutu , oku , ramos , namalwa are burkitt s lymphoma cell lines . su - dhl4 and lam53(oci - ly8-lam53 ) are diffuse large b - cell lymphoma lines . hct116 ( colon ) , mcf7 ( breast ) , nci - h460 ( lung ) , and ovcar3 ( ovarian ) are epithelial cancer cell lines . cambinol , ( 5-[(2-hydroxy-1-naphthyl)methyl]-6-phenyl-2-thioxo-2,3-dihydro-4(1h)-pyrimidinone ) 1 , is a nonselective sirtuin inhibitor with equivalent inhibitory activity against sirt1 and sirt2 . it also is a reasonably potent antitumor agent in vitro and in vivo with particular activity against burkitt s lymphoma cell lines . in an effort to delineate the contribution of sirt1 and sirt2 inhibition in this antitumor activity have partly addressed the structure activity relationships of six - membered pyrimidinedione - containing ( i.e. , cambinol - like ) compounds . in an effort to investigate an alternative chemical space , we prepared a series of five - membered pyrazolone- and 5-isoxaolone - containing compounds . the five - membered ring pyrazolone compounds ( i.e. , 223 ) exhibit a range of specificities but generally favor sirt1 inhibition . the most selective of these compounds , 14 and 17 , exhibit a greater than 5-fold and 7-fold , respectively , preference for sirt1 over sirt2 . the 5-isoxazolone compounds ( i.e. , 2426 ) favor sirt2 inhibition with a greater than 15-fold preference shown by 24 . in order to gain a molecular understanding of the observed selectivity , we sought to identify portions of the ligand involved in the interaction with sirt1 protein . the std nmr experiment with recombinant sirt1 and 20 , a relatively nonselective but highly soluble ligand , has identified positions of the ligand as potential sites for improving selectivity and potency ( figure 2 ) . the efficiency of saturation transfer was lowest at c-8 of the quinolone ring system ( naphthalene numbering ) and at the 4-methyl group of the aryl substituent suggesting that these portions of the ligand interact with the protein surface but in a suboptimal manner . consistent with this finding , compounds with large substituents at the c-4 position yielded more potent inhibitors ( e.g. , 20 versus 23 , table 2 ) in agreement with the structural model proposed by medda et al . saturation transfer was most efficient at c-4 and c-7 of the quinoline ring suggesting close ligand / protein interactions ; however , the c-8 proton showed a weaker interaction . examination of the structural variation at these positions suggests a reasonable explanation of the observed selectivities . the binding site for the c-4 phenyl position appears to tolerate medium - sized substituents equally well in sirt1 and sirt2 , with bulkier groups such as isopropyl showing an improved preference for sirt1 . the strongest determinant of selectivity in the current compound series is the hydrogen bond donor ( i.e. , n h at the pyrazolone 2 position in 17 ) or hydrogen bond acceptor ( i.e. , o at the dihydro 1,2-oxazol-5-one 1 position in 24 ) . remarkably , the reversal of directionality of the hydrogen bond donor / acceptor pair leads to a 7.8-fold sirt1 selectivity in the case of the former 24 and a 15.4-fold sirt2 selectivity for the latter , with approximately equipotent overall inhibitory activity against the preferred isoform : 26 m ic50 for 17 against sirt1 and 13 m ic50 for 24 against sirt2 . placement of a bulky hydrophobic phenyl group at the naphthalene c-6 position yielded 8 , a potent although relatively nonselective sirt3 inhibitor . the sar trends observed in the current compound series provide a strong starting point for development of potent isoform - selective as well as pan - sirtuin inhibitors . while both sirt1 and sirt2-selective compounds exhibit some degree of cytotoxicity , a strong correlation between sirt2 inhibition and namalwa cytotoxicity ( r = 0.56 , p = 0.0014 ) suggests that sirt2 inhibition may be primarily responsible for the observed antilymphoma activity . lack of correlation between sirt1 inhibition and cytotoxicity observed in our study is consistent with the findings that ex-527 , a potent and selective sirt1 inhibitor , does not exhibit activity against b - cell lymphoma cell lines , as reported previously by others for epithelial tumor cells . it is possible , however , that inhibition of sirt1 may play a role in other tumor contexts , as suggested by the findings by rotili et al . in addition to burkitt s lymphoma cell lines , we tested 8 , 17 , and 24 against normal immortalized lymphoblastic lines ( b1 and b2 ) , diffuse large b - cell lymphoma lines as well as a panel of solid tumor cell lines ( table 5 ) . interestingly , colon ( hct116 ) breast ( mcf7 ) and nonsmall cell lung carcinoma ( nci - h460 ) also showed significant sensitivity to sirt2 inhibition . the data presented in this manuscript make a strong case for further development of sirtuin inhibitors as anticancer agents . human sirt1 cdna was cloned into the pet-4a vector and transformed into bl21-de3 escherichia coli cells . expression of sirt1 protein fusion with hexa - histidine tag ( c - terminal ) was induced with iptg ( 5 mm ) . recombinant protein was purified from bacterial lysates using the ni - nta column ( clontech laboratories inc . sirt2 and sirt3 were purchased from cayman chemical ( ann arbor , mi ) . enzyme inhibition assays were performed in 96-well plates using the sirt - glo assay ( promega corp . compounds were dissolved in 100% dmso and were tested over a five 3-fold dilution concentration range ( final dmso concentration 0.25% ) . ic50 values were determined in triplicate and reported values are averages of two independent experiments . cell lines were obtained from atcc ( manasas , va ) and were grown under standard conditions . for viability assays , cells were dispensed into 96-well plates and treated with test compounds in 100% dmso ( final dmso concentration 0.25% ) . cells were incubated with test compounds ( or dmso controls ) for 72 h , and viability was determined using the celltiterglo assay ( promega corp . , madison wi ) according to manufacturer s instructions . assays were carried out in triplicate reported values are averages of two independent experiments . nci - h460 human lung carcinoma cells ( atcc ) were grown under standard conditions . louis , mo ) , -tubulin ( clone dm1a , calbiochem , san diego , ca ) , actin ( roche , indianapolis , in ) , acetylated p53 ( cell signaling , danvers , ma ) , p53 ( cell signaling , danvers , ma ) and acetylated lysine ( polyclonal antibody ; cell signaling , danvers , ma ) were used at dilutions recommended by the manufacturer . namalwa cells were treated with 10 and 25 m ( 24 ) or solvent ( dmso ) for 16 h , washed in phosphate buffered saline , and suspended at approximately 200 000 cells in 200 l of binding buffer containing 5 l of annexin v - pe ( bd biosciences , san jose , ca ) for measuring phosphatidylserine , and 4,6-diamidino-2-phenylindole ( dapi ) at a concentration of 2.5 after 15 min incubation at room temperature , cell fluorescence was analyzed on a facscanr ( becton dickinson , franklin lakes , nj ) . unless specified otherwise , reagents were obtained from sigma - aldrich and used without additional purification . 3-(4-isopropyl - phenyl)-3-oxo - propionic acid ethyl ester was obtained from vitas m laboratories . thin layer chromatography was carried out using merck 60 f254 silica gel plates using appropriate solvent mixtures . solvents were acs reagent grade and anhydrous solvents ( aldrich and acros ) were used as received . lcms was performed using an agilent 1100 hplc system equipped with a waters xterra ms c18 5 um , 4.6 50 mm column , an agilent photodiode array detector and an in - line agilent 6130 single quadrupole mass spectrometer . the lc method involved gradient elution from 0 to 95% acetonitrile in water ( 0.1% formic acid ) over 6 min . final purity of compounds was determined using ace 3 , c8300 , 150 3.0 mm column with the above - mentioned gradient over 15 min . all nmr experiments were performed at 25 c on a varian unity plus 300 spectrometer or varian unity - inova 500 mhz spectrometer equipped with a 5 mm triple - resonance h(c / n ) , z - axis pulsed - field gradient probe head . for characterization purposes , samples consisted of a 5 mm solution of each compound in chloroform - d ( 99.8% d , cambridge isotopes ) , dimethyl sulfoxide - d6 ( 99.9% d , cambridge isotopes ) , benzene - d6 ( 99.5% d , cambridge isotopes ) or acetone - d6 ( 99.9% d , cambridge isotopes ) , and the spectra were referenced to residual solvent peaks at 7.27 , 2.50 , 7.16 , and 2.05 ppm , respectively . h-1d spectra were acquired at a resolution of 16k complex points in the time domain with 32 accumulations each ( sw = 6000 hz , d1 = 3 s ) . all std experiments were performed in d2o solution to eliminate the influence of exchangeable protein protons . the purified sirt1 stock solution ( 12 m ) was dialyzed against deuterated phosphate buffered saline ( pbs ) . the exchange buffer was prepared by lyophilizing a pbs solution at ph 7.4 and redissolving it in d2o ( 99.9% d , cambridge isotopes ) . nmr samples contained 1.5 m sirt1 and 200 m compound 20 in pbs in d2o ph 7.4 ( uncorrected for d2o ) with 1% dimethyl sulfoxide - d6 as a cosolvent . briefly , two free induction decay ( fid ) data sets were collected in an interleaved manner to minimize temporal fluctuations with the protein irradiation frequency set on - resonance ( 0.5 ppm ) and off - resonance ( 40 ppm ) , respectively ( sw = 6000 hz , 16 steady state scans , 2048 transients , 4k complex points , d1 = 3 s ) . protein saturation was obtained using a train of individual 50 ms long , frequency selective gaussian radio frequency ( rf ) pulses separated by an interpulse delay of 1 ms . the number of selective pulses was set to 50 , leading to a total saturation time ( sat ) of 2.5 s. gradient tailored excitation ( watergate ) scheme was employed to suppress the residual water signal . the fid acquired with off - resonance irradiation generated the reference spectrum ( ioff ) whereas the difference fid ( off - resonance on - resonance ) yielded the std spectrum ( istd = ioff ion ) . spectra were processed with an exponential apodization function ( 1 hz line - broadening ) and zero - filling to 8k complex points before fourier transformation and baseline correction with a third order bernstein polynomial fit . the std measurements were done in duplicate , and all data were processed and analyzed using mnova 8.1 processing software ( mestrelab research , santiago de compostela , spain ) . the procedure was adapted from the protocol for formylation of electron rich phenols reported by garcia et al . a solution of ticl4 ( 18 mmol ) and dichloromethyl ether ( 9 mmol ) in anhydrous dichloromethane ( 10 ml ) was stirred at 0 c for 15 min . a solution of the corresponding substituted naphthol ( 9 mmol ) in either ch2cl2 or 1,2-dichloroethane ( 30 ml ) was added dropwise , and the reaction was warmed to room temperature . the reaction was allowed to stir overnight after which it was quenched by adding 1 n hcl ( 10 ml ) . the aqueous layer was extracted with ch2cl2 ( 3 10 ml ) , and the organic layers were then combined , dried with na2so4 , and reduced to dryness to afford a residue which was further purified using medium pressure chromatography to yield the pure aldehyde . a reddish brown residue initially obtained was further purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 110% etoac ) yielding 1.5 g ( 6 mmol , 67% ) as a white powder . h nmr ( 500 mhz , chloroform - d ) 13.12 ( s , 1h ) , 10.79 ( s , 1h ) , 8.24 ( d , j = 8.8 hz , 1h ) , 7.97 ( d , j = 2.4 hz , 1h ) , 7.91 ( d , j = 9.3 hz , 1h ) , 7.70 ( dd , j = 8.8 , 2.4 hz , 1h ) , 7.19 ( d , j = 9.3 hz , 1h ) . lrms : m / z = 249.1 ( m h ) . 6-bromo-2-hydroxynaphthalene-1-carbaldehyde ( 1.39 mmol ) , phenyl boronic acid ( 2.09 mmol ) , bis(dibenzylideneacetone)palladium(0 ) ( 0.0139 mmol ) , and tricyclohexylphosphine ( 0.033 mmol ) were suspended in dioxane ( 7 ml ) . to this mixture was added potassium phosphate tribasic ( 1.27 m ) , and the solution was heated at 100 c for 6 h. the reaction was cooled , loaded onto a small silica column , and eluted with etoac ( 20 ml ) , and the filtrate was collected . the extracts were washed with water , dried using na2so4 , and reduced to dryness to afford a dark brown residue . the residue was then purified by medium pressure chromatography using gradient etoac / hexane solvent system ( 110% etoac ) yielding 140 mg ( 0.56 mmol , 40% ) as a white powder . h nmr ( 500 mhz , chloroform - d ) 13.16 ( s , 1h ) , 10.85 ( s , 1h ) , 8.42 ( d , j = 8.8 hz , 1h ) , 8.05 ( d , j = 8.8 hz , 1h ) , 8.00 ( d , j = 2.4 hz , 1h ) , 7.90 ( d , j = 8.8 hz , 1h ) , 7.71 ( d , j = 7.3 hz , 2h ) , 7.51 ( t , j = 7.8 hz , 2h ) , 7.42 ( t , j = 7.8 hz , 1h ) , 7.19 ( d , j = 8.8 hz , 1h ) , lrms [ es ] : m / z = 249.1 ( m + h ) . a black residue was obtained which further purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 110% etoac ) yielding 500 mg ( 2.47 mmol , 43% ) . h nmr ( 500 mhz , chloroform - d ) 12.90 ( s , 1h ) , 10.79 ( s , 1h ) , 8.27 ( d , j = 9.3 hz , 1h ) , 7.90 ( d , j = 8.8 hz , 1h ) , 7.30 ( dd , j = 9 , 2.7 hz , 1h ) , 7.16 ( d , j = 8.3 hz , 1h ) , 7.13 ( d , j = 2.7 hz , 1h ) , 3.93 ( s , 3h ) . the reaction was carried out in 1,2-dichloroethane . the residue obtained after extraction was purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 110% etoac ) yielding 72 mg ( 0.365 mmol , 42% ) . h nmr ( 500 mhz , chloroform - d ) 13.36 ( s , 1h ) , 10.82 ( s , 1h ) , 8.45 ( d , j = 8.8 hz , 1h ) , 8.19 ( d , j = 2 hz , 1h ) , 8.05 ( d , j = 9.3 hz , 1h ) , 7.80 ( dd , j = 8.8 , 2 hz , 1h ) , 7.30 ( d , j = 9.3 hz , 1h ) . lrms [ es ] : m / z = 198.1 the reaction residue was purified using a gradient etoac / hexane solvent system ( 1025% etoac ) yielding 386 mg ( 2.07 mmol , 81% ) as a pale yellow solid . h nmr ( 500 mhz , chloroform - d ) 13.03 ( s , 1h ) , 10.81 ( s , 1h ) , 8.26 ( d , j = 8.8 hz , 1h ) , 7.92 ( d , j = 9.3 hz , 1h ) , 7.59 ( d , j= 2.0 , 1h ) , 7.46 ( dd , j = 8.8 , 2 hz , 1h ) , 7.12 ( d , j = 9.3 hz , 1h ) , 2.51 ( s , 3h ) . lrms [ es ] : m / z = 187.1 ( m + h ) . the residue obtained was purified using a gradient etoac / hexane solvent system ( 1025% etoac ) yielding 322 mg ( 1.61 mmol , 67% ) . h nmr ( 500 mhz , chloroform - d ) 13.07 ( s , 1h ) , 10.85 ( s , 1h ) , 8.28 ( d , j = 7.8 hz , 1h ) , 7.94 ( d , j = 9.3 hz , 1h ) , 7.60 ( d , j= 2.0 , 1h ) , 7.51 ( dd , j = 7.8 , 2 hz , 1h ) , 7.12 ( d , j = 9.3 hz , 1h ) , 2.82 ( q , j = 7.8 hz , 2h ) , 1.34 ( t , j = 7.8 hz , 3h ) . lrms [ es ] : m / z = 201.1 ( m + h ) . a reddish brown residue was initially obtained which further purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 110% etoac ) yielding 860 mg ( 3.42 mmol , 76% ) as a white powder . chloroform - d ) 13.19 ( s , 1h ) , 10.74 ( s , 1h ) , 8.50 ( s , 1h ) , 7.95 ( d , j = 9.3 hz , 1h ) , 7.67 ( d , j = 8.8 hz , 1h ) , 7.54 ( dd , j = 8.8 , 1.7 hz , 1h ) , 7.17 ( d , j = 9.3 hz , 1h ) . lrms : m / z = 249.1 ( m h ) . a reddish brown residue was initially obtained was further purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 110% etoac ) yielding 850 mg ( 3.38 mmol , 76% ) as a white powder . h nmr ( 500 mhz , chloroform - d ) 13.82 ( s , 1h ) , 10.76 ( s , 1h ) , 8.35 ( d , j = 8.8 hz , 1h ) , 8.31 ( s , 1h ) , 7.76 ( d , j = 8.4 hz , 1h ) , 7.66 ( ddd , j = 8.8 , 7.0 , 1.2 hz , 1h ) , 7.48 ( ddd , j = 8.8 , 7.0 , 1.2 hz , 1h ) . lrms [ es ] : m / z = 251.0 ( m + h ) . a reddish brown residue was initially obtained which further purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 110% etoac ) yielding 75 mg ( 0.37 mmol , 36% ) as a white powder . h nmr ( 500 mhz , chloroform - d ) 13.46 ( s , 1h ) , 10.74 ( s , 1h ) , 8.23 ( d , j = 8.3 hz , 1h ) , 7.69 ( d , j = 7.8 hz , 1h ) , 7.48 ( ddd , j = 8.3 , 7.6 , 1.2 hz , 1h ) , 7.41 ( ddd , j = 8.3 , 7.6 , 1.2 hz , 1h ) , 7.30 ( s , 1h ) , 4.01 ( s , 3h ) . lrms [ es ] : m / z = 203.1 ( m + h ) . the compounds were synthesized using the established synthetic route for cambinol and its analogues reported earlier . to a solution of substituted hydroxyl naphthaldehydes ( 5 mmol ) in ethanol ( 5 ml ) were added the corresponding ethyl benzoyl acetates ( 5 mmol ) . piperidine ( 5 drops ) was added , and the reaction was heated under reflux for 2 h. the reaction was allowed to cool , and the yellowish precipitate obtained was collected by filtration and washed with ethanol several times to get the condensation product . h nmr ( 500 mhz , chloroform - d ) 9.00 ( d , j = 0.6 hz , 1h ) , 8.348.28 ( m , 1h ) , 8.188.12 ( m , 1h ) , 8.088.03 ( m , 1h ) , 8.017.95 ( m , 1h ) , 7.867.73 ( m , 3h ) , 7.65 ( ddd , j = 8.1 , 7.0 , 1.1 hz , 1h ) , 7.587.52 ( m , 1h ) , 7.437.35 ( m , 1h ) . lrms [ es ] : m / z = 379.09 ( m + h ) . yield 500 mg ( 1.32 mmol , 72% ) . h nmr ( 500 mhz , chloroform - d ) 8.98 ( d , j = 0.7 hz , 1h ) , 8.338.27 ( m , 1h ) , 8.178.11 ( m , 1h ) , 8.007.94 ( m , 1h ) , 7.827.72 ( m , 3h ) , 7.687.59 ( m , 3h ) , 7.577.51 ( m , 1h ) . lrms [ es ] : m / z = 379.09 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 8.89 ( s , 1h ) , 8.26 ( d , j = 8.4 hz , 1h ) , 8.10 ( d , j = 9.0 hz , 1h ) , 7.987.90 ( m , 1h ) , 7.877.80 ( m , 2h ) , 7.72 ( ddd , j = 8.4 , 7.0 , 1.3 hz , 1h ) , 7.61 ( ddd , j = 8.2 , 7.0 , 1.1 hz , 1h ) , 7.52 ( d , j = 9.0 hz , 1h ) , 7.30 ( d , j = 7.9 hz , 1h ) , 7.18 ( s , 1h ) , 2.44 ( s , 3h ) . lrms [ es ] : m / z = 315.10 ( m + h ) . the precipitate was recrystallized overnight from ethyl acetate and dried under vacuum yielding 490 mg ( 1.57 mmol , h nmr ( 500 mhz , chloroform - d ) 8.95 ( s , 1h ) , 8.29 ( d , j = 8.4 hz , 1h ) , 8.13 ( d , j = 9.0 hz , 1h ) , 8.007.92 ( m , 3h ) , 7.74 ( ddd , j = 8.4 , 7.0 , 1.3 hz , 1h ) , 7.63 ( ddd , j = 8.2 , 7.0 , 1.1 hz , 1h ) , 7.53 ( d , j = 9.0 hz , 1h ) , 7.227.13 ( m , 2h ) . lrms [ es ] : m / z = 319.1 ( m + h ) . the precipitate was recrystallized from ethyl acetate and dried under a vacuum to yield 450 mg ( 1.21 mmol , 53% ) . h nmr ( 500 mhz , chloroform - d ) 9.06 ( s , 1h ) , 8.32 ( d , j = 8.4 hz , 1h ) , 8.15 ( d , j = 9.0 hz , 1h ) , 7.99 ( ddt , j = 13.6 , 8.1 , 0.8 hz , 3h ) , 7.817.73 ( m , 3h ) , 7.65 ( ddd , j = 8.2 , 7.0 , 1.1 hz , 1h ) , 7.54 ( dd , j = 9.0 , 0.7 hz , 1h ) . lrms [ es ] : m / z = 369.1 ( m + h ) . yield 204 mg ( 0.51 mmol , 68% ) . h nmr ( 500 mhz , chloroform - d ) 8.91 ( s , 1h ) , 8.34 ( d , j = 8.7 hz , 1h ) , 8.198.12 ( m , 2h ) , 7.99 ( dd , j = 8.7 , 1.9 hz , 1h ) , 7.887.82 ( m , 2h ) , 7.767.70 ( m , 2h ) , 7.597.48 ( m , 3h ) , 7.477.39 ( m , 1h ) , 7.31 ( d , j = 7.9 hz , 2h ) , 2.45 ( s , 3h ) . lrms [ es ] : m / z = 391.1 ( m + h ) . yield 200 mg ( 0.52 mmol , 74% ) h nmr ( 500 mhz , chloroform - d ) 10.771 ( s , 1h ) , 8.23 ( d , j = 9.0 hz , 1h ) , 7.95 ( d , j = 2.0 hz , 1h ) , 7.89 ( d , j = 9.1 hz , 1h ) , 7.707.71 ( m , j = 9.0 hz , 3h ) , 7.68 ( d , j = 2.0 hz , 1h ) , 7.317.22 ( m , 2h ) , 7.17 ( d , j = 9.1 hz , 1h ) . lrms [ es ] : m / z = 379.09 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 8.82 ( s , 1h ) , 8.15 ( d , j = 9.2 hz , 1h ) , 7.99 ( d , j = 9.0 hz , 1h ) , 7.867.80 ( m , 2h ) , 7.50 ( dd , j = 9.0 , 0.7 hz , 1h ) , 7.37 ( dd , j = 9.2 , 2.6 hz , 1h ) , 7.337.23 ( m , 3h ) , 3.96 ( s , 3h ) , 2.44 ( s , 3h ) . lrms [ es ] : m / z = 345.1 ( m + h ) . h nmr ( 500 mhz , benzene - d6 ) 8.01 ( s , 1h ) , 7.917.82 ( m , 2h ) , 7.24 ( d , j = 1.7 hz , 1h ) , 7.03 ( dd , j = 8.7 , 1.7 hz , 1h ) , 6.94 ( d , j = 8.7 hz , 1h ) , 6.926.88 ( m , 3h ) , 6.796.75 ( m , 1h ) , 1.96 ( s , 3h ) . lrms [ es ] : m / z = 340.1 ( m + h ) . yield 260 mg ( 0.76 mmol , 70% ) . h nmr ( 500 mhz , chloroform - d ) 8.87 ( s , 1h ) , 8.18 ( d , j = 8.6 hz , 1h ) , 8.04 ( d , j = 9.0 hz , 1h ) , 7.887.80 ( m , 2h ) , 7.73 ( d , j = 2.0 hz , 1h ) , 7.58 ( dd , j = 8.6 , 1.8 hz , 1h ) , 7.49 ( d , j = 9.0 hz , 1h ) , 7.30 ( d , j = 8.0 hz , 2h ) , 2.86 ( q , j = 7.6 hz , 2h ) , 2.44 ( s , 3h ) , 1.35 ( t , j = 7.6 hz , 3h ) . lrms [ es ] : m / z = 343.1 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 8.87 ( s , 1h ) , 8.15 ( d , j = 8.6 hz , 1h ) , 8.02 ( d , j = 9.0 hz , 1h ) , 7.877.80 ( m , 2h ) , 7.72 ( d , j = 1.8 hz , 1h ) , 7.55 ( dd , j = 8.6 , 1.7 hz , 1h ) , 7.49 ( dd , j = 9.0 , 0.7 hz , 1h ) , 7.337.26 ( m , 2h ) , 2.582.54 ( m , 3h ) , 2.44 ( s , 3h ) . lrms [ es ] : m / z = 329.1 ( m + h ) . h nmr ( 500 mhz , dmso - d6 ) 9.16 ( s , 1h ) , 8.57 ( d , j = 9.0 hz , 1h ) , 8.40 ( d , j = 2.1 hz , 1h ) , 8.28 ( d , j = 9.1 hz , 1h ) , 7.917.81 ( m , 3h ) , 7.71 ( d , j = 9.1 hz , 1h ) , 7.387.32 ( m , 2h ) , 2.40 ( s , 3h ) . lrms [ es ] : m / z = 393.0 ( m + h ) . h nmr ( 500 mhz , acetone - d6 ) 9.11 ( s , 1h ) , 8.84 ( dd , j = 1.8 , 0.8 hz , 1h ) , 8.348.28 ( m , 1h ) , 8.06 ( d , j = 8.7 hz , 1h ) , 7.977.90 ( m , 2h ) , 7.78 ( dd , j = 8.7 , 1.9 hz , 1h ) , 7.63 ( dd , j = 9.0 , 0.7 hz , 1h ) , 7.37 ( ddt , j = 8.0 , 1.5 , 0.7 hz , 2h ) , 2.44 ( s , 3h ) . lrms [ es ] : m / z = 393.1 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 8.85 ( s , 1h ) , 8.38 ( s , 1h ) , 8.278.21 ( m , 1h ) , 7.917.81 ( m , 3h ) , 7.73 ( ddd , j = 8.4 , 6.9 , 1.3 hz , 1h ) , 7.63 ( ddd , j = 8.1 , 6.9 , 1.1 hz , 1h ) , 7.347.27 ( m , 2h ) , 2.45 ( s , 3h ) . lrms [ es ] : m / z = 393.1 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 8.86 ( s , 1h ) , 8.208.14 ( m , 1h ) , 7.83 ( dq , j = 8.3 , 2.0 hz , 3h ) , 7.617.53 ( m , 2h ) , 7.44 ( s , 1h ) , 7.29 ( d , j = 7.9 hz , 2h ) , 4.10 ( s , 3h ) , 2.44 ( s , 3h ) . lrms [ es ] : m / z = 345.1 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 8.79 ( s , 1h ) , 8.168.09 ( m , 2h ) , 8.01 ( d , j = 9.1 hz , 1h ) , 7.967.89 ( m , 2h ) , 7.79 ( dd , j = 8.9 , 2.1 hz , 1h ) , 7.597.53 ( m , 1h ) , 7.016.94 ( m , 2h ) , 3.90 ( s , 3h ) . lrms [ es ] : m / z = 409.0 ( m + h ) . the corresponding 2-benzoyl ketocoumarin ( 0.5 mmol ) was dissolved in dry pyridine ( 3 ml ) . to this solution was added nabh4 ( 0.625 mmol ) , and the reaction was stirred at room temp for 2 h. the mixture was then poured in cold 2 m hydrochloric acid ( 10 ml ) , which resulted in a white precipitate . the precipitate was washed several times with water , dried under a vacuum to yield the corresponding 2-benzoyl-1,2-dihydrocoumarin which was taken to the next step without purification . to a stirring solution of 2-benzoyl-1,2 dihydrocoumarin ( 0.1 mmol ) in dmf ( 0.2 ml ) was added hydrazine ( 0.125 mmol ) , and the mixture was stirred for 1.5 h at room temperature . water ( 5 ml ) was then added , and the aqueous layer was extracted with etoac ( 15 ml ) . the organic layer was separated , dried with na2so4 , and reduced to dryness to afford a yellowish brown crude product , which was further purified using medium pressure chromatography . the solid product was recrystallized from a mixture of ethyl acetate and n - heptane . h nmr ( 300 mhz , acetone - d6 ) 7.64 ( dd , j = 8.0 , 1.4 hz 1h ) , 7.53 ( d , 8.8 hz , 1h ) , 7.517.46 ( m , 3h ) , 7.407.32 , ( m , 3h ) , 7.157.01 ( m , 3h ) , 4.11 ( s , 2h ) . lrms [ es ] : m / z = 317.1 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient meoh : etoac solvent system ( 110% meoh ) . h nmr ( 500 mhz , acetone - d6 ) 7.837.56 ( m , 4h ) , 7.567.44 ( m , 2h ) , 7.39 ( t , j = 7.9 hz , 1h ) , 7.207.04 ( m , 3h ) , 4.27 ( s , 2h ) . lrms [ es ] : m / z = 395.01 ( m + h ) . h nmr ( 500 mhz , dmso - d6 ) 11.67 ( s , 1h ) , 9.88 ( s , 1h ) , 7.687.60 ( m , 2h ) , 7.51 ( dd , j = 20.3 , 8.5 hz , 3h ) , 7.35 ( d , j = 8.5 hz , 2h ) , 7.14 ( dt , j = 6.8 , 4.0 hz , 2h ) , 7.06 ( d , j = 8.8 hz , 1h ) , 4.11 ( s , 2h ) . lrms [ es ] : m / z = 395.01 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient meoh : etoac solvent system ( 110% meoh ) . h nmr ( 500 mhz , dmso - d6 ) 11.54 ( s , 1h ) , 10.13 ( s , 2h ) , 7.687.62 ( m , 1h ) , 7.54 ( d , j = 8.8 hz , 1h ) , 7.48 ( t , j = 8.1 hz , 1h ) , 7.38 ( d , j = 7.9 hz , 2h ) , 7.21 ( d , j = 7.7 hz , 2h ) , 7.167.01 ( m , 3h ) , 4.10 ( s , 2h ) , 2.34 ( s , 3h ) . h nmr ( 500 mhz , dmso - d6 ) 10.00 ( s , 1h ) , 9.89 ( s , 1h ) , 7.687.61 ( m , 2h ) , 7.597.46 ( m , 2h ) , 7.41 ( dd , j = 8.4 , 5.6 hz , 2h ) , 7.267.20 ( m , 2h ) , 7.14 ( tt , j = 6.3 , 2.5 hz , 2h ) , 7.05 ( d , j = 8.8 hz , 1h ) , 4.09 ( s , 2h ) . lrms [ es ] : m / z = 335.10 ( m + h ) . yield 60 mg ( 0.15 mmol , 57% ) . h nmr ( 500 mhz , dmso - d6 ) 11.82 ( s , 1h ) , 9.78 ( s , 1h ) , 7.677.56 ( m , 6h ) , 7.51 ( d , j = 8.8 hz , 1h ) , 7.14 ( td , j = 6.9 , 4.6 hz , 2h ) , 7.04 ( d , j = 8.8 hz , 1h ) , 4.16 ( s , 2h ) . lrms [ es ] : m / z = 385.10 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 110% etoac ) yielding 20 mg ( 0.05 mmol , 50% ) . h nmr ( 500 mhz , dmso - d6 ) 8.28 ( d , j = 1.9 hz , 1h ) , 8.107.97 ( m , 4h ) , 7.937.77 ( m , 3h ) , 7.50 ( t , j = 7.6 hz , 2h ) , 7.447.33 ( m , 4h ) , 4.16 ( s , 2h ) , 2.39 ( s , 3h ) . lrms [ es ] : m / z = 407.1 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 110% etoac ) yielding 10 mg ( 0.027 , 39% ) . h nmr ( 500 mhz , dmso - d6 ) 9.87 ( s , 1h ) , 9.62 ( s , 1h ) , 7.43- 7.40 ( m , 4h ) , 7.20 ( d , j = 7.5 hz , 1h ) , 7.117.02 ( m , 3h ) , 6.736.67 ( m , 1h ) , 4.06 ( s , 2h ) , 2.34 ( s , 3h ) . lrms [ es ] : m / z = 361.2 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 110% etoac ) yielding 10 mg ( 0.028 , 40% ) . h nmr ( 500 mhz , dmso - d6 ) 9.49 ( s , 1h ) , 8.28 ( d , j = 1.8 hz , 1h ) , 7.70 ( d , j = 8.8 hz , 1h ) , 7.34 ( m , 4h ) , 7.18 ( m , 3h ) , 4.11 ( s , 2h ) , 2.31 ( s , 3h ) . lrms [ es ] : m / z = 356.2 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 1025% etoac ) yielding 42 mg ( 0.12 mmol , 80% ) . h nmr ( 300 mhz , dmso - d6 ) 7.527.35 ( m , 3h ) , 7.16 ( m , 3h ) , 6.86 ( d , j = 8.9 hz , 1h ) , 4.07 ( s , 2h ) , 2.622.51 ( m , 3h ) , 2.32 ( s , 3h ) . lrms [ es ] : m / z = 345.2 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient etoac / hexane solvent system ( 10- 25% etoac ) . h nmr ( 300 mhz , dmso - d6 ) 7.547.35 ( m , 4h ) , 7.16 ( m , 4h ) , 6.92 ( dd , j = 8.9 , 1.8 hz , 1h ) , 4.08 ( s , 2h ) , 2.60 ( q , j = 7.5 hz , 2h ) , 2.34 ( s , 3h ) , 1.16 ( t , j = 7.5 hz , 3h ) . lrms [ es ] : m / z = 359.2 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient meoh / etoac solvent system ( 110% meoh ) yielding 16 mg ( 0.045 mmol , 58% ) . h nmr ( 300 mhz , dmso - d6 ) 7.90 ( s , 1h ) , 7.53 ( d , j = 8.8 hz , 1h ) , 7.43 ( m , 2h ) , 7.35 ( m , 3h ) , 7.18 ( d , j = 8.8 hz , 1h ) , 7.11 ( d , j = 8.8 hz , 1h ) , 4.10 ( s , 2h ) , 2.32 ( s , 3h ) . lrms [ es ] : m / z = 394.93 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient meoh / etoac solvent system ( 110% meoh ) yielding 45 mg ( 0.110 mmol , 62% ) . h nmr ( 500 mhz , dmso - d6 ) 7.89 ( d , j = 2.1 hz , 1h ) , 7.52 ( d , j = 8.8 hz , 1h ) , 7.46 ( s , 1h ) , 7.33 ( d , j = 7.8 hz , 2h ) , 7.13 ( m , 4h ) , 4.06 ( s , 2h ) , 2.31 ( s , 3h ) . lrms [ es ] : m / z = 409.10 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient meoh / etoac solvent system ( 110% meoh ) yielding 16 mg ( 0.110 mmol , 52% ) . h nmr ( 300 mhz , dmso - d6 ) 7.547.35 ( m , 4h ) , 7.16 ( m , 4h ) , 6.92 ( dd , j = 8.9 , 1.8 hz , 1h ) , 4.08 ( s , 2h ) , 2.34 ( s , 3h ) . lrms [ es ] : m / z = 409.10 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient meoh / etoac solvent system ( 110% meoh ) yielding 20 mg ( 0.110 mmol , 65% ) . h nmr ( 300 mhz , dmso - d6 ) 8.01 ( s , 1h ) , 7.67 ( d , j = 8.1 hz , 1h ) , 7.517.33 ( m , 4h ) , 7.15 ( t , j = 7.5 hz , 1h ) , 7.01 ( d , j = 9.0 hz , 1h ) , 6.87 ( t , j = 7.4 hz , 1h ) , 4.08 ( s , 2h ) , 2.44 ( s , 3h ) . lrms [ es ] : m / z = 409.1 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient meoh / etoac solvent system ( 110% meoh ) yielding 25 mg ( 0.069 mmol , 48% ) . h nmr ( 500 mhz , chloroform - d ) 7.587.53 ( m , 1h ) , 7.47 ( d , j = 8.0 hz , 2h ) , 7.41 ( d , j = 7.8 hz , 2h ) , 7.197.12 ( m , 1h ) , 7.006.91 ( m , 2h ) , 6.81 ( ddd , j = 8.3 , 7.0 , 1.3 hz , 1h ) , 4.18 ( s , 2h ) , 2.54 ( s , 3h ) . lrms [ es ] : m / z = 361.2 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient meoh / etoac solvent system ( 110% meoh ) yielding 15 mg ( 0.035 mmol , 48% ) . h nmr ( 300 mhz , dmso - d6 ) 7.92 ( d , j = 2.1 hz , 1h ) , 7.597.43 ( m , 2h ) , 7.38 ( d , j = 8.4 hz , 2h ) , 7.14 ( dd , j = 19.4 , 8.9 hz , 2h ) , 6.93 ( d , j = 8.3 hz , 2h ) , 4.06 ( s , 2h ) , 3.78 ( s , 3h ) . lrms [ es ] : m / z = 427.1 ( m + h ) . the corresponding 6-hydroxy-2(1h)-quinolinone ( 0.6 mmol ) was taken up in trifluoroacetic acid ( 1 ml ) . to this solution was added hexamethylenetetramine ( 1.2 mmol ) , and the resulting mixture was then heated at 100 c for 2 h. the reaction was allowed to cool , methanol ( 10 ml ) was added , and the solvent was evaporated to afford a dark residue . water ( 15 ml ) was added , and the resulting precipitate was filtered and dried . the precipitate was dissolved in dmf ( 0.8 ml ) , the solution cooled to 0 c , and phosphorus oxyhalide ( pocl3 or pobr3 ) ( 1.8 mmol ) was added dropwise . the light gray colored suspension ice - cold water ( 15 ml ) was added , and the precipitate was filtered and dried under a vacuum to yield the corresponding aldehyde as a pure product . synthesis of 6-hydroxyquinoline-5-carbaldehyde ( 60 ) has been reported previously by using reimer - teimann conditions . h nmr ( 500 mhz , dmso - d6 ) 11.06 ( s , 1h ) , 8.23 ( d , j = 8.8 hz , 1h ) , 7.75 ( d , j = 8.8 hz , 1h ) , 7.39 ( d , j = 9.3 , 1h ) , 7.36 ( d , j = 9.3 , 1h ) . lrms [ es ] : m / z = 208.2 ( m + h ) . the suspension was heated to 50 c and stirred for 18 h. ice - cold water ( 15 ml ) was then added , and resulting light brown precipitate was filtered and dried under a vacuum to afford the pure product . yield 60 mg ( 0.23 mmol , 45% ) . h nmr ( 500 mhz , dmso - d6 ) 11.16 ( s , 1h ) , 8.70 ( d , j = 8.8 hz , 1h ) , 7.82 ( d , j = 8.8 hz , 1h ) , 7.42 ( d , j = 9.3 , 1h ) , 7.39 ( d , j = 9.3 , 1h ) . 1h nmr ( 500 mhz , dmso - d6 ) 12.71 ( s , 1h ) , 10.74 ( s , 1h ) , 8.07 ( d , j = 9.3 hz , 1h ) , 7.58 ( s , 1h ) , 7.48 ( d , j = 9.3 , 1h ) , 2.69 ( s , 3h ) . lrms [ es ] : m / z = 222.1 ( m + h ) . compounds were prepared using general procedure ( ii ) knoevenagel condensation as described above . yield 28 mg ( 0.093 mmol , 54% ) . h nmr ( 500 mhz , chloroform - d ) 9.069.01 ( m , 1h ) , 8.81 ( s , 1h ) , 8.628.56 ( m , 1h ) , 8.38 ( d , j = 9.3 hz , 1h ) , 7.867.75 ( m , 2h ) , 7.64 ( dd , j = 8.6 , 4.2 hz , 1h ) , 7.347.24 ( m , 2h ) , 2.45 ( s , 3h ) . lrms [ es ] : m / z = 316.1 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 8.72 ( d , j = 0.6 hz , 1h ) , 8.558.49 ( m , 1h ) , 8.29 ( dd , j = 9.3 , 0.8 hz , 1h ) , 7.857.76 ( m , 2h ) , 7.63 ( d , j = 8.8 hz , 1h ) , 7.347.20 ( m , 2h ) , 2.45 ( s , 3h ) . lrms [ es ] : m / z = 350.0 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 8.72 ( d , j = 0.7 hz , 1h ) , 8.448.38 ( m , 1h ) , 8.30 ( dd , j = 9.3 , 0.7 hz , 1h ) , 7.857.72 ( m , 2h ) , 7.31 ( ddt , j = 7.1 , 1.3 , 0.7 hz , 2h ) , 7.257.19 ( m , 1h ) , 2.45 ( s , 3h ) . lrms [ es ] : m / z = 396.0 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 9.17 ( s , 1h ) , 8.29 ( d , j = 9.3 hz , 1h ) , 7.80 ( dd , j = 23.1 , 8.5 hz , 3h ) , 7.43 ( s , 1h ) , 7.31 ( d , j = 7.9 hz , 2h ) , 2.97 ( s , 3h ) , 2.45 ( s , 3h ) . lrms [ es ] : m / z = 364.1 ( m + h ) . h nmr ( 500 mhz , chloroform - d ) 8.71 ( s , 1h ) , 8.29 ( dd , j = 9.3 , 0.8 hz , 1h ) , 7.897.83 ( m , 2h ) , 7.80 ( d , j = 9.3 hz , 1h ) , 7.63 ( d , j = 8.8 hz , 1h ) , 7.397.33 ( m , 2h ) , 3.00 ( p , j = 6.9 hz , 1h ) , 1.29 ( d , j = 6.9 hz , 6h ) . lrms [ es ] : m / z = 378.1 ( m + h ) . the compounds were obtained as white precipitates , which were purified by medium pressure chromatography using a gradient meoh / dcm solvent system ( 110% meoh ) unless otherwise noted . h nmr ( 500 mhz , d2o ) 11.60 ( s , 1h ) , 8.56 ( m , 2h ) , 8.04 ( s , 1h ) , 7.67 ( d , j = 9.0 hz , 1h ) , 7.34 ( dd , j = 17.7 , 8.5 hz , 4h ) , 7.17 ( d , j = 7.7 hz , 1h ) , 4.11 ( s , 3h ) , 2.32 ( s , 3h ) . lrms [ es ] : m / z = 332.1 ( m + h ) . yield 20 mg ( 0.054 mmol , 65% ) . h nmr ( 500 mhz , dmso - d6 ) 11.57 ( s , 1h ) , 10.25 ( s , 1h ) , 8.08 ( d , j = 8.7 hz , 1h ) , 7.58 ( d , j = 9.0 hz , 1h ) , 7.367.30 ( m , 4h ) , 7.22 ( d , j = 9.0 hz , 1h ) , 7.15 ( d , j = 8.7 hz , 1h ) , 4.09 ( s , 2h ) , 2.31 ( s , 3h ) . lrms [ es ] : m / z = 366.1 ( m + h ) . h nmr ( 300 mhz , dmso - d6 ) 7.96 ( d , j = 8.9 hz , 1h ) , 7.59 ( d , j = 9.0 hz , 1h ) , 7.32 ( dd , j = 8.5 , 5.2 hz , 4h ) , 7.16 ( d , j = 7.8 hz , 1h ) , 4.08 ( s , 2h ) , 2.31 ( s , 3h ) . lrms [ es ] : m / z = 411.1 ( m + h ) . the crude product was purified by medium pressure chromatography using a gradient etoac / hexanes solvent system ( 1025% etoac ) . h nmr ( 500 mhz , dmso - d6 ) 11.54 ( s , 1h ) , 8.97 ( s , 1h ) , 7.57 ( d , j = 8.9 hz , 1h ) , 7.28 ( d , j = 9.0 hz , 2h ) , 7.20 ( d , j = 7.7 hz , 2h ) , 7.08 ( d , j = 8.9 hz , 1h ) , 4.28 ( s , 2h ) , 2.72 ( s , 3h ) , 2.28 ( s , 3h ) . lrms [ es ] : m / z = 380.1 ( m + h ) . h nmr ( 500 mhz , dmso - d6 ) 11.48 ( s , 1h ) , 8.03 ( d , j = 9.0 hz , 1h ) , 7.56 ( d , j = 9.0 hz , 1h ) , 7.31 ( m , 3h ) , 7.18 ( m , 3h ) , 4.07 ( s , 2h ) , 2.87 ( p , j = 6.9 hz , 1h ) , 1.20 ( d , j = 6.9 hz , 6h ) . lrms [ es ] : m / z = 394.1 ( m + h ) . 2-(4-methylbenzoyl)-3h - benzo[f]chromen-3-one ( 0.06 mmol ) was taken in dmf ( 0.2 ml ) to which was added the corresponding hydroxylamine ( 0.12 mmol ) , and the reaction mixture was stirred at 60 c for 18 h. the solvent was evaporated , and the residue was dissolved in etoac ( 5 ml ) . the organic layer was then washed with water , dried with na2so4 , and reduced to dryness to yield an oily product . the oil was further purified by medium pressure chromatography using gradient etoac / hexanes solvent system ( 050% etoac ) to afford the pure product . yield 13 mg ( 0.031 mmol , 50% ) of 1:1 mixture of imine and enamine tautomers . h nmr ( 300 mhz , dmso - d6 ) 9.88 ( s , 1h ) , 8.047.85 ( m , 4h ) , 7.65 ( d , j = 8.9 hz , 1h ) , 7.537.42 ( m , 2h ) , 7.24 ( dd , j = 21.9 , 8.4 hz , 3h ) , 7.056.91 ( m , 1h ) , 4.65 ( dd , j = 8.6 , 5.0 hz , 1h ) , 2.35 ( s , 3h ) . lrms [ es ] : m / z = 412.1 ( m + h ) . pyridine ( 1.5 equiv ) was added to the reaction mixture as methyl hydroxylamine was purchased as a hydrochloride salt . h nmr ( 300 mhz , dmso - d6 ) 9.74 ( s , 1h ) , 7.93 ( d , j = 2.1 hz , 1h ) , 7.54 ( dd , j = 9.0 , 2.3 hz , 2h ) , 7.37 ( dd , j = 9.1 , 2.2 hz , 1h ) , 7.287.15 ( m , 4h ) , 7.03 ( d , j = 8.9 hz , 1h ) , 3.92 ( s , 2h ) , 2.97 ( s , 3h ) , 2.33 ( s , 3h ) . . pyridine ( 1.5 equiv ) was added to the reaction mixture as benzyl hydroxylamine was purchased as a hydrochloride salt . h nmr ( 500 mhz , d2o ) 9.73 ( s , 1h ) , 7.94 ( d , j = 2.1 hz , 1h ) , 7.52 ( m , 4h ) , 7.407.22 ( m , 6h ) , 7.147.07 ( m , 2h ) , 7.04 ( d , j = 8.9 hz , 1h ) , 3.90 ( s , 2h ) , 2.51 ( s , 2h ) , 2.36 ( s , 3h ) . lrms [ es ] : m / z = 500.1 ( m + h ) .

sirtuins are a family of nad+-dependent protein deacetylases that play critical roles in epigenetic regulation , stress responses , and cellular aging in eukaryotic cells . in an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity , we previously identified a nonselective sirtuin inhibitor called cambinol ( ic50 50 m for sirt1 and sirt2 ) with in vitro and in vivo antilymphoma activity . in the current study , we used saturation transfer difference ( std ) nmr experiments with recombinant sirt1 and 20 to map parts of the inhibitor that interacted with the protein . our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues : 17 with > 7.8-fold selectivity for sirt1 , 24 with > 15.4-fold selectivity for sirt2 , and 8 with 6.8- and 5.3-fold selectivity for sirt3 versus sirt1 and sirt2 , respectively . in vitro cytotoxicity studies with these compounds as well as ex527 , a potent and selective sirt1 inhibitor , suggest that antilymphoma activity of this compound class may be predominantly due to sirt2 inhibition .

Introduction Results Discussion and Conclusions Experimental Section

in the past decade , human sirtuins ( homologues of yeast silent information regulator two or sir-2 ) have emerged as targets for cancer chemotherapy as well as for neurodegenerative and aging - related disorders such as huntington s disease , alzheimer s disease , and diabetes . although strong evidence exists for sirtuins having a central role in these debilitating diseases , their validation as targets for therapeutic intervention using small molecule modulators has been controversial . the most publicized efforts at modulation of sirtuin activity have been with the plant polyphenol resveratrol . ex-527 , a potent and selective sirt1 inhibitor ( sirt1 : human sirtuin isoform 1 ) , was found to be devoid of chemotherapeutic effect ; however , cambinol , tenovin-1 , tenovin-6 , and salermide , nonselective sirt1/sirt2 inhibitors , were found to have significant antitumor activity . combined use of a nonselective sirtuin inhibitor niacinamide ( nicotinamide ) and a pan - type i / ii hdac ( i.e. additionally , srt1720 , a potent direct sirt1 activator that was originally developed for its potential in lifespan extension or antiaging activity , was later found to be beneficial in a rat diabetes model employing a mechanism which may involve indirect activation of sirt1 . the recent functional characterization of other sirtuin isoforms such as sirt3 , sirt5 , sirt6 , and sirt7 has further complicated the field as it is becoming increasingly clear that in addition to sirt1 and 2 , these isoforms may also play major roles in aging ( sirt3 , sirt6 ) as well as in cell - proliferation disorders ( sirt7 ) . additional controversies regarding artifacts of popular in vitro assays to identify novel small molecule modulators of sirtuin activity have also hampered the validation of these enzymes for pharmacological intervention . previously , in an attempt to identify isoform selective sirtuin inhibitors , we carried out a phenotypic screen using an nci chemical library that resulted in discovery of cambinol ( 5-[(2-hydroxy-1-naphthyl)methyl]-6-phenyl-2-thioxo-2,3-dihydro-4(1h)-pyrimidinone ) 1 . titration experiments using sirt2 and an acetyl - peptide substrate showed that cambinol was competitive with the peptide substrate but noncompetitive with nad suggesting that its binding site partially overlaps with that of the substrate . cambinol inhibits sirt1 and sirt2 with mid - micromolar ic50 and induces hyperacetylation of p53 , a known sirt1 target , and -tubulin , a sirt2 target , in cell - based deacetylation assays . cambinol however suffers from major drawbacks including ( 1 ) moderate potency , as it did not induce tumor regression , but a decrease in tumor growth rate , and ( 2 ) poor solubility . since the discovery of cambinol , we have focused on optimization of cambinol s potency and validation of specific sirtuin isoforms ( e.g. , sirt1 and sirt2 ) as targets for b - cell lymphoma chemotherapy . the -naphthol nucleus is absolutely essential for activity , and the aryl ring is also important as replacing it with five - membered or a heterocyclic ring results in significant loss of potency . to further develop the structure activity relationship ( sar ) , we investigated substituting the pyrimidinedione ring ( heteroaryl group , figure 1b ) with five - membered heterocycles ( pyrazolone 1c and isoxazolone 1d ) as well as altering the functional groups on the naphthyl and aryl rings . to guide us with the specific positioning of the functional group to observe increased potency , we conducted saturation transfer difference ( std ) nmr experiments using 20 ( figure 2 ) . the data obtained from std nmr experiments identified portions of the ligand that interact with the receptor and suggest sites available for additional elaboration . cell - based toxicity assays show a significant correlation between sirt2 inhibition and cytotoxicity in the namalwa burkitt s lymphoma cell line . syntheses on new cambinol analogues closely parallel that of the parent compound , cambinol ( scheme 1 ) . for naphthyl and quinolyl derivatives of cambinol , hydroxy - naphthaldehyde and hydroxy - quinaldehyde starting materials were prepared by titanium - catalyzed reiche formylation reaction ( 1-carbaldehyde-2-hydroxynaphthalene derivatives ) from the corresponding phenols , and the duff reaction of hexamethylenetetramine ( hmta ) with 6-hydroxy-2-quinolones in the presence of trifluoroacetic acid ( tfa ) afforded the corresponding 5-carbaldehyde-6-hydroxy-2-quinolones , respectively ( schemes 1 and 2 ) . the pyrazolone analogues were prepared by reacting the appropriate -ketoester with hydrazine , while syntheses of the isoxazolone analogues used corresponding hydroxylamine compounds as nucleophiles ( scheme 1 ) . reagents and conditions : ( a ) cl2chome , ticl4 , ch2cl2 , 020 c , 24 h ; ( b ) aryloyl ethyl acetoacetate , piperidine , etoh , reflux , 2 h ; ( c ) nabh4 , pyridine , 20 c , 2 h ; ( d ) thiourea , naoet , etoh , reflux , 18 h ; ( e ) hydrazine , dmf , 20 c , 1.5 h ; ( f ) hydroxylamine , dmf , 60 c , 18 h ; reagents and conditions : ( a ) hmta , tfa , 100 c , 2 h ; ( b ) pox3 , dmf , temp , time x = cl , br ; ( c ) aryloyl ethyl acetoacetate , piperidine , etoh , reflux , 2 h ; ( d ) nabh4 , pyridine , 20 c , 24 h ; ( e ) hydrazine , dmf , 20 c , 1.5 h. sirt1 , sirt2 , and sirt3 enzyme inhibition studies , carried out using the sirt - glo assay ( promega corp . , madison , wi ) at a single inhibitor concentration ( 50 m ) , revealed a range of activities against each target ( tables 1 , 2 , and 3 ) . cambinol , the parent of this class of agents , was found to be an nonselective sirt1 , sirt2 inhibitor with 54% sirt1 inhibition , 46% sirt2 and 16% sirt3 inhibition at a single 50 m concentration ( table 1 ) , and dose response titration against these enzymes with cambinol gave ic50 values very similar to published values ( sirt1 ic50 = 56 m , sirt2 ic50 = 51 m ) and > 200 m sirt3 ic50 . we carried out an std nmr experiment using recombinant sirt1 and compound 20 ( figure 2 ) . the relative saturation transfer enhancement at positions we were able to evaluate is shown in figure 2 . the results show that c-7 proton ( naphthalene numbering is used for clarity since most of the compounds discussed in this section are naphthalene derivatives ) gives the strongest std signal ( 100% relative intensity ) and forms a close contact with the protein . the aryl c-4 methyl protons ( 66% signal intensity ) and c-8 proton ( 74% signal intensity ) form contacts with the protein , but these may be suboptimal . consistent with this , compounds 1 ( cambinol ) , 2 , 6 , 13 , and 22 that contain a small substituent at c-4 ( i.e. , hydrogen or fluorine ) are relatively poor and nonselective sirtuin inhibitors . the most selective compounds 17 and 24 have methyl groups at c-4 suggesting that sirt1 and sirt2 residues that interact with this group are well conserved . , methyl or isopropyl ) substituents at the c-4 position of the aryl moiety tend to favor sirt1 inhibition with the bulkiest substituent ( i.e. the std values for the -methylene protons and the nhs of the pyrazolone moiety could not be determined because of the overlap with the water signal and the fast exchange with solvent deuterons , respectively . std values for the c-3 hydrogen and for protons in the phenyl ring ( c-2 , c-3 , c-5 , c-6 ) could not be accurately determined due to overlapping signals . the most significant determinant of the selectivity between sirt1 and sirt2 in the present compound series is the h - bond donor or acceptor at the 2 position of the heteroaryl unit ( i.e. the reversal of hydrogen bond directionality results in a reversal of selectivity with the hydrogen - bond donor compounds 17 displaying up to a 7.8-fold sirt1 selectivity and the hydrogen bond acceptor 24 a 15.4-fold sirt2 selectivity with approximately similar overall inhibitory activity against the preferred isoform : 26 m ic50 for 17 against sirt1 and 13 m ic50 for 24 against sirt2 . this compound however was relatively nonselective with sirt1 and sirt2 ic50 of 41 and 32 m , respectively . we treated nci - h460 cells with 1 m etoposide in the presence or absence of cambinol 1 or 14 , a sirt1-selective inhibitor ( tables 1 and 4 ) . part of the motivation behind these studies was to determine whether sirt1 , sirt2 or sirt3 inhibition , or possibly a subset of these enzymes , was responsible for cambinol s antilymphoma activity . cell - based sirtuin activity assays were carried out by determining the acetylation status of well established sirt1 ( p53 ) and sirt2 ( -tubulin ) in the presence of isoform selective inhibitors . cells were plated in 96-well plates , treated with the test compound or dmso control for 72 h , and viable cells were quantified by luminescence using celltiterglo ( promega corp . cells entering an early phase of apoptosis are present in the upper left panel . cambinol , ( 5-[(2-hydroxy-1-naphthyl)methyl]-6-phenyl-2-thioxo-2,3-dihydro-4(1h)-pyrimidinone ) 1 , is a nonselective sirtuin inhibitor with equivalent inhibitory activity against sirt1 and sirt2 . it also is a reasonably potent antitumor agent in vitro and in vivo with particular activity against burkitt s lymphoma cell lines . in an effort to delineate the contribution of sirt1 and sirt2 inhibition in this antitumor activity have partly addressed the structure activity relationships of six - membered pyrimidinedione - containing ( i.e. in an effort to investigate an alternative chemical space , we prepared a series of five - membered pyrazolone- and 5-isoxaolone - containing compounds . the most selective of these compounds , 14 and 17 , exhibit a greater than 5-fold and 7-fold , respectively , preference for sirt1 over sirt2 . in order to gain a molecular understanding of the observed selectivity , we sought to identify portions of the ligand involved in the interaction with sirt1 protein . the std nmr experiment with recombinant sirt1 and 20 , a relatively nonselective but highly soluble ligand , has identified positions of the ligand as potential sites for improving selectivity and potency ( figure 2 ) . the efficiency of saturation transfer was lowest at c-8 of the quinolone ring system ( naphthalene numbering ) and at the 4-methyl group of the aryl substituent suggesting that these portions of the ligand interact with the protein surface but in a suboptimal manner . saturation transfer was most efficient at c-4 and c-7 of the quinoline ring suggesting close ligand / protein interactions ; however , the c-8 proton showed a weaker interaction . the binding site for the c-4 phenyl position appears to tolerate medium - sized substituents equally well in sirt1 and sirt2 , with bulkier groups such as isopropyl showing an improved preference for sirt1 . the strongest determinant of selectivity in the current compound series is the hydrogen bond donor ( i.e. remarkably , the reversal of directionality of the hydrogen bond donor / acceptor pair leads to a 7.8-fold sirt1 selectivity in the case of the former 24 and a 15.4-fold sirt2 selectivity for the latter , with approximately equipotent overall inhibitory activity against the preferred isoform : 26 m ic50 for 17 against sirt1 and 13 m ic50 for 24 against sirt2 . placement of a bulky hydrophobic phenyl group at the naphthalene c-6 position yielded 8 , a potent although relatively nonselective sirt3 inhibitor . the sar trends observed in the current compound series provide a strong starting point for development of potent isoform - selective as well as pan - sirtuin inhibitors . while both sirt1 and sirt2-selective compounds exhibit some degree of cytotoxicity , a strong correlation between sirt2 inhibition and namalwa cytotoxicity ( r = 0.56 , p = 0.0014 ) suggests that sirt2 inhibition may be primarily responsible for the observed antilymphoma activity . lack of correlation between sirt1 inhibition and cytotoxicity observed in our study is consistent with the findings that ex-527 , a potent and selective sirt1 inhibitor , does not exhibit activity against b - cell lymphoma cell lines , as reported previously by others for epithelial tumor cells . in addition to burkitt s lymphoma cell lines , we tested 8 , 17 , and 24 against normal immortalized lymphoblastic lines ( b1 and b2 ) , diffuse large b - cell lymphoma lines as well as a panel of solid tumor cell lines ( table 5 ) . interestingly , colon ( hct116 ) breast ( mcf7 ) and nonsmall cell lung carcinoma ( nci - h460 ) also showed significant sensitivity to sirt2 inhibition . namalwa cells were treated with 10 and 25 m ( 24 ) or solvent ( dmso ) for 16 h , washed in phosphate buffered saline , and suspended at approximately 200 000 cells in 200 l of binding buffer containing 5 l of annexin v - pe ( bd biosciences , san jose , ca ) for measuring phosphatidylserine , and 4,6-diamidino-2-phenylindole ( dapi ) at a concentration of 2.5 after 15 min incubation at room temperature , cell fluorescence was analyzed on a facscanr ( becton dickinson , franklin lakes , nj ) . for characterization purposes , samples consisted of a 5 mm solution of each compound in chloroform - d ( 99.8% d , cambridge isotopes ) , dimethyl sulfoxide - d6 ( 99.9% d , cambridge isotopes ) , benzene - d6 ( 99.5% d , cambridge isotopes ) or acetone - d6 ( 99.9% d , cambridge isotopes ) , and the spectra were referenced to residual solvent peaks at 7.27 , 2.50 , 7.16 , and 2.05 ppm , respectively . nmr samples contained 1.5 m sirt1 and 200 m compound 20 in pbs in d2o ph 7.4 ( uncorrected for d2o ) with 1% dimethyl sulfoxide - d6 as a cosolvent . briefly , two free induction decay ( fid ) data sets were collected in an interleaved manner to minimize temporal fluctuations with the protein irradiation frequency set on - resonance ( 0.5 ppm ) and off - resonance ( 40 ppm ) , respectively ( sw = 6000 hz , 16 steady state scans , 2048 transients , 4k complex points , d1 = 3 s ) . a solution of the corresponding substituted naphthol ( 9 mmol ) in either ch2cl2 or 1,2-dichloroethane ( 30 ml ) was added dropwise , and the reaction was warmed to room temperature . to this solution was added nabh4 ( 0.625 mmol ) , and the reaction was stirred at room temp for 2 h. the mixture was then poured in cold 2 m hydrochloric acid ( 10 ml ) , which resulted in a white precipitate . to a stirring solution of 2-benzoyl-1,2 dihydrocoumarin ( 0.1 mmol ) in dmf ( 0.2 ml ) was added hydrazine ( 0.125 mmol ) , and the mixture was stirred for 1.5 h at room temperature . water ( 5 ml ) was then added , and the aqueous layer was extracted with etoac ( 15 ml ) .

chemokines ( cks ) are low molecular weight chemoattractant peptides , belonging to the cytokine family . differently from interleukins , cks act via g protein - coupled receptors ( gpcrs ) , controlling cell migration and trafficking throughout the body , during immune response and development [ 3 , 4 ] . cks are also critical mediators of several physiological mechanisms such as wound - healing and tissue homeostasis [ 3 , 5 ] ; moreover , cks are expressed in the central nervous system ( cns ) [ 6 , 7 ] where they not only act as mediators of development , intercellular communication , and inflammatory processes but also function as neurotransmitters or neuromodulators , mainly involved in neuroendocrine regulations . recently , it has been shown that cks play a relevant role in tumorigenesis , neoangiogenesis , tumor progression , and metastasization [ 9 , 10 ] . evidence for autocrine / paracrine regulatory mechanisms in different normal and cancer cell types , driven by chemokine / receptors interaction on the same or a nearby cell , supports the potential role of cks in the control of physiological or tumoral endocrine functions . in particular , the chemokine ( c - x - c motif ) ligand 12 ( cxcl12 ) and its receptors , cxcr4 and cxcr7 , have been involved in cancer cell proliferation , migration , and invasion [ 1113 ] . they are classified by size ( microadenoma , < 10 mm or macroadenoma , > 10 mm ) and on the basis of their ability to produce hormones , as secreting or functioning tumors ( about 50% of adenomas ) or as clinically nonfunctioning pituitary adenomas ( nfpa ) that do not release hormones or , more often , secrete clinically nonrelevant ( i.e. , gonadotropins ) or nonbioactive hormones ( -subunit of glycoproteic hormones ) [ 14 , 15 ] . almost all pituitary tumors display a benign clinical course being slow growing and show low incidence of metastasis ; however , they are frequently associated with high morbidity and mortality due to mass - related effects and paraneoplastic syndromes related to hormone hypersecretion . functioning pituitary adenoma leads to hypersecretion of hormones that results in classic clinical syndromes , mainly acromegaly ( overproduction of gh ) , hyperprolactinemia ( excess of prl ) , and cushing 's disease ( overproduction of acth ) and , more rarely , secondary hyperthyroidism ( increased tsh secretion ) . nfpas do not secrete sufficient hormones ( mainly fsh or lh ) to be detectable in the blood or to cause hormonal manifestations ; in other cases only biologically inactive -subunit is released and more rarely they are classified as true nonsecreting adenomas . importantly , all pituitary adenomas may induce hypopituitarism and neurological symptoms ( for example compression of the optic chiasma ) due to mass effect [ 16 , 17 ] . clinical relevance and recent advances in the comprehension of their molecular pathogenesis suggest that pituitary adenomas should be considered a more critical disease than a benign endocrine pathology . thus , a deeper evaluation of the mechanisms at the basis of their tumorigenesis and better prognostic markers to identify tumors with a high risk of recurrence are most awaited to improve pituitary adenoma clinical outcome . this review will focus on the diverse role of cxc chemokines and their receptors in normal pituitary cell functions and pituitary tumor development and progression , summarize recent progress in cxcr7 functions , and discuss the present issues and future perspectives . cks are classified , according to the number and spacing of the first two cysteine residues of a conserved cysteine motif , into four groups : ( 1 ) cxc ( with a single nonconserved amino acid residue -x- between the first n - terminal c residues : cxcl1 - 17 ) ; ( 2 ) cc ( two adjacent cysteine residues : ccl1 - 28 ) ; ( 3 ) xc ( only one n - terminal cysteine : xcl1 - 2 ) ; ( 4 ) cx3c ( three nonconserved amino acid residues separating the n - terminal c residues : cx3cl1 ) [ 18 , 19 ] . ck receptors are typical gpcrs , all of them signaling through heterotrimeric g proteins ( g , and g- subunits ) , with the remarkable exception of cxcr7 which is exclusively biased towards -arrestin - mediated signaling . upon ligand binding , many ck receptors may form homo- and heterodimers that activate distinct intracellular signaling pathways from individual receptors . the classification of ck receptors is based on the class of their ligands ( e.g. , cxc ligands bind cxc receptors ) . cks may also bind a small group of so - called atypical chemokine receptors ( ackrs ) , which are unable to initiate downstream conventional g - protein - dependent signaling , resulting , from a functional point of view , in the inability to induce directional cell migration . these receptors bind distinct and complementary range of cks and likely control ck networks during development and physiopathological processes by scavenging cks . ackrs , including duffy antigen receptor for chemokines ( darc , ackr1 ) , c6 ( ackr2 ) , cxcr7 ( ackr3 ) , and ccx - ckr1 ( ackr4 ) ( for classification and nomenclature see ) , were proposed to serve as decoy receptors to scavenge inflammatory cks from the extracellular microenvironment , inhibiting their signaling . indeed , recent evidence supports the ability of ackrs to transport , internalize , and degrade cks , leading to the formation of ck gradients in normal and cancer tissues , responsible for the functional modulation of their signaling . cks belonging to the cxc family can be further grouped according to the presence or absence of a the tripeptide motif glu - leu - arg ( elr ) preceding the cxc domain ( elr or elr ) , which affects receptor binding specificity and biological effects . notably , elr cxc chemokines ( i.e. , cxcl9 , -10 and -11 ) are interferon -inducible and act as potent angiostatic factors to impair angiogenic stimuli induced by growth factors . conversely , elr cks ( cxcl1 , -2 , -3 , -5 , -6 , -7 , -8 ) are proangiogenic [ 24 , 25 ] . cxcl12 ( previously known as stromal cell - derived factor-1 , sdf-1 ) is an exception to this characterization , since it is elr but mediates tumor - promoting angiogenesis via its receptor cxcr4 [ 9 , 24 , 26 ] . common features shared by all cks are pleiotropism , promiscuity , and redundancy , with a single ck able to bind several receptors , whereas multiple cks bind the same receptor resulting in the same functional outcome . upon ligand binding , ck receptors undergo conformational change that activates the g subunits sensitive to bordetella pertussis toxin ( ptx ) . their activation dissociates the gtp - bound g subunit from the g- dimer , and both these active components trigger intracellular signals , such as activation of phospholipase c ( plc)/inositol triphosphate ( ip3)-ca / diacyl glycerol ( dag)/protein kinase c ( pkc ) and inhibition of adenylyl cyclase ( ac)-camp / protein kinase a ( pka ) . moreover , these receptors control the activity of different kinases , including extracellular regulated kinases ( erk1/2 ) , c - jun n - terminal kinase ( jnk ) , p38 , phosphatidyl inositol 3 kinase ( pi3k)-akt , and the focal adhesion kinase ( fak ) . the distinct transductional cascades regulated by ck receptors mainly depend on the g subfamily which they activate : gi inhibits ac but also activates tyrosine kinases of the src family , favoring signal integration ; gq increases plc activity , to cleave pip2 to form dag and ip3 . in turn , dag activates pkc , whereas ip3 binds specific receptors on the endoplasmic reticulum inducing ca release from intracellular stores . finally , g12 controls the activity of the small g protein rhoa , via rho - gef . on the other hand , ck receptor activation of g subunits results in the activation of pi3k leading , through the phosphoinositide - dependent kinase 1 and 2 ( pdk1 - 2 ) , to akt phosphorylation and subsequent activation of its downstream signal proteins such as glycogen synthase kinase 3 ( gsk3 ) , mammalian target of rapamycin ( mtor ) , and fak , which control migration in different types of normal and tumor cells . after stimulatory responses , the inactivation of ck receptor signaling occurs after the hydrolysis of gtp to gdp by the intrinsic gtpase activity of g subunit , followed by its reassociation with g/ in an inactive complex . moreover , receptor desensitization , internalization , and lysosomal degradation are mediated by g protein - coupled receptor kinases ( grks ) and arrestins . cks are constitutively secreted by leukocytes , fibroblasts , endothelial , and epithelial cells to mediate cell activation , trafficking , and homing [ 5 , 30 ] . beside their basal expression , most cks are highly induced during inflammatory or infective processes driving different phases of immune response via a ck gradient which directs leukocyte recruitment to the site of inflammation . furthermore , cks directly activate specialized effector lymphocytes during the different steps of immune response , for example , cxcl8 ( formerly named il8 ) recruits neutrophils , basophils , and eosinophils expressing its receptors , cxcr1 and cxcr2 . adaptive immune responses are mediated by cks ( cxcl9-l10-l11 ) secreted by macrophages activated by inf- released by natural killer and t helper 1 ( th1 ) cells that express cxcr3 , the receptor for cxcl9-l11 , thus amplifying leukocyte recruitment and , finally , inflammation . cks also play a key role in embryogenesis , organogenesis , angiogenesis , and germ cell migration , especially during neural development . the constitutive expression of cks and their receptors in adult normal brain was initially identified in the immune - like competent cell populations such as microglia and astrocytes . the subsequent detection of their expression in neurons [ 3234 ] broadened ck role as neuromodulators / neurotransmitters in neurological processes such as thermoregulation , pain perception , and stress conditions , as well as in pituitary functions . focusing on the cxcl12/cxcr4-r7 network , it exerts a variety of functions in cns development as well as in mature brain . cxcl12 directs the migration of embryonic and adult stem cells in the developing central and peripheral nervous system [ 35 , 36 ] , controlling the formation of cerebellum , cerebral cortex , hippocampus , and dorsal root and sympathetic ganglia [ 25 , 37 , 38 ] . postnatally , cxcr4 expression , while downregulated in many brain areas , persists in the hypothalamus where it modulates the hypothalamic - pituitary system and the hypothalamic - pituitary - gonadal axis , in particular , cooperating to the regulation of neuroendocrine and reproductive systems [ 3941 ] . as far as cns development is concerned , the pivotal role of cxcl12/cxcr4 emerged from studies using knockout mice for either the ligand or the receptor . both models exhibited a superimposable abnormal neuron migration in the cerebellum , dentate gyrus , and dorsal root ganglia [ 35 , 36 , 42 , 43 ] . furthermore , cxcl12/cxcr4 axis controls migration and homing of cajal - retzius cells [ 44 , 45 ] , postmitotic neurons , cortical interneurons [ 40 , 4749 ] , and dopaminergic neurons . cxcl12/cxcr4 regulation of stem cell positioning and migration persists in adults , in the neurogenic niches of brain and in the bone marrow , where hematopoietic progenitors cells are retained by the interaction between ligand and receptor that also promotes their survival and proliferation . interestingly , a similar homing mechanism has been demonstrated for adult neural progenitor cells ( npcs ) or neural stem cells ( nscs ) . cxcr7 , the second cxcl12 receptor , has a 10-fold higher binding affinity than cxcr4 but also binds cxcl11 ( formerly known as ifn - inducible t cell chemoattractant , i - tac , ) , which , in turn , interacts with cxcr3 . presently , the function of cxcr7 is still controversial . cxcr7 does not mediate cxcl12-dependent cell migration [ 20 , 52 , 54 ] and displays atypical signaling pathways , failing to induce intracellular ca mobilization and inhibition of camp production , since this receptor does not seem to be coupled to gi. based on its ability to rapidly sequester and degrade cxcl12 and thus to suppress cxcr4 activity , cxcr7 was firstly proposed to be a decoy receptor [ 5457 ] ; currently , this activity is considered only a part of the possible mechanisms by which cxcr7 modulates cellular functions . indeed , emerging evidence suggests that cxcr7 can promote cell motility [ 5860 ] and trigger intracellular signals in different human normal and cancer cell types [ 6164 ] . in particular , cxcr7 activates akt , map kinase ( mapk ) , and jak / stat3 cascades , either by direct modulation , through a -arrestin - dependent pathway [ 20 , 65 ] , or after heterodimerization with cxcr4 [ 59 , 6669 ] . preferential signaling through g - proteins or -arrestin is influenced by both cxcr4-cxcr7 dimer formation and the oligomerization state of cxcl12 [ 70 , 71 ] . cxcr7 was recently shown to activate mtor in human renal cancer cells through the modulation of erk1/2 and p38 activities , further suggesting that it is a fully signaling receptor although independent from g proteins . however , cxcr7 knockout mice display a lethal phenotype due to a heart valve and vascular defects , a very similar scenario observed in mice with targeted disruption of the genes encoding cxcr4 and cxcl12 [ 67 , 74 ] . in the adult rat brain , cxcr7 is expressed at high levels in vessels , pyramidal cells , and mature dentate gyrus granule cells , overlapping cxcl12 expression pattern [ 75 , 76 ] , and a functional role for cxcr7 in the control of neuronal migration to the subventricular and intermediate zone was suggested [ 69 , 77 ] . in rat mature neurons and blood vessels , cxcr7 appears to be the preponderant cxcl12 receptor , likely contributing to cxcl12-dependent neuronal development . moreover , cxcr7 acts as scavenger on brain microvessel endothelium and it is essential for inflammatory leukocytes to infiltrate the cns . cxcr7 is also expressed in neural tube and brain of mice embryos . in rat cortex , cxcr7 is localized in gabaergic neuron precursors , and cajal - retzius cells and , unlike cxcr4 , it has been identified in neurons forming the cortical plate and in the developing dentate gyrus and cerebellar external germinal layer [ 75 , 80 ] . migrating immature cortical interneurons co - express cxcr4 ( membrane surface expression ) and cxcr7 ( intracellular expression , mainly endosomes ) . cxcr7 rapidly recycles from membrane to intracellular pools of interneurons , and its trafficking mediates cxcl12 endocytosis . it is essential for the regulation of interneuron migration in the developing cerebral cortex since its removal causes an increase in extracellular cxcl12 content , which favors its binding to cxcr4 and consequently induces the endocytosis and degradation of cxcr4 . thus , cxcr7 regulates cxcr4 expression and likely controls cxcl12 signaling to drive successful migration in the developing cerebral cortex . in addition , since cxcr7 and cxcr4 mutant mice displayed opposite defects in interneuron motility and positioning , cxcr4 and cxcr7 were proposed to have distinct roles and signal transduction to regulate interneuron movement . this fine tuning of cxcl12 response induced by cxcr7 occurs either directly modulating -arrestin - mediated signaling cascades or scavenging local cxcl12 availability . deletion of one of the cxcl12 receptors is sufficient to generate a migration phenotype that corresponds to the cxcl12-deficient pathway and interfering with the cxcl12-scavenging activity of cxcr7 causes loss of cxcr4 function . for example , during development , cxcl12 regulates the migration of gonadotropin - releasing hormone ( gnrh ) neurons , through cxcr4-mediated activation of the girk channel , but this effect is modulated by cxcr7 which controls cxcl12 content availability acting as a scavenger along the migratory path . the relevance of cxcl12 and cxcr4-r7 system in cns ontogeny and functions is even more crucial in the view of their expression in both embryonic and adult brain stem cells , a subset of undifferentiated cells characterized by self - renewal through asymmetric division , differentiation into multiple lineages , and constant proliferation that in adults acts in tissue maintenance and repair . the role of cxcl12 and cxcr4 in stemness maintenance has gained much attention also in the neuroendocrinology field due to the proposed role of stem cells in pituitary plasticity [ 84 , 85 ] . both cxcl12 and cxcr4 are expressed in different anterior pituitary cell subtypes , as well as in nonhormonal cell types [ 8688 ] . the chemotactic activity of this ck could be also relevant in folliculostellate ( fs ) cell , non - fs nestin cell , and stem cell migration [ 86 , 89 ] . therefore , understanding the ck - dependent mechanisms associated with candidate stem cells within pituitary might help to clarify their activity in development or in normal mature hormone - producing and tumor pituitary cells [ 84 , 90 , 91 ] . the stem cell concept applied to cancer has radically changed the research approach to tumorigenesis and treatment , since the subset of cancer cells , namely , cancer stem cells ( cscs ) , seems responsible for tumor initiation , metastasis , and resistance to therapy . although , at present , all factors and signals that regulate cscs are not completely clarified , accumulating evidence suggests a key role of the cxcl12/cxcr4 axis in csc maintenance and growth [ 62 , 93 ] . moreover , interactions between cscs and tumor microenvironment through secreted cks ( e.g. , cxcl12 ) , possibly occurring also in pituitary adenomas , may act as chemoattractant to recruit fibroblasts , endothelial , mesenchymal , and inflammatory cells to the tumor , via cxcr4 . through the release of growth factors ( bfgf , egf , and vegf ) , cyto / chemokines , and neuroendocrine proteins ( steroid hormones , prolactin , growth hormone , ghrelin , erythropoietin , catecholamines , etc . ) neuronal and neuroendocrine pathways regulate fundamental functions within the cns and its interaction with the immune system . complex autocrine / paracrine signals through neuropeptides ( e.g. , egf and vip ) , neurotransmitters , cytokines ( il-1 , il-6 ) , and cks occur also in pituitary regulation , differently from the classical hypothalamic input and feedback signals from the periphery [ 9597 ] . egf expression has been observed at all stages of pituitary development and in the adult pituitary , and the egfr pathway contributes to pituitary physiology and tumorigenesis . il-1 receptors were detected in pituitary cells , and their activation inhibits prolactin ( prl ) secretion from dispersed rat pituitary cells through the regulation of ac and plc activities , and ca fluxes [ 100103 ] . il-6 and its receptors are also expressed in the pituitary gland [ 104 , 105 ] where their interaction regulates apoptosis and proliferation of endocrine cells in vitro . il-6 is mainly produced by the fs cells and activates a paracrine loop on the hormone - secreting cells [ 107 , 108 ] regulating acth , prl , lh , and gh secretion [ 110112 ] via the modulation of ac and plc activities . interestingly , il-6 exerts opposite effects on normal and adenomatous pituitary cells : it is inhibitory for normal anterior pituitary and stimulatory for adenoma cells . il-18 was also proposed to exert paracrine effects in pig anterior pituitary being the ligand and its receptor expressed by different subsets of gh secreting cells . finally , interleukins ' regulation of the hypothalamic - pituitary - adrenal axis also involves the modulation of vasoactive intestinal peptide- ( vip- ) secreting pituitary cells to control , in a paracrine manner , prl release . cks can affect pituitary hormone secretion via the hypothalamic - pituitary axis or autocrine / paracrine regulation . cxcl1 is expressed in the posterior pituitary , in the paraventricular nucleus ( pvn ) of the hypothalamus and the median eminence . in response to stressful stimuli , this ck is released in the median eminence to reach its receptor ( cxcr2 ) expressed in pituitary cells and induce the release of prl and gh and the inhibition of lh and fsh secretion . the generation of transgenic rats ( s100-gfp rats ) that express green fluorescent protein in s100-positive pituitary fs cells in the anterior pituitary led to the characterization of s100-positive cells [ 86 , 121 ] and transcripts of cxcl10 ( ifn- inducible protein 10 kda , ip-10 ) were identified in a subpopulation of these cells . importantly , cxcr3 , the receptor for cxcl10 , was shown to be expressed in corticotrophs , suggesting a possible autocrine / paracrine effect of cxcl10 , released from fs cells , on acth - producing cells . cxcl12/cxcr4 is the major regulatory axis not only connecting the immune and nervous systems , but also playing a role in neuroimmune regulation of the anterior pituitary physiological functions . cxcl12 was detected in both rat pituitary and hypothalamus and its expression in hypothalamic neurons , concomitant with cxcr4 positivity at pituitary level , corroborated the hypothesis that this ck could represent a hypothalamic regulatory factor of anterior pituitary function . as a consequence , the chemokinergic regulation of anterior pituitary cells might derive from coordinate activity of cxcl12 originating from both hypothalamic neurons and systemic circulation . a regionalized constitutive expression of cxcl12 was reported in adult rat brain , particularly in arginine vasopressin- ( avp- ) expressing neurons where its interaction with cxcr4 leads to modulates induced plasma avp release in vivo . the expression pattern of this chemokine and its receptor in the rat hypothalamo - neurohypophyseal system was further investigated : they colocalize within avp - expressing neurons in both supraoptic ( son ) and paraventricular ( pvn ) nucleus as well as in dense core vesicles of avp - positive nerve terminals in the posterior pituitary , showing a similar distribution . since avp controls body fluid homeostasis , the interaction between cxcl12 and avp was studied in avp - deficient brattleboro rats that show low expression of both cxcl12 and cxcr4 , correlated with avp protein expression level in son , pvn , and posterior pituitary . however , since cxcl12 mrna is increased , it was hypothesized that cxcl12 synthesis is present in these cells but , being costored with avp , a concomitant massive release of both peptides is responsible for their low content at both hypothalamic and posterior pituitary levels . avp and cxcl12 expression is dependent on water balance and is centrally regulated , further strengthening the role of cxcl12 in neuroendocrine functions . however , cxcl12 and cxcr4 are also coexpressed in rat pituitary cells and a further autocrine / paracrine regulation of pituitary functioning was hypothesized . complete colocalization between cxcr4 and gh was reported in normal rat pituitary , suggesting that cxcr4 is a rather specific regulator of somatotroph activity , in rats . indeed , cxcl12 stimulates gh transcription and secretion in both primary rat anterior pituitary cells and the gh - producing pituitary adenoma cell line , gh3 . interestingly , rat fs cells also express cxcr4 and secrete cxcl12 , which acts as a potent chemoattractant for these cells . the activation of this autocrine loop facilitates the formation of f - actin in fs cells and the subsequent directional extension of their cytoplasmic processes toward other fs cells . cxcl12/cxcr4 interaction induces invasion and interconnection of fs cells to near lobular structures likely forming a circuit that causes or maintains local cellular arrangement in the anterior pituitary . in humans , scattered expression of both cxcr4 and cxcl12 within the anterior lobe was detected by immunohistochemistry , revealing a nonhomogeneous positivity for both proteins throughout the tissue , including large negative areas , others showing few positive cells and rare zones with higher expression ( figure 1 ) . interestingly , in all these areas , cxcr4 expression resulted largely higher than its ligand , although all the cxcl12-positive cells express cxcr4 , as well . cxcr4-expressing cells do not belong to specific secreting cell type , being present in gh , prl , or acth - secreting cells , while no expression was observed in human fs cells . however , some cxcr4-expressing cells do not coexpress any hormones and no colocalization of either cxcr4 or cxcl12 was observed in fs cells ; thus it was proposed that cxcl12/cxcr4 system may also label undifferentiated / progenitor cells . notably , this ck - receptor pair was undetectable in human posterior pituitary lobe , contrarily to what was observed in rats . thus , from these data it is evident that , in normal pituitary , cxcl12 is secreted by cell subpopulations that , cooperating with hypothalamic factors ( including cxcl12 itself ) , may contribute to paracrine modulation of pituitary functioning ( figure 2 ) . consequently , alterations of the endocrine regulatory pathways due to upregulation of hypothalamic / pituitary cxcl12/cxcr4 axis might lead to the development of pituitary adenomas [ 127 , 129 ] . the activity of cxcr7 in normal pituitary deserves further investigation ; however its expression in pituitary adenoma tissues [ 130 , 131 ] suggests possible involvement in pituitary function regulation . beside direct cxcr4-dependent activation of erk1/2 , transactivation of tyrosine kinase receptors is currently a relevant mechanism in tumor cell responses . mainly , the transactivation of epidermal growth factor receptor ( egfr ) family members mediates the mitogenic activity of different cks in human cancer . a cross - talk between cxcl12 and egfr and/or her2/neu phosphorylation was demonstrated in breast and ovarian cancer cells through g protein - dependent activation of kinases of the src family [ 132134 ] . moreover , in breast cancer , cxcr4 interacts with the egfr variant , egfr viii , a constitutively active mutant highly expressed in cancer stem cells , to regulate invasion via p38 mapk . cxcr4 signaling is negatively regulated by protein - tyrosine phosphatases ( ptps ) , such as the src homology - containing protein - tyrosine phosphatase 1 ( shp1 ) and the sh2 domain - containing inositol 5-phosphatases ( ship ) , while shp2 , constitutively associated with cxcr4 , potentiates ck signaling [ 137 , 138 ] . these observations are particularly relevant since they highlight possible direct antagonisms between cxcr4 and somatostatin receptors ( sstr ) that are powerful activators of ptps [ 139141 ] . this antagonistic activity could acquire clinical relevance in light of the fact that sstr agonists are the main pharmacological tool available for the treatment of pituitary adenomas [ 142 , 143 ] . the concomitant expression of ligand - receptor pair in the same tumor cells , responsible of autocrine / paracrine activation , is one of the leading causes of clinical aggressive behavior in various cancer types [ 144 , 145 ] . exploiting the coexpression of cks and their receptors , cancer cells and cells of the tumor microenvironment as previously described , this autocrine mechanism is maximally effective for cxcr4 , the most widely expressed ck receptor in human solid and hemopoietic malignancies . the autocrine / paracrine loop of the cxcl12/cxcr4 pair has been deeply investigated in brain tumors in both in vitro and in vivo models : cxcl12 stimulates proliferation and migration of glioblastoma cells and xenografted tumors inducing erk1/2 and akt phosphorylation [ 147149 ] . the mitogenic activity mediated by cxcl12-cxcr4 was also reported in meningioma , in which cxcr4 activation increased dna synthesis through activation of erk1/2 in primary cultures and its expression level significantly correlated with ki-67 proliferation index of the original tumor tissue [ 150 , 151 ] , while cxcr7 was mainly localized in tumor endothelia . similar cxcr4 tumor - promoting effects were observed in breast carcinoma , suggesting cxcl12 as possible autocrine / paracrine growth factor . interestingly , in breast cancer cells the synthesis and release of cxcl12 is under the control of 17-estradiol contributing to its proliferative effects and mediating , via a src - dependent mechanism , egfr transactivation [ 133 , 154 ] . furthermore , cxcl12 is also indirectly implicated in tumor pathogenesis , acting as chemoattractant for cxcr4-positive cells , directing tumor cell migration [ 155157 ] and controlling invasive and metastatic properties of cxcr4-expressing cancer cells to distant organs [ 158 , 159 ] . the invasive behavior of cancer cells might indirectly depend on locally released cxcl2 that , via an autocrine mechanism , binds to cxcr4 impairing chemotaxis towards cxcl12-producing target organs and metastatic spread . in addition , normal cells forming tumor stroma ( i.e. , macrophages , lymphocytes , fibroblasts , and endothelial cells ) concur to cancer development and progression through cxcl12 secretion . concentration gradient directs cancer cell motility in several tumors [ 10 , 161 ] , including aggressive solid neoplasms ( breast , colon , brain ovarian , prostate , renal cell and oral squamous cell carcinomas , and melanoma ) . importantly , as observed in a rat mammary adenocarcinoma cell line overexpressing both receptors , cxcr4 and cxcr7 play opposing roles in breast cancer metastasis : cxcr4 mediates cancer cell invasion allowing cells to follow the cxcl12 gradient generated by metastatic targets whereas cxcr7 favors tumor growth increasing angiogenesis but impairs cell migration scavenging the chemokine . raf / mek / erk and pi3k / akt pathway dysregulation is a common alteration responsible of tumor initiation and progression . while the pathways are classically activated by growth factors , cross - talks and transactivation mechanisms with neuropeptide - cytokine - ck / gpcrs have been increasingly recognized . this cross - talk activates erk1/2 , which is directly responsible for cell growth and differentiation , depending on the cellular context and represents one of the major proliferative pathways in cancer [ 168 , 169 ] . the overexpression or constitutive activation of receptors for growth factors , cytokines , and cks potentiates the activation of ras / raf / mek / erk pathway also in pituitary adenoma . mapk phosphorylation is relevant in different pituitary cell types such as att20 cells , where it regulates crh - induced pomc transcription , gonadotrophs for gnrh signaling , gh - secreting cells for ghrh - dependent cyclin d1 expression , and gh4c1 somatotroph cell line in which the gsp oncogene impairs ras / erk1/2-dependent prl gene regulation . pi3k / akt / mtor pathway , activated by a variety of growth factors and hormones , when dysregulated , leads to aberrant growth of pituitary adenoma cells . akt is overexpressed and hyperphosphorylated in nfpa , in a mouse model of tsh - oma , and in gh3 cells in which the inhibition of pi3k / akt signaling by octreotide increases the expression of the tumor suppressor gene zac1 [ 176 , 177 ] . similarly to cell proliferation , survival mechanisms also sustain pituitary development and tumorigenesis : in particular the balance of pro- and antiapoptotic factors , physiologically contributing to normal pituitary cell plasticity , when unbalanced , favors pituitary cell transformation . for example , in pituitary adenomas , antiapoptotic mediators , such as the bcl-2 protein family , are upregulated , while fas , a major apoptotic factor in different cell types , activates apoptosis in both normal rat lactotrophs and somatotrophs and in pituitary adenoma cell lines . several pituitary - related genes may exert a role in apoptosis of secretory pituitary cells , as the developmental factor pitx2 and the transcription factor pit-1 [ 183 , 184 ] , although their mechanisms are not yet fully understood . recent studies , however , highlighted that , when considering the complexity of regulatory pathways involved in pituitary cell survival and proliferation , it should take into account not only apoptosis but also senescence , an alternative process acting during tumor - suppressive cell fate . importantly , senescence is gaining biological significance also in pituitary adenomas , whose typical benign nature could result from protective antiproliferative mechanisms . several transformation events ( e.g. , dna damage , loss of tumor - suppressor gene , oncogene activation , and growth factor overexpression ) induce preventive cellular senescence , characterized by cell cycle exit and subsequent irreversible proliferation arrest . thus , pituitary tumors may be more prone to activate senescence - associated pathways , maintaining their benign behavior , preventing malignant transformation , and regulating their development . interestingly , il-6 has been shown to participate in oncogene - induced senescence in pituitary gp130 overexpressing tumor cells . moreover , angiogenic and apoptotic processes cooperate in determining tumor aggressiveness , and this regulation might also be involved in the pathogenesis of pituitary transformation . the pituitary gland is highly vascularized but , unlike other solid malignancies , conflicting results are available on angiogenic factors associated with pituitary adenoma progression and recurrence [ 186 , 187 ] . vegf was proposed as pituitary proangiogenic factor and possible therapeutic target , and the hypoxia - inducible factor- ( hif- ) , a key molecule in hypoxic pathways triggering vessel formation , was detected in pituitary tumor tissues [ 189 , 190 ] and may favor hemorrhage in pituitary macroadenomas . cks , particularly cxcl12 signaling via cxcr4 and cxcr7 , represent candidate mediators of the above described intracellular pathways , determining proliferative , antiapoptotic , and angiogenic signals , thus possibly concurring to pituitary tumor development and aggressiveness . pituitary adenomas are common intracranial tumors of the adenohypophysis causing serious morbidity , due to excessive hormonal secretion , mass effects , and local invasion of surrounding structures . at present , the understanding of biological and molecular pathogenesis and mechanisms of progression of these tumors is largely incomplete ( for review see [ 14 , 192 , 193 ] ) . emerging evidence reports that multiple factors might contribute to pituitary tumorigenesis , such as frequently altered gene expression , genetic ( aryl hydrocarbon receptor interacting protein , aip ; multiple endocrine neoplasia syndrome type 1 , men1 ; guanine nucleotide - activating alpha subunit , gnas , ) , and epigenetic ( cyclin - dependent kinase inhibitor 2a , cdkn2a , or p16 ; fgfr2/melanoma associated antigen -mage-3 pathway ) mutations , and abnormal micrornas . the improvement of this knowledge is even more helpful taking into account the peculiar properties of pituitary adenomas as compared to other malignancies : they commonly grow slowly but with local invasive behavior and occasionally develop into high aggressive tumors . this often prevents the efficacy of surgical and systemic medical treatments , the latter hampered by the lack of definite mechanisms underlying pituitary cell transformation and potential therapeutic targets . however , few studies addressed the potential role of components of this peptide family in regulating human pituitary tumorigenesis . cxcl8 mrna was identified in a small percentage of anterior pituitary adenomas [ 199 , 200 ] , altogether with the expression of cxcr2 , the cxcl8 receptor that also binds other cxc cks ( cxcl1 , cxcl7 ) , confirming the potentiality of autocrine stimulation in pituitary adenomas . indeed , a consistent release of cxcl8 was observed in primary cultures derived from human somatotroph adenomas , induced by stimulation with interleukin-1 and inhibited by gh releasing hormone ( ghrh ) . thus , a further assessment of the possible role of this ck in the pathogenesis of pituitary tumors is required , likely being based on cxcl8 ability to recruit active neutrophil within the adenoma , influencing the inflammatory response or acting as mitogen for normal and transformed cells . however , the majority of studies focused on the analysis of cxcl12 and cxcr4 expression in human neoplastic pituitary tissues and their role in adenoma cell proliferation [ 128 , 129 , 203205 ] . cxcr4 mrna is expressed in almost all gh - secreting pituitary adenomas and in the great majority of nfpas , whilst cxcl12 was identified in about 2/3 of these tumors . notably , most cxcl12-positive cells also express cxcr4 strongly suggesting an autocrine / paracrine regulation of tumor cell proliferation [ 129 , 205 ] . this hypothesis was further confirmed measuring the in vitro basal secretion of cxcl12 by human pituitary adenoma primary cultures resulting in an autocrine constitutive stimulation of dna synthesis ( figure 2 ) and , indirectly , by the absence of cxcr4 activating mutations in gh - secreting and nfpa able to sustain adenoma cell proliferation . this observation was confirmed by a high percentage of different types of secreting pituitary adenomas showing expression of both cxcl12 and cxcr4 [ 129 , 203 , 204 ] . finally , the evaluation of cxcr4 and cxcl12 expression in invasive and noninvasive pituitary adenoma specimens , by flow cytometry and immunohistochemical staining , demonstrated that the percentage of cxcr4- and cxcl12-positive cells was significantly higher in invasive pituitary adenomas . thus , the correlation of cxcr4 and cxcl12 expression levels and tumor invasiveness was proposed to be exploited as potential early diagnostic biomarkers , one of the major challenges in diagnosis and treatment of invasive tumors . among the mechanisms that occur in pituitary tumorigenesis interestingly , while controversial findings on the role of vegf were reported [ 207209 ] , cxcl12 has been proposed as a better defined proangiogenic and proliferative factor in pituitary adenomas . in fact , cxcl12 and cxcr4 are concomitantly upregulated in hypoxic foci within pituitary tumor tissues , and one of the main cxcl12 effects in pituitary adenomas is to mobilize cd34- ( and cxcr4- ) expressing endothelial progenitors and promote their homing in ischemic foci activating the proangiogenic program . moreover , in gh3 rat pituitary adenoma cells , hypoxia - activated cxcl12-cxcr4 signaling interacts with the endocrine pathways resulting in upregulation of gh synthesis and secretion and cell proliferation . thus , in pathological conditions ( i.e. , hypoxia ) , on one hand , increased cxcl12 and cxcr4 expression and signalling may promote neoangiogenesis by recruiting endothelial progenitor cells and/or inducing proliferation of endothelial cells and , on the other , directly favouring hormone hypersecretion and pituitary cell proliferation . interestingly , gh4c1 rat pituitary adenoma cell line expresses cxcr4 but not cxcl12 , thus it was proposed as a suitable model to characterize the molecular pathways regulated by this receptor in pituitary adenomas , without the interference of the endogenously released ck [ 127 , 211 , 212 ] . in these cells cxcl12 exerts a powerful secretagogue and mitogenic activity , as well as promotes cell migration . interestingly , these effects are induced by different and independent intracellular mechanisms , although all of them were ptx - dependent . gh secretion is a ca - dependent event , in which increased ion concentration resulted from ip3-mediated ca release from intracellular stores . conversely , gh4c1 proliferation is induced by cxcl12 through the activation of erk1/2 through the classical mek - dependent pathway and via the activation of the cytosolic ca - dependent tyrosine kinase , pyk2 , that , in turn , activates the large - conductance ca - activated k channels ( bkca ) [ 95 , 127 , 211 , 212 ] . similarly , , the role of cxcl12/cxcr4 as potential pharmacological target in acromegalic patients has been scantily investigated . it was shown that a synthetic antagonist of cxcr4 , d - arg3fc131 , is able to inhibit the growth of gh3 tumor cells and trigger apoptosis both in vitro and in mice xenografts . similar results were obtained using phidianidine a , an indole alkaloid isolated from the marine opisthobranch mollusc phidiana militaris , which reduced gh4c1 proliferation , migration , and erk1/2 phosphorylation . cxcr7 , the second cxcl12 receptor , was reported to be expressed in the att20 mouse corticotroph pituitary adenoma cell line , but the characterization of its possible role in pituitary adenoma development or progression will require further evaluation . studies on human pituitary adenoma cells derived from postsurgical specimens are limited , mostly due to their low proliferative activity in vitro . however , such generalized cxcr4 and cxcl12 overexpression in human pituitary adenomas , as compared to normal pituitary , strongly suggests that , in conditions of deregulation , this receptor system could be a relevant factor for pituitary adenoma development and/or progression . however , cxcl12 effects of on cell proliferation were directly evaluated in vitro on a small number of primary cultures of adenoma cell derived from ghoma , nfpa , and acth - secreting adenomas . to avoid interference with the cxcl12 released from fibroblasts a specific protocol to obtain adenoma cell cultures highly purified was used . cxcl12 induced a statistically significant increase in dna synthesis in the majority ( 65% ) of the adenoma tested . interestingly , in few adenomas , the blockade of cxcr4 with amd3100 , a known cxcr4 antagonist , caused , beside the abolishment of cxcl12-mediated increase in cell proliferation , also a reduction of basal dna synthesis . measuring cxcl12 levels in the culture medium , it was shown that these tumors retained in vitro a significant basal secretion of cxcl12 causing a constitutive , autocrine stimulation of dna synthesis . thus , these data suggest that pituitary adenoma cells over - express cxcl12 and cxcr4 as compared to normal tissue and that an autocrine activation of this pathway ( figure 2 ) , actually , occurs in vivo . overall , these observations imply that cxcl12/cxcr4 axis might play an important biological role in pituitary adenoma as potential growth and angiogenic factor for pituitary cells . the increased cxcr4 activation may result from either endocrine ( increased cxcl12 levels may reach pituitary through the blood stream or being released in the portal pituitary system from hypothalamus ) and/or autocrine / paracrine mechanisms ( figure 2 ) . importantly , the latter mechanism seems to be active mainly in pituitary tumors rather than in normal gland , where most of the cxcr4-expressing normal cells do not express cxcl12 . chemokines are key factors in cns physiology and pathology , being relevant mediators in cancer development . the cxcl12/cxcr4-r7 signaling pathway plays a unique role in the regulation of a variety of cell types , including embryonic and cancer cells . in particular , deregulation of this chemokinergic system is strictly related to tumor initiation and progression , and the balance of its activity within the tumor microenvironment is highly complex phenomenon . multiple factors cooperate to pituitary tumor pathogenesis , but , although to date not explored in depth , a pivotal role of ck and their receptors seems to be important . several mechanisms by which cxcl12/cxcr4 modulates pituitary function and promotes adenoma cell proliferation and their target as potential therapeutic approach have been suggested . cxcl12 is overexpressed in pituitary cells where it can directly influence adenoma formation , through autocrine mechanisms resulting in constitutive cxcr4 activation . this pathway grants pituitary cells with a proliferative advantage that triggers clonal expansion of transformed cells and sustains tumor cell survival . increasing knowledge about pituitary cell origin and development might provide significant insights into deregulated pathways in pituitary tumorigenesis . finally , cxcr4 is an easily druggable target and the characterization of its role in pituitary adenomas could pave the way for novel pharmacological approaches , especially for those adenoma subtypes , ( i.e. , tsh and acth secreting tumors , as well as nfpa ) still waiting for efficacious drugs .

chemokines are chemotactic regulators of immune surveillance in physiological and pathological conditions such as inflammation , infection , and cancer . several chemokines and cognate receptors are constitutively expressed in the central nervous system , not only in glial and endothelial cells but also in neurons , controlling neurogenesis , neurite outgrowth , and axonal guidance during development . in particular , the chemokine cxcl12 and its receptors , cxcr4 and cxcr7 , form a functional network that controls plasticity in different brain areas , influencing neurotransmission , neuromodulation , and cell migration , and the dysregulation of this chemokinergic axis is involved in several neurodegenerative , neuroinflammatory , and malignant diseases . cxcr4 primarily mediates the transduction of proliferative signals , while cxcr7 seems to be mainly responsible for scavenging cxcl12 . importantly , the multiple intracellular signalling generated by cxcl12 interaction with its receptors influences hypothalamic modulation of neuroendocrine functions , although a direct modulation of pituitary functioning via autocrine / paracrine mechanisms was also reported . both cxcl12 and cxcr4 are constitutively overexpressed in pituitary adenomas and their signalling induces cell survival and proliferation , as well as hormonal hypersecretion . in this review we focus on the physiological and pathological functions of immune - related cyto- and chemokines , mainly focusing on the cxcl12/cxcr4 - 7 axis , and their role in pituitary tumorigenesis . accordingly , we discuss the potential targeting of cxcr4 as novel pharmacological approach for pituitary adenomas .

1. Background 2. Chemokine Classification and Receptor Interactions 3. Physiological Functions of CKs: Focus on the CXCL12/CXCR4-R7 Axis in the CNS 4. Chemokine Functions in Normal Pituitary 5. CXCL12/CXCR4-R7 in Cancer Development and Progression: Autocrine/Paracrine Loops 6. A Network Map of Proliferative Signaling in Pituitary Tumor Development 7. Chemokines in Pituitary Tumorigenesis 8. Conclusions

cks are also critical mediators of several physiological mechanisms such as wound - healing and tissue homeostasis [ 3 , 5 ] ; moreover , cks are expressed in the central nervous system ( cns ) [ 6 , 7 ] where they not only act as mediators of development , intercellular communication , and inflammatory processes but also function as neurotransmitters or neuromodulators , mainly involved in neuroendocrine regulations . evidence for autocrine / paracrine regulatory mechanisms in different normal and cancer cell types , driven by chemokine / receptors interaction on the same or a nearby cell , supports the potential role of cks in the control of physiological or tumoral endocrine functions . in particular , the chemokine ( c - x - c motif ) ligand 12 ( cxcl12 ) and its receptors , cxcr4 and cxcr7 , have been involved in cancer cell proliferation , migration , and invasion [ 1113 ] . they are classified by size ( microadenoma , < 10 mm or macroadenoma , > 10 mm ) and on the basis of their ability to produce hormones , as secreting or functioning tumors ( about 50% of adenomas ) or as clinically nonfunctioning pituitary adenomas ( nfpa ) that do not release hormones or , more often , secrete clinically nonrelevant ( i.e. this review will focus on the diverse role of cxc chemokines and their receptors in normal pituitary cell functions and pituitary tumor development and progression , summarize recent progress in cxcr7 functions , and discuss the present issues and future perspectives . cks may also bind a small group of so - called atypical chemokine receptors ( ackrs ) , which are unable to initiate downstream conventional g - protein - dependent signaling , resulting , from a functional point of view , in the inability to induce directional cell migration . indeed , recent evidence supports the ability of ackrs to transport , internalize , and degrade cks , leading to the formation of ck gradients in normal and cancer tissues , responsible for the functional modulation of their signaling . common features shared by all cks are pleiotropism , promiscuity , and redundancy , with a single ck able to bind several receptors , whereas multiple cks bind the same receptor resulting in the same functional outcome . on the other hand , ck receptor activation of g subunits results in the activation of pi3k leading , through the phosphoinositide - dependent kinase 1 and 2 ( pdk1 - 2 ) , to akt phosphorylation and subsequent activation of its downstream signal proteins such as glycogen synthase kinase 3 ( gsk3 ) , mammalian target of rapamycin ( mtor ) , and fak , which control migration in different types of normal and tumor cells . furthermore , cks directly activate specialized effector lymphocytes during the different steps of immune response , for example , cxcl8 ( formerly named il8 ) recruits neutrophils , basophils , and eosinophils expressing its receptors , cxcr1 and cxcr2 . cks also play a key role in embryogenesis , organogenesis , angiogenesis , and germ cell migration , especially during neural development . the constitutive expression of cks and their receptors in adult normal brain was initially identified in the immune - like competent cell populations such as microglia and astrocytes . the subsequent detection of their expression in neurons [ 3234 ] broadened ck role as neuromodulators / neurotransmitters in neurological processes such as thermoregulation , pain perception , and stress conditions , as well as in pituitary functions . focusing on the cxcl12/cxcr4-r7 network , it exerts a variety of functions in cns development as well as in mature brain . cxcl12 directs the migration of embryonic and adult stem cells in the developing central and peripheral nervous system [ 35 , 36 ] , controlling the formation of cerebellum , cerebral cortex , hippocampus , and dorsal root and sympathetic ganglia [ 25 , 37 , 38 ] . postnatally , cxcr4 expression , while downregulated in many brain areas , persists in the hypothalamus where it modulates the hypothalamic - pituitary system and the hypothalamic - pituitary - gonadal axis , in particular , cooperating to the regulation of neuroendocrine and reproductive systems [ 3941 ] . cxcl12/cxcr4 regulation of stem cell positioning and migration persists in adults , in the neurogenic niches of brain and in the bone marrow , where hematopoietic progenitors cells are retained by the interaction between ligand and receptor that also promotes their survival and proliferation . cxcr7 , the second cxcl12 receptor , has a 10-fold higher binding affinity than cxcr4 but also binds cxcl11 ( formerly known as ifn - inducible t cell chemoattractant , i - tac , ) , which , in turn , interacts with cxcr3 . in particular , cxcr7 activates akt , map kinase ( mapk ) , and jak / stat3 cascades , either by direct modulation , through a -arrestin - dependent pathway [ 20 , 65 ] , or after heterodimerization with cxcr4 [ 59 , 6669 ] . in addition , since cxcr7 and cxcr4 mutant mice displayed opposite defects in interneuron motility and positioning , cxcr4 and cxcr7 were proposed to have distinct roles and signal transduction to regulate interneuron movement . the role of cxcl12 and cxcr4 in stemness maintenance has gained much attention also in the neuroendocrinology field due to the proposed role of stem cells in pituitary plasticity [ 84 , 85 ] . both cxcl12 and cxcr4 are expressed in different anterior pituitary cell subtypes , as well as in nonhormonal cell types [ 8688 ] . , cxcl12 ) , possibly occurring also in pituitary adenomas , may act as chemoattractant to recruit fibroblasts , endothelial , mesenchymal , and inflammatory cells to the tumor , via cxcr4 . , egf and vip ) , neurotransmitters , cytokines ( il-1 , il-6 ) , and cks occur also in pituitary regulation , differently from the classical hypothalamic input and feedback signals from the periphery [ 9597 ] . egf expression has been observed at all stages of pituitary development and in the adult pituitary , and the egfr pathway contributes to pituitary physiology and tumorigenesis . il-1 receptors were detected in pituitary cells , and their activation inhibits prolactin ( prl ) secretion from dispersed rat pituitary cells through the regulation of ac and plc activities , and ca fluxes [ 100103 ] . il-6 and its receptors are also expressed in the pituitary gland [ 104 , 105 ] where their interaction regulates apoptosis and proliferation of endocrine cells in vitro . il-6 is mainly produced by the fs cells and activates a paracrine loop on the hormone - secreting cells [ 107 , 108 ] regulating acth , prl , lh , and gh secretion [ 110112 ] via the modulation of ac and plc activities . in response to stressful stimuli , this ck is released in the median eminence to reach its receptor ( cxcr2 ) expressed in pituitary cells and induce the release of prl and gh and the inhibition of lh and fsh secretion . importantly , cxcr3 , the receptor for cxcl10 , was shown to be expressed in corticotrophs , suggesting a possible autocrine / paracrine effect of cxcl10 , released from fs cells , on acth - producing cells . cxcl12/cxcr4 is the major regulatory axis not only connecting the immune and nervous systems , but also playing a role in neuroimmune regulation of the anterior pituitary physiological functions . the expression pattern of this chemokine and its receptor in the rat hypothalamo - neurohypophyseal system was further investigated : they colocalize within avp - expressing neurons in both supraoptic ( son ) and paraventricular ( pvn ) nucleus as well as in dense core vesicles of avp - positive nerve terminals in the posterior pituitary , showing a similar distribution . since avp controls body fluid homeostasis , the interaction between cxcl12 and avp was studied in avp - deficient brattleboro rats that show low expression of both cxcl12 and cxcr4 , correlated with avp protein expression level in son , pvn , and posterior pituitary . however , cxcl12 and cxcr4 are also coexpressed in rat pituitary cells and a further autocrine / paracrine regulation of pituitary functioning was hypothesized . interestingly , in all these areas , cxcr4 expression resulted largely higher than its ligand , although all the cxcl12-positive cells express cxcr4 , as well . thus , from these data it is evident that , in normal pituitary , cxcl12 is secreted by cell subpopulations that , cooperating with hypothalamic factors ( including cxcl12 itself ) , may contribute to paracrine modulation of pituitary functioning ( figure 2 ) . cxcr4 signaling is negatively regulated by protein - tyrosine phosphatases ( ptps ) , such as the src homology - containing protein - tyrosine phosphatase 1 ( shp1 ) and the sh2 domain - containing inositol 5-phosphatases ( ship ) , while shp2 , constitutively associated with cxcr4 , potentiates ck signaling [ 137 , 138 ] . the concomitant expression of ligand - receptor pair in the same tumor cells , responsible of autocrine / paracrine activation , is one of the leading causes of clinical aggressive behavior in various cancer types [ 144 , 145 ] . exploiting the coexpression of cks and their receptors , cancer cells and cells of the tumor microenvironment as previously described , this autocrine mechanism is maximally effective for cxcr4 , the most widely expressed ck receptor in human solid and hemopoietic malignancies . the mitogenic activity mediated by cxcl12-cxcr4 was also reported in meningioma , in which cxcr4 activation increased dna synthesis through activation of erk1/2 in primary cultures and its expression level significantly correlated with ki-67 proliferation index of the original tumor tissue [ 150 , 151 ] , while cxcr7 was mainly localized in tumor endothelia . , macrophages , lymphocytes , fibroblasts , and endothelial cells ) concur to cancer development and progression through cxcl12 secretion . importantly , as observed in a rat mammary adenocarcinoma cell line overexpressing both receptors , cxcr4 and cxcr7 play opposing roles in breast cancer metastasis : cxcr4 mediates cancer cell invasion allowing cells to follow the cxcl12 gradient generated by metastatic targets whereas cxcr7 favors tumor growth increasing angiogenesis but impairs cell migration scavenging the chemokine . the overexpression or constitutive activation of receptors for growth factors , cytokines , and cks potentiates the activation of ras / raf / mek / erk pathway also in pituitary adenoma . mapk phosphorylation is relevant in different pituitary cell types such as att20 cells , where it regulates crh - induced pomc transcription , gonadotrophs for gnrh signaling , gh - secreting cells for ghrh - dependent cyclin d1 expression , and gh4c1 somatotroph cell line in which the gsp oncogene impairs ras / erk1/2-dependent prl gene regulation . for example , in pituitary adenomas , antiapoptotic mediators , such as the bcl-2 protein family , are upregulated , while fas , a major apoptotic factor in different cell types , activates apoptosis in both normal rat lactotrophs and somatotrophs and in pituitary adenoma cell lines . several pituitary - related genes may exert a role in apoptosis of secretory pituitary cells , as the developmental factor pitx2 and the transcription factor pit-1 [ 183 , 184 ] , although their mechanisms are not yet fully understood . recent studies , however , highlighted that , when considering the complexity of regulatory pathways involved in pituitary cell survival and proliferation , it should take into account not only apoptosis but also senescence , an alternative process acting during tumor - suppressive cell fate . importantly , senescence is gaining biological significance also in pituitary adenomas , whose typical benign nature could result from protective antiproliferative mechanisms . moreover , angiogenic and apoptotic processes cooperate in determining tumor aggressiveness , and this regulation might also be involved in the pathogenesis of pituitary transformation . vegf was proposed as pituitary proangiogenic factor and possible therapeutic target , and the hypoxia - inducible factor- ( hif- ) , a key molecule in hypoxic pathways triggering vessel formation , was detected in pituitary tumor tissues [ 189 , 190 ] and may favor hemorrhage in pituitary macroadenomas . cks , particularly cxcl12 signaling via cxcr4 and cxcr7 , represent candidate mediators of the above described intracellular pathways , determining proliferative , antiapoptotic , and angiogenic signals , thus possibly concurring to pituitary tumor development and aggressiveness . emerging evidence reports that multiple factors might contribute to pituitary tumorigenesis , such as frequently altered gene expression , genetic ( aryl hydrocarbon receptor interacting protein , aip ; multiple endocrine neoplasia syndrome type 1 , men1 ; guanine nucleotide - activating alpha subunit , gnas , ) , and epigenetic ( cyclin - dependent kinase inhibitor 2a , cdkn2a , or p16 ; fgfr2/melanoma associated antigen -mage-3 pathway ) mutations , and abnormal micrornas . the improvement of this knowledge is even more helpful taking into account the peculiar properties of pituitary adenomas as compared to other malignancies : they commonly grow slowly but with local invasive behavior and occasionally develop into high aggressive tumors . however , few studies addressed the potential role of components of this peptide family in regulating human pituitary tumorigenesis . cxcl8 mrna was identified in a small percentage of anterior pituitary adenomas [ 199 , 200 ] , altogether with the expression of cxcr2 , the cxcl8 receptor that also binds other cxc cks ( cxcl1 , cxcl7 ) , confirming the potentiality of autocrine stimulation in pituitary adenomas . thus , a further assessment of the possible role of this ck in the pathogenesis of pituitary tumors is required , likely being based on cxcl8 ability to recruit active neutrophil within the adenoma , influencing the inflammatory response or acting as mitogen for normal and transformed cells . however , the majority of studies focused on the analysis of cxcl12 and cxcr4 expression in human neoplastic pituitary tissues and their role in adenoma cell proliferation [ 128 , 129 , 203205 ] . cxcr4 mrna is expressed in almost all gh - secreting pituitary adenomas and in the great majority of nfpas , whilst cxcl12 was identified in about 2/3 of these tumors . this observation was confirmed by a high percentage of different types of secreting pituitary adenomas showing expression of both cxcl12 and cxcr4 [ 129 , 203 , 204 ] . finally , the evaluation of cxcr4 and cxcl12 expression in invasive and noninvasive pituitary adenoma specimens , by flow cytometry and immunohistochemical staining , demonstrated that the percentage of cxcr4- and cxcl12-positive cells was significantly higher in invasive pituitary adenomas . thus , the correlation of cxcr4 and cxcl12 expression levels and tumor invasiveness was proposed to be exploited as potential early diagnostic biomarkers , one of the major challenges in diagnosis and treatment of invasive tumors . among the mechanisms that occur in pituitary tumorigenesis interestingly , while controversial findings on the role of vegf were reported [ 207209 ] , cxcl12 has been proposed as a better defined proangiogenic and proliferative factor in pituitary adenomas . in fact , cxcl12 and cxcr4 are concomitantly upregulated in hypoxic foci within pituitary tumor tissues , and one of the main cxcl12 effects in pituitary adenomas is to mobilize cd34- ( and cxcr4- ) expressing endothelial progenitors and promote their homing in ischemic foci activating the proangiogenic program . , hypoxia ) , on one hand , increased cxcl12 and cxcr4 expression and signalling may promote neoangiogenesis by recruiting endothelial progenitor cells and/or inducing proliferation of endothelial cells and , on the other , directly favouring hormone hypersecretion and pituitary cell proliferation . in these cells cxcl12 exerts a powerful secretagogue and mitogenic activity , as well as promotes cell migration . cxcr7 , the second cxcl12 receptor , was reported to be expressed in the att20 mouse corticotroph pituitary adenoma cell line , but the characterization of its possible role in pituitary adenoma development or progression will require further evaluation . however , such generalized cxcr4 and cxcl12 overexpression in human pituitary adenomas , as compared to normal pituitary , strongly suggests that , in conditions of deregulation , this receptor system could be a relevant factor for pituitary adenoma development and/or progression . thus , these data suggest that pituitary adenoma cells over - express cxcl12 and cxcr4 as compared to normal tissue and that an autocrine activation of this pathway ( figure 2 ) , actually , occurs in vivo . overall , these observations imply that cxcl12/cxcr4 axis might play an important biological role in pituitary adenoma as potential growth and angiogenic factor for pituitary cells . the increased cxcr4 activation may result from either endocrine ( increased cxcl12 levels may reach pituitary through the blood stream or being released in the portal pituitary system from hypothalamus ) and/or autocrine / paracrine mechanisms ( figure 2 ) . importantly , the latter mechanism seems to be active mainly in pituitary tumors rather than in normal gland , where most of the cxcr4-expressing normal cells do not express cxcl12 . the cxcl12/cxcr4-r7 signaling pathway plays a unique role in the regulation of a variety of cell types , including embryonic and cancer cells . in particular , deregulation of this chemokinergic system is strictly related to tumor initiation and progression , and the balance of its activity within the tumor microenvironment is highly complex phenomenon . multiple factors cooperate to pituitary tumor pathogenesis , but , although to date not explored in depth , a pivotal role of ck and their receptors seems to be important . finally , cxcr4 is an easily druggable target and the characterization of its role in pituitary adenomas could pave the way for novel pharmacological approaches , especially for those adenoma subtypes , ( i.e.

in recent years , various uses of wax and fat microspheres in the pharmaceutical field have come into forefront , involving the microspheres technology . the goal of any drug delivery system is to provide a therapeutic amount of drug(s ) to the proper site in the body in order to promptly achieve and thereby to maintain the desired drug concentrations during treatment . this idealized objective can be achieved by targeting the drugs to a specific organ or tissue with the help of controlling the release rate of the drug during the transit time in gastrointestinal tract . poorly water - soluble drugs , which are lipophilic in nature mix , easily with fat and show good absorption rate . among the reported conventional methods different strategies have been developed in recent years to design different types of wax microspheres loaded with hydrophilic and lipophilic drugs using toxic solvents . the use of such solvents during formulation is of environmental concern and also faces challenge to human safety . to overcome these problems , in the present investigation , water is used to prepare wax microspheres by meltable dispersed emulsified cooling - induced solidification method . furthermore , the process was optimized to produce microspheres to give better yield with spherical geometry and predictable dissolution pattern . cetyl alcohol ( ca ) , used in the current study , has good pharmaceutical and biological properties . ca is hard , but oily to the touch , and is devoid of taste or smell , making it very useful as an ingredient in cosmetics , as a pharmaceutical excipient . it is an innocuous material generally regarded as essentially nontoxic and nonirritant , biodegradable , biocompatible , nonimmunogenic , gastroresistant , of high carrier capacity , and having controlled release of drug , low production costs , reproducible properties , and good shelf life . but the hypersensitivity reported in some cases may be due to impurities in commercial grades of ca . in pharmaceutical formulations , it is used in the preparation of suppositories , delayed release solid dosage forms , emulsions , lotions , creams , and ointment . regulatory status : it is included in the fda as inactive ingredient and in nonparenteral medicines licensed in the uk . i m is a nonsteroidal , anti - inflammatory agent with antipyretic , analgesic properties and is an indole derivative designated chemically as 1-(p - chlorobenzoyl)-5-methoxy-2-methyl-1h - indole-3-acetic acid . it is lipid - soluble , practically insoluble in water and sparingly soluble in alcohol . i m has a pka of 4.5 and is stable in neutral or slightly acidic media and decomposes in strong alkali . the suspension has a ph of 4.05.0 and it has a melting point between 155c and 161c and has molecular weight of 357.8 . i m is widely used in the treatment of active stages of moderate - to - severe stages of rheumatoid arthritis . due to its narrow therapeutic index , the frequency of adverse effects is dosing related . considering the long therapeutic regimen of osteoarthritis therapy , the administration of i m may induce adverse side effects on gastrointestinal tract ( git ) as well as central nervous system ( cns ) , renal and cardiac systems . the occurrence of these adverse effects can be reduced by the use of controlled release formulations . oral conventional dosage forms are administered 2 - 3 times a day to maintain adequate and effective therapeutic concentration in blood ; however , it fails to protect the patients against morning stiffness . development of controlled release formulation of i m has several advantages over the other conventional dosage forms , such as reduction in occurrence of high initial peak plasma concentrations , protection against morning stiffness , prolonged duration of action , improved bioavailability , patient compliance and reduction in adverse effects . the side effects could be lowered by controlling the drug release and by adjusting the absorption rate . previous experimental results have demonstrated that the waxes are biocompatible , nonimmunogenic material used for the entrapment of drug and its controlled drug release in the intestinal tract . delivering the drug in the intestinal environment from fatty ca microspheres could be manipulated by suitable coating techniques . the chief characteristics of enteric coating are their impermeability to gastric juice , but susceptibility to intestinal juice [ 13 , 14 ] . desired plasma levels can be achieved without the risk of side effects using once - a - day dose of controlled release preparation . these findings suggested that the kinetic control is an effective route for preventing the toxicity of i m . the aim of the present study was to formulate , characterize , and study the in vitro release of i m from microspheres and compare with commercially available oral formulation microcid sr ( 75 mg capsule ) furthermore , to investigate the pharmacokinetics and bioavailability of two different oral i m formulation ( optimized microsphere formulation and microcid sr 75 mg capsule ) following a single dosing in healthy albino sheeps in order to prove the bioequivalence between the preparations . indomethacin ( i m ) and mefanamic acid ( ma ) , the internal standard , were kindly donated by micro labs ( bangalore , india ) . cetyl alcohol ( ca - melting point 4951c ) , tween 80 , and all other chemicals and solvents used were of analytical grade and purchased from ranbaxy fine chemicals ( new delhi , india ) . commercially available oral capsule formulation ( microcid sr 75 mg , micro labs ltd . , india ) was used for the present study . 9 gm of ca was melted in a china dish kept on water bath . to the melted wax mixture , i m ( 3 gm ) previously passed through sieve no . 100 was dispersed in melted wax mass and stirred to obtain a homogeneous mixture . the resultant mixture was then poured into 150 ml of phthalate buffer solution ( ph 4.5 ) , previously heated to a temperature higher than melting point of ca ( > 50c ) . the surfactant , tween 80 ( 0.3% w / w ) , was added to the above mixture and stirred mechanically at 900 rpm using a stirrer ( rq-127a , remi , india ) . the mixture was stirred continuously above the melting point of ca at 900 rpm for 5 min . the temperature of the reaction mixture was cooled rapidly and brought down to 10c by the addition of cold water . the resultant solid spheres were collected by filtration and washed with water to remove surfactant residue . air - drying was carried out at room temperature for 48 h to give discrete , solid , and free flowing microspheres . a total of five formulations were prepared by varying the ca to drug ratios ( table 1 ) . tap density of the prepared ca microspheres was determined using tap density tester , and percentage carr 's index ( % i ) was calculated using the formula : ( 1)carr 's index(% i ) = [ ( tapped densitybulk density)tapped density]100 . angle of repose ( h ) was assessed to know the flow ability of ca microspheres , by a fixed funnel method : ( 2)tan()=heightradius . scanning electron microscope ( sem ) photomicrographs were recorded using joel - lv-5600 sem , usa . the obtained images were processed by image analysis software to characterize each individual microsphere by mean feret diameter ( fd ) ( average of 180 caliper measurements with an angle of rotation of 1 ) , aspect ratio ( ar ) ( ratio of longest feret diameter and its longest perpendicular diameter ) , and two - dimensional shape factor ( er ) by the following equation : ( 3)er=2rpm ( bl)2 , where r is the radius , pm is the perimeter , l is length ( longest feret diameter ) , and b is width ( longest perpendicular diameter to the longest feret diameter ) of the microspheres . all dynamic dsc studies were carried out on dupont thermal analyzer with 2010 dsc module . calorimetric measurements were made with the help of an empty cell ( high purity alpha alumina discs of dupont company ) as the reference . ftir spectra of pure drug , empty microspheres , and drug - loaded microspheres were obtained using kbr pellet method ( applying 6000 kg / cm ) . spectral measurements were obtained by powder diffuse reflectance on an ftir spectrophotometer ( shimadzu , ftir 8400s , japan ) in the wave number region of 4004000 cm to study drug excipient interactions . drug was extracted from ca microspheres using methanol , filtered and analyzed for drug content after suitable dilution . estimation of i m was accomplished by uv / visible spectroscopy ( shimadzu1601 , japan ) at 319 nm after sufficient dilution with ph 7.2 phosphate buffer . usp xxii dissolution apparatus type ii was employed to study percentage of drug release from various formulations prepared . accurately weighed quantities of drug - loaded microspheres ( im - equivalent to 75 mg ) of each batch were taken in 900 ml dissolution medium ( 2 h in ph 1.2 hydrochloric acid buffer and 6 h in ph 7.2 phosphate buffer ) and stirred at 100 rpm by maintaining at a temperature of 37 0.5c . the drug concentrations were determined by withdrawing the 10 ml of aliquots using guarded sample collectors periodically at an interval of 30 min for first 4 h and at 60 min interval for the next 4 h. release studies were carried out in triplicate . the optimized formulation was subjected for stability studies and stored in glass bottles at 25c/60% rh ( relative humidity ) , 30c/65% rh , and 40c/75% rh for a period of 90 days . 100 mg of microspheres from each batch of formulations was taken at the end of 30th , 60th , and 90th days and was subjected for drug content studies . in vivo release studies the sheep ages were in the range 57 years and their body weight ranged between 25 and 28 kg . a written approval was obtained from the institutional ethical committee of jss college of pharmacy , mysore , india . detailed verbal and written information on the study was provided to the veterinary surgeon , central animal facility , jss medical college hospital , and permission was obtained . the study was conducted as an open , randomized complete crossover design in which a single 75 mg dose of i m ( microcid sr 75 mg capsule and formulation f3 ) was administered to fasted , healthy adult males and females on two different occasions , separated by a wash - out period of 2 weeks between dosing interval . the content uniformity of marketed product and optimized formulation has been estimated as per usp specification . the contents of 5 units of microcid sr 75 mg capsule and formulation f3 were individually combined and weighed to the average weight of each unit . all the animals were shifted to the clinical trial laboratory from animal house at 6.00 am after overnight fast of 10 h. after shaving near the neck , an 18 gauge ( 1.3 45 mm , 96 ml / min ) cannula was inserted into a jugular vein and kept with heparinised saline lock for ensuing 24 h blood sampling . test medications ( marketed product and optimized formulation ) were administered to the sheeps , fed with banana and 200 ml water . light food was provided at 3rd h followed by two standard meals at 7th and 11th h following drug administration . blood samples ( 5 ml ) were collected at 0 h ( pre dose interval ) and at 0.5 , 1 , 1.5 , 2 , 2.5 , 3 , 3.5 , 4 , 5 , 6 , 7 , 8 , 12 , 16 , 20 , and 24 h postdose intervals . blood samples were centrifuged ( eltek tc 4100 d centrifuge , elektroshaft , india ) at 1500 rpm for 10 min . any other type of food was not permitted after 12 h administration of test medication . all subjects remained ambulatory and strenuous physical activity was prohibited during the first 12 h of blood sampling . plasma concentration of drug from the collected samples was quantified by modified hplc method . internal standard mefanamic acid ( ma ) ( 100 l ) and citrate buffer ( ph 3.0 , 500 l ) were added to 10 ml screw capped glass tubes containing 500 l of spiked plasma . the tubes were extracted gently with 7 ml of petroleum ether : dichloromethane ( 50 : 50 ) for 5 min on a rotary shaker and centrifuged at 900 rpm for 5 min . the organic phase was transferred to a watch glass and evaporated to dryness at 40c . the residue was resuspended in 100 l of mobile phase and 25 l was injected to the column . quantification was achieved by the measurement of the peak area ratio of the i m to the internal standard ( mefanamic acid ) . the limit of detection of i m in plasma was 100 ng / ml ( 500 l of plasma injected ) . the i m concentrations in plasma were assayed using a fully validated high performance liquid chromatography with ultraviolet detection ( hplc - uv ) method , with respect to adequate sensitivity , specificity , linearity , recovery , accuracy , and precision ( both within and between days ) . the hplc system consisted of hplc - shimadzu ( tokyo , japan ) lc-6a model , fitted with a -bondapack c18 ( 4.6 250 mm ) column of particle size 5 m ( supelco , bellefonte , pa ) . the flow rate was maintained at 1 ml / min , and the drug concentration was detected using a uv / visible detector ( spd-6av ) . the mobile phase consisted of 80% methanol and 0.02 m sodium acetate buffer ( 60 : 40 v / v ) . the stability of the samples under frozen conditions , at room temperature , and during freeze - thaw cycle was also determined . the peak plasma concentration ( cmax ) and time needed to reach peak plasma concentration ( tmax ) were computed directly from plasma level profiles as a measure of the rate of absorption of the drug from each product . the elimination rate constant ( kel ) was calculated from the terminal elimination phase of logarithm of drug concentrations against time curve by the method of least square regression analysis . the biological halflife ( t1/2 ) was determined by the relation : ( 4)t1/2 = 0.693k . the extent of absorption for the drug ( microcid sr 75 mg capsule and formulation f3 ) in different subjects from the area under the plasma concentration time curve from zero to 24 h ( auc024 ) were calculated by the trapezoidal rule method . area under the plasma concentration time curve from zero to infinity ( auc0 ) was calculated using the formula ; ( 5)auc0=auc0t+c24k , where c24 is drug concentrations in plasma at 24 h. the drug plasma concentration and pharmacokinetic parameters were analyzed by paired t - test and analysis of variance ( anova ) at 95% confidence limit . difference between two related means was considered statistically significant when their p values were equal to or less than 0.05 . evidence has [ 1012 ] shown in the recent years that wax / fatty materials have the physical properties and behavior suitable to prepare gastroresistant , biocompatible , biodegradable microspheres to release the entrapped drug in the intestinal lumen [ 14 , 15 , 18 ] . in the present study , a modified novel meltable dispersion emulsified cooling - induced solidification method was employed using inert fat ( ca ) and nontoxic solvents to entrap the drug . in the present study , various parameters were studied such as drug and ca ratio , stirring speed and time , amount of surfactant added , volume of the aqueous phase used , effect of ph on drug entrapment , temperature of the aqueous phase , and rapid cooling studies . therefore the influence of the above parameters was highlighted . when the ph value of the external aqueous phase was acidic , the solubility of the drug was reduced and the encapsulated amount of the drug increased . the maximum drug load was obtained at ph 4.2 ( phthalate buffer ) . as the ph increased from 4.2 to 7.0 , the percent of i m loading was reduced from 21.52 to 3.92% . in the present study , it was found that 150 ml of aqueous phase is suitable for producing the spherical microspheres . as the volume of external phase increased , the yield was reduced and the resultant microspheres were irregularly shaped . when the volume of the aqueous phase was less than 150 ml , the resultant microspheres were highly aggregated in nature and highly impossible to distinguish as an individual microsphere . in order to avoid the formation of irregularly shaped larger particles , in the present method , incorporation of drug into ca microspheres required the addition of tween 80 as a surfactant , at an optimum concentration to reduce the interfacial tension between the hydrophobic material and external aqueous phase . an attempt was made to incorporate drug in the ca microspheres without the addition of a surfactant . but the process has a failed , as it resulted in an aggregate cake - like mass during the solidification of ca . this may be due to repulsion resulting from high interfacial tension between the hydrophobic ca material and external aqueous phase . it was found that tween 80 having an hlb value of 15 was suitable to increase substantially dispersion of ca in external aqueous phase and promote drug incorporation in the ca microspheres . to obtain an optimal surfactant concentration , various concentrations ranging from 0.1 to 0.6% ( w / w ) of the total formulation discrete microspheres with good flow properties using an optimum concentration of surfactant 0.3% w / w ( tween 80 ) were used . concentrations of tween 80 ranging from 0.1 to 0.2% w / w failed to produce reproducible microspheres . the resultant ca microspheres were composed of irregular masses , which were not possible to distinguish as individual microspheres [ 1012 ] . temperature of the aqueous phase was maintained at 5c higher than the melting point of the ca in the corresponding formulations . from sem studies it was observed that the resultant microspheres were free from surface irregularities , except some wrinkles . it was also observed that when the temperature of the aqueous phase was less than 5c than the melting point of the ca , big flakes were produced . in the present study , to produce the spherical discrete microspheres , an optimum drug to ca ratio of 1 : 3 w / w was used ( table 1 ) . it was found that higher amount of drug to ca ratio ( 2 : 3 ) produces aggregate masses during the cooling process . sem photographs also indicated the presence of the crystals on the surface of the microspheres . hence an optimum 1 : 3 ratio was used to prepare microspheres [ 10 , 12 , 14 ] . it was observed that the average size of the microspheres ranged between 12 to 32 m as presented in table 2 . the important factor that influences a stirring speed of 900 rpm and stirring time of 3 min were used to obtain reproducible microspheres . it was observed that with the increase in the stirring speed from 900 to 1100 rpm there was a decrease in the average size of the spheres and recovery yield of the microspheres . it is due to small - sized wax microspheres , which were lost during successive washings . when the stirring speed was lower than 900 rpm , larger pellets were formed . it was also found that an increase in stirring time , from 2 to 4 min ( at a stirring speed of 900 rpm ) , there was a decrease in the recovery yield of microspheres . when the stirring time was lower than 2 min , it was observed that some amount of melted material adhered to the sides of the beaker during the cooling process resulting in lower recovery of yield . microparticulate drug delivery systems are formulated as single unit dosage forms in the form of capsule or tablet . the values of angle of repose were well within the range , indicating reasonable good flow potential for the microspheres . the tapped density values ranged between 0.42 g / cm and 0.51 g / cm . the results of % compressibility index ranged from 10.19% to 13.98% , suggests good flow characteristics of the microspheres ( table 2 ) . sem photographs showed that the ca microspheres were spherical in nature and had a smooth surface with inward dents and shrinkage , which is due to the collapse of the wall of the microspheres ( figure 1 ) . microphotography reveals the absence of crystals of the drug on the surface of microsphere , indicating uniform distribution of the drug within the microspheres . the rate of solvent removal from the microspheres exerts an influence on the morphology of the final product . interestingly , for microspheres dried at room temperature for 24 h , the sphericity values were nearer to the value 1 , whereas for microspheres cured for 24 h at 4c , the obtained sphericity values ranged between 1.131.19 . the removal of residual moisture content from microspheres during curing exerts an influence on the morphology of the final product . dsc studies were performed on pure drug , empty , and drug - loaded microspheres , have shown sharp endothermic peaks . it was observed that presence of endothermic peak of the drug at 161.4c in the drug - loaded ca microspheres indicates that the drug is uniformly distributed in the wall of the microspheres . the characteristic ir absorption peaks of i m comparing the ir spectra at 3413 ( aromatic c h stretching ) , 2618 ( carboxylic acid stretching ) , 1695 ( c = o stretching ) , 1600 ( c = c stretching ) , 1452 ( o ch3 deformation ) , and 1236 cm ( o h deformation ) were not altered after successful encapsulation of drug , indicating no chemical interactions between the drug and ca used . a comparison and an interpretation of this region in our spectra agree with their conclusions [ 14 , 18 ] . the percent of drug loading in the formulations was found to be in the range of 18.6721.52% . it was low in the formulation f4 ( 19.46 ) and more in f3 ( 23.52 ) . the encapsulation efficiency ( % ) was found to be more in formulation f3 ( 94.34% ) as compared to f1 ( 89.54% ) , f2 ( 89.63% ) , f5 ( 88.98% ) , and f4 ( 87.67% ) . from this result , it can be concluded that the formulation f3 had more encapsulation efficiency . from the release studies depicted in figure 4 , it was observed that there is no significant release of drug at gastric ph from fat microspheres . at the end of 8th h , in vitro drug release from f3 ( 96.32% ) was slower than microcid sr ( 98.98% ) in the intestinal environment . drug was released in a biphasic manner consisting of initial fast release followed by a slow release in intestinal ph from the ca microspheres [ 2 , 10 , 12 , 18 ] . the decreased in vitro drug release from ca microspheres might be due to more hydrophobicity and influence of molecular weight of ca . in vitro drug release was considerably retarded from the ca microspheres when compared to microcid sr . the rate of drug release followed first order release kinetics and numerical data fitted into peppas model showed that the mechanism of drug release from ca microspheres was fickian diffusion ( f1 - 0,412 , f2 - 0.415 , f3 - 0.398 , f4 - 0.421 , f5 - 0.431 , and microcid sr-0.476 ) . after an initial burst effect , the subsequent release of drug from microspheres was slow . microcid sr 75 mg capsule and formulation f3 were subjected for stability studies for 90 days . it was observed that in vitro drug release from microcid sr 75 mg capsule and formulation f3 at the end of 90 days ( 8th h ) were 99.43 and 95.09% , respectively . however , no significant change in in vitro drug release from both the products was noticed after the study period , indicating good stability for the prepared formulation . drug content uniformity for microcid sr 75 mg capsule and formulation f3 was found to be 74.83 mg and 74.80 mg , respectively . the percents of drug content uniformity of microcid sr 75 mg capsule and formulation f3 are 99.76 and 99.49% , respectively . hence , the percent of drug content uniformity in both products were well within the limits as per united state pharmacopoeia and national formulary specification . recovery of the i m from the plasma was calculated by comparison of peak height ratio after direct injection of i m or ma to the peak height of the same concentrations of the analytes extracted from plasma . in both cases the extraction solvent selected in this investigation gave higher recoveries and clean extracts than other solvents tested . plasma spiked with 500 ng / ml of i m and 1000 ng / ml of ma the retention times for i m and ma were 5.52 and 8.23 min , respectively . sensitivity of hplc assay qualitative confirmation of the purity of i m and ma peaks were obtained ( table 3 ) . the limit of quantification was 50 ng / ml of i m in plasma when 0.5 ml plasma was placed . the obtained mean correlation coefficients for the standard curves ( n = 6 ) was 0.998 . assay was shown to be sensitive , capable of reliably detecting i m concentrations in plasma as low as 50 ng / ml . interferences from endogenous compounds were overcome by using an acidic buffer ( citrate buffer ph 3.0 ) to alter the ph of the aqueous phase before extraction . to prevent the substantial interferences from endogenous compounds , strong acid - like hcl was employed . the mean plasma concentration as a function of time is shown in figure 5 and the calculated pharmacokinetic parameters of microcid sr and f3 formulations are given in table 4 . after oral administration of both products , more mean cmax value was observed for microcid sr 75 mg capsule ( 2134 29.6 ng / ml ) than formulation f3 ( 1989 20.30 ng / ml ) . however , the difference in the cmax values obtained for microcid sr 75 mg capsule and formulation f3 was statistically insignificant . mean plasma concentrations of i m for both products in all experimental conditions were within the therapeutic concentration range ( 3003000 ng / ml ) . the cmax values for both products do not exceed the above limit in all animals . it was observed the plasma concentration of i m falls below detection limit ( 50 ng / ml ) after 24 h in all animals following administration of either product . on the basis of the therapeutic concentration range of i m , it could be concluded that the therapeutic effects of both formulations would be probably be maintained for about 12 h following a single dose administration . thus it could be predicted that the two controlled release formulations included in this study are associated with a similar onset of therapeutic response following a single dose administration under fasting conditions . furthermore , it could be predicted that both controlled release formulations in this study are associated with a similar onset of therapeutic response , following a single dose administration under fasting conditions . the time taken to reach peak plasma concentration tmax of i m was little higher in case of microcid sr compared to formulation f3 , but no statistical significance differences between two products are observed ( table 4 ) . the calculated mean t1/2 values for microcid sr and formulation f3 were 2.62 0.02 h and 2.67 0.02 h , respectively . there was not much difference in the t1/2 for i m between both formulations , and no statistical significance differences were observed between both products . mean rate of absorption ka for microcid sr was 0.3934 0.002 h and for formulation f30.3856 0.002 h and mean elimination rate constants kel for microcid sr and for formulation f3 were 0.2843 0.004 h and 0.2678 0.004 h , respectively . however a small difference between both products related to cmax , tmax , t1/2 and reduced fluctuations ( peak to trough ratios ) of the plasma concentrations . all these effects probably may be due to the dissolution rate limited drug release and hence absorption . from the study it was observed that reduced fluctuations combined with the elevated mean plasma concentration from both products offer advantage in protecting patients against morning stiffness . the systematic availability of i m can be determined by comparison of the area under the plasma concentration ( auc ) versus time curves . the mean auc024 values for microcid sr and formulation f3 were 12478 104.21 ng / ml h and 12145 87.32 ng / ml h , respectively . slow in vitro release of i m from the products microcid sr and f3 formulations may be responsible for the decreased auc values when compared to the reported conventional dosage forms . the average value of the individual and mean auc024 ratio at 95% confidence limit ( 0.81.25 ) was within acceptable limits for bioequivalent products . in order to obtain in vitro - in vivo correlation , drug absorption profiles were compared for microcid sr and formulation f3 using the cumulative fraction of the drug absorbed in vivo against cumulative fraction of the drug dissolved in vitro up to 8 h. from the study it was noticed that both products showed an adequate correlation . currently accepted criteria in the us for bioequivalence for most dosage forms require that the mean pharmacokinetic parameters of the test dosage forms should be within 80120% of the reference dosage form using 90% confidence interval . pharmacokinetic parameters clearly indicate that the parameters of f3 are in good agreement with microcid sr . the observed mean auc0values for microcid sr and formulation f3 were 12632 132.12 ng / ml.h and 12452 96.32 ng / ml.h which does not show any significant statistical difference between the products . on the basis of fda recommendation , the two products , microcid sr and formulation f3 , no untoward effects were observed by any of the subjects after the administration of either product . thus , the two formulations can be considered similar , because all the subjects are very well tolerated . these observations clearly indicate the absence of high peak plasma concentrations ( > 5000 ng / ml ) , which are very often associated with adverse effects due to drug accumulation , because of the accumulation effect . the products microcid sr and formulation f3 investigated in the present study were found to be bioequivalent . the objective of the study was to prepare and evaluate wax microspheres loaded with i m by optimized meltable dispersion emulsified cooling - induced solidification method for controlled release . the method employed was simple , rapid , and economical and does not imply the use of toxic organic solvents . the results of the drug entrapment and micromeritic properties exhibited fairly good spherical nature as evidenced by sem photograph . the compatible state of the drug - loaded wax microspheres was evaluated by ftir and dsc . both formulations were found to be bioequivalent and both formulations showed an adequate correlation between cumulative fractions dissolved in vitro and cumulative fractions absorbed in vivo . optimized formulation f3 and marketed product microcid sr showed similarity in drug release profiles and in vivo bioequivalent behavior . from the present work , it can be concluded that the prepared wax microspheres demonstrate the potential use of cetyl alcohol for the development of controlled drug delivery systems for water insoluble or lipophilic drug .

the purpose of the present study was to compare the in vitro release and to find out whether the bioavailability of a 75 mg indomethacin capsule ( microcid sr ) was equivalent to optimized formulation ( indomethacin - loaded cetyl alcohol microspheres ) . indomethacin - loaded cetyl alcohol microspheres were prepared by meltable emulsified cooling - induced technique . surface morphology of microspheres has been evaluated using scanning electron microscopy . a single dose , randomized , complete cross over study of i m microspheres was carried out on 10 healthy male and female albino sheep 's under fasting conditions . the plasma was separated and the concentrations of the drug were determined by hplc - uv method . plasma indomethacin concentrations and other pharmacokinetic parameters obtained were statistically analyzed . the sem images revealed the spherical shape of fat microspheres , and more than 98.0% of the isolated microspheres were in the size range 1232 m . dsc , ftir spectroscopy and stability studies indicated that the drug after encapsulation with fat microspheres was stable and compatible . both formulations were found to be bioequivalent as evidenced by in vivo studies . based on this study , it can be concluded that cetyl alcohol microspheres and microcid sr capsule are bioequivalent in terms of the rate and extent of absorption .

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

in recent years , various uses of wax and fat microspheres in the pharmaceutical field have come into forefront , involving the microspheres technology . this idealized objective can be achieved by targeting the drugs to a specific organ or tissue with the help of controlling the release rate of the drug during the transit time in gastrointestinal tract . to overcome these problems , in the present investigation , water is used to prepare wax microspheres by meltable dispersed emulsified cooling - induced solidification method . cetyl alcohol ( ca ) , used in the current study , has good pharmaceutical and biological properties . in pharmaceutical formulations , it is used in the preparation of suppositories , delayed release solid dosage forms , emulsions , lotions , creams , and ointment . i m is widely used in the treatment of active stages of moderate - to - severe stages of rheumatoid arthritis . considering the long therapeutic regimen of osteoarthritis therapy , the administration of i m may induce adverse side effects on gastrointestinal tract ( git ) as well as central nervous system ( cns ) , renal and cardiac systems . development of controlled release formulation of i m has several advantages over the other conventional dosage forms , such as reduction in occurrence of high initial peak plasma concentrations , protection against morning stiffness , prolonged duration of action , improved bioavailability , patient compliance and reduction in adverse effects . the side effects could be lowered by controlling the drug release and by adjusting the absorption rate . previous experimental results have demonstrated that the waxes are biocompatible , nonimmunogenic material used for the entrapment of drug and its controlled drug release in the intestinal tract . delivering the drug in the intestinal environment from fatty ca microspheres could be manipulated by suitable coating techniques . these findings suggested that the kinetic control is an effective route for preventing the toxicity of i m . the aim of the present study was to formulate , characterize , and study the in vitro release of i m from microspheres and compare with commercially available oral formulation microcid sr ( 75 mg capsule ) furthermore , to investigate the pharmacokinetics and bioavailability of two different oral i m formulation ( optimized microsphere formulation and microcid sr 75 mg capsule ) following a single dosing in healthy albino sheeps in order to prove the bioequivalence between the preparations . cetyl alcohol ( ca - melting point 4951c ) , tween 80 , and all other chemicals and solvents used were of analytical grade and purchased from ranbaxy fine chemicals ( new delhi , india ) . commercially available oral capsule formulation ( microcid sr 75 mg , micro labs ltd . , india ) was used for the present study . air - drying was carried out at room temperature for 48 h to give discrete , solid , and free flowing microspheres . a total of five formulations were prepared by varying the ca to drug ratios ( table 1 ) . tap density of the prepared ca microspheres was determined using tap density tester , and percentage carr 's index ( % i ) was calculated using the formula : ( 1)carr 's index(% i ) = [ ( tapped densitybulk density)tapped density]100 . angle of repose ( h ) was assessed to know the flow ability of ca microspheres , by a fixed funnel method : ( 2)tan()=heightradius . the obtained images were processed by image analysis software to characterize each individual microsphere by mean feret diameter ( fd ) ( average of 180 caliper measurements with an angle of rotation of 1 ) , aspect ratio ( ar ) ( ratio of longest feret diameter and its longest perpendicular diameter ) , and two - dimensional shape factor ( er ) by the following equation : ( 3)er=2rpm ( bl)2 , where r is the radius , pm is the perimeter , l is length ( longest feret diameter ) , and b is width ( longest perpendicular diameter to the longest feret diameter ) of the microspheres . all dynamic dsc studies were carried out on dupont thermal analyzer with 2010 dsc module . ftir spectra of pure drug , empty microspheres , and drug - loaded microspheres were obtained using kbr pellet method ( applying 6000 kg / cm ) . spectral measurements were obtained by powder diffuse reflectance on an ftir spectrophotometer ( shimadzu , ftir 8400s , japan ) in the wave number region of 4004000 cm to study drug excipient interactions . estimation of i m was accomplished by uv / visible spectroscopy ( shimadzu1601 , japan ) at 319 nm after sufficient dilution with ph 7.2 phosphate buffer . accurately weighed quantities of drug - loaded microspheres ( im - equivalent to 75 mg ) of each batch were taken in 900 ml dissolution medium ( 2 h in ph 1.2 hydrochloric acid buffer and 6 h in ph 7.2 phosphate buffer ) and stirred at 100 rpm by maintaining at a temperature of 37 0.5c . the drug concentrations were determined by withdrawing the 10 ml of aliquots using guarded sample collectors periodically at an interval of 30 min for first 4 h and at 60 min interval for the next 4 h. release studies were carried out in triplicate . the optimized formulation was subjected for stability studies and stored in glass bottles at 25c/60% rh ( relative humidity ) , 30c/65% rh , and 40c/75% rh for a period of 90 days . in vivo release studies the sheep ages were in the range 57 years and their body weight ranged between 25 and 28 kg . detailed verbal and written information on the study was provided to the veterinary surgeon , central animal facility , jss medical college hospital , and permission was obtained . the study was conducted as an open , randomized complete crossover design in which a single 75 mg dose of i m ( microcid sr 75 mg capsule and formulation f3 ) was administered to fasted , healthy adult males and females on two different occasions , separated by a wash - out period of 2 weeks between dosing interval . the content uniformity of marketed product and optimized formulation has been estimated as per usp specification . quantification was achieved by the measurement of the peak area ratio of the i m to the internal standard ( mefanamic acid ) . the limit of detection of i m in plasma was 100 ng / ml ( 500 l of plasma injected ) . the i m concentrations in plasma were assayed using a fully validated high performance liquid chromatography with ultraviolet detection ( hplc - uv ) method , with respect to adequate sensitivity , specificity , linearity , recovery , accuracy , and precision ( both within and between days ) . the flow rate was maintained at 1 ml / min , and the drug concentration was detected using a uv / visible detector ( spd-6av ) . the stability of the samples under frozen conditions , at room temperature , and during freeze - thaw cycle was also determined . the peak plasma concentration ( cmax ) and time needed to reach peak plasma concentration ( tmax ) were computed directly from plasma level profiles as a measure of the rate of absorption of the drug from each product . the biological halflife ( t1/2 ) was determined by the relation : ( 4)t1/2 = 0.693k . the extent of absorption for the drug ( microcid sr 75 mg capsule and formulation f3 ) in different subjects from the area under the plasma concentration time curve from zero to 24 h ( auc024 ) were calculated by the trapezoidal rule method . area under the plasma concentration time curve from zero to infinity ( auc0 ) was calculated using the formula ; ( 5)auc0=auc0t+c24k , where c24 is drug concentrations in plasma at 24 h. the drug plasma concentration and pharmacokinetic parameters were analyzed by paired t - test and analysis of variance ( anova ) at 95% confidence limit . in the present study , a modified novel meltable dispersion emulsified cooling - induced solidification method was employed using inert fat ( ca ) and nontoxic solvents to entrap the drug . in the present study , various parameters were studied such as drug and ca ratio , stirring speed and time , amount of surfactant added , volume of the aqueous phase used , effect of ph on drug entrapment , temperature of the aqueous phase , and rapid cooling studies . when the ph value of the external aqueous phase was acidic , the solubility of the drug was reduced and the encapsulated amount of the drug increased . as the ph increased from 4.2 to 7.0 , the percent of i m loading was reduced from 21.52 to 3.92% . in the present study , it was found that 150 ml of aqueous phase is suitable for producing the spherical microspheres . as the volume of external phase increased , the yield was reduced and the resultant microspheres were irregularly shaped . when the volume of the aqueous phase was less than 150 ml , the resultant microspheres were highly aggregated in nature and highly impossible to distinguish as an individual microsphere . in order to avoid the formation of irregularly shaped larger particles , in the present method , incorporation of drug into ca microspheres required the addition of tween 80 as a surfactant , at an optimum concentration to reduce the interfacial tension between the hydrophobic material and external aqueous phase . an attempt was made to incorporate drug in the ca microspheres without the addition of a surfactant . temperature of the aqueous phase was maintained at 5c higher than the melting point of the ca in the corresponding formulations . from sem studies it was observed that the resultant microspheres were free from surface irregularities , except some wrinkles . in the present study , to produce the spherical discrete microspheres , an optimum drug to ca ratio of 1 : 3 w / w was used ( table 1 ) . it was observed that the average size of the microspheres ranged between 12 to 32 m as presented in table 2 . it was observed that with the increase in the stirring speed from 900 to 1100 rpm there was a decrease in the average size of the spheres and recovery yield of the microspheres . it was also found that an increase in stirring time , from 2 to 4 min ( at a stirring speed of 900 rpm ) , there was a decrease in the recovery yield of microspheres . when the stirring time was lower than 2 min , it was observed that some amount of melted material adhered to the sides of the beaker during the cooling process resulting in lower recovery of yield . sem photographs showed that the ca microspheres were spherical in nature and had a smooth surface with inward dents and shrinkage , which is due to the collapse of the wall of the microspheres ( figure 1 ) . microphotography reveals the absence of crystals of the drug on the surface of microsphere , indicating uniform distribution of the drug within the microspheres . the rate of solvent removal from the microspheres exerts an influence on the morphology of the final product . the removal of residual moisture content from microspheres during curing exerts an influence on the morphology of the final product . dsc studies were performed on pure drug , empty , and drug - loaded microspheres , have shown sharp endothermic peaks . it was observed that presence of endothermic peak of the drug at 161.4c in the drug - loaded ca microspheres indicates that the drug is uniformly distributed in the wall of the microspheres . the characteristic ir absorption peaks of i m comparing the ir spectra at 3413 ( aromatic c h stretching ) , 2618 ( carboxylic acid stretching ) , 1695 ( c = o stretching ) , 1600 ( c = c stretching ) , 1452 ( o ch3 deformation ) , and 1236 cm ( o h deformation ) were not altered after successful encapsulation of drug , indicating no chemical interactions between the drug and ca used . the percent of drug loading in the formulations was found to be in the range of 18.6721.52% . it was low in the formulation f4 ( 19.46 ) and more in f3 ( 23.52 ) . the encapsulation efficiency ( % ) was found to be more in formulation f3 ( 94.34% ) as compared to f1 ( 89.54% ) , f2 ( 89.63% ) , f5 ( 88.98% ) , and f4 ( 87.67% ) . from this result , it can be concluded that the formulation f3 had more encapsulation efficiency . from the release studies depicted in figure 4 , it was observed that there is no significant release of drug at gastric ph from fat microspheres . at the end of 8th h , in vitro drug release from f3 ( 96.32% ) was slower than microcid sr ( 98.98% ) in the intestinal environment . in vitro drug release was considerably retarded from the ca microspheres when compared to microcid sr . the rate of drug release followed first order release kinetics and numerical data fitted into peppas model showed that the mechanism of drug release from ca microspheres was fickian diffusion ( f1 - 0,412 , f2 - 0.415 , f3 - 0.398 , f4 - 0.421 , f5 - 0.431 , and microcid sr-0.476 ) . microcid sr 75 mg capsule and formulation f3 were subjected for stability studies for 90 days . it was observed that in vitro drug release from microcid sr 75 mg capsule and formulation f3 at the end of 90 days ( 8th h ) were 99.43 and 95.09% , respectively . drug content uniformity for microcid sr 75 mg capsule and formulation f3 was found to be 74.83 mg and 74.80 mg , respectively . the percents of drug content uniformity of microcid sr 75 mg capsule and formulation f3 are 99.76 and 99.49% , respectively . recovery of the i m from the plasma was calculated by comparison of peak height ratio after direct injection of i m or ma to the peak height of the same concentrations of the analytes extracted from plasma . plasma spiked with 500 ng / ml of i m and 1000 ng / ml of ma the retention times for i m and ma were 5.52 and 8.23 min , respectively . sensitivity of hplc assay qualitative confirmation of the purity of i m and ma peaks were obtained ( table 3 ) . the limit of quantification was 50 ng / ml of i m in plasma when 0.5 ml plasma was placed . assay was shown to be sensitive , capable of reliably detecting i m concentrations in plasma as low as 50 ng / ml . the mean plasma concentration as a function of time is shown in figure 5 and the calculated pharmacokinetic parameters of microcid sr and f3 formulations are given in table 4 . after oral administration of both products , more mean cmax value was observed for microcid sr 75 mg capsule ( 2134 29.6 ng / ml ) than formulation f3 ( 1989 20.30 ng / ml ) . however , the difference in the cmax values obtained for microcid sr 75 mg capsule and formulation f3 was statistically insignificant . mean plasma concentrations of i m for both products in all experimental conditions were within the therapeutic concentration range ( 3003000 ng / ml ) . it was observed the plasma concentration of i m falls below detection limit ( 50 ng / ml ) after 24 h in all animals following administration of either product . on the basis of the therapeutic concentration range of i m , it could be concluded that the therapeutic effects of both formulations would be probably be maintained for about 12 h following a single dose administration . thus it could be predicted that the two controlled release formulations included in this study are associated with a similar onset of therapeutic response following a single dose administration under fasting conditions . furthermore , it could be predicted that both controlled release formulations in this study are associated with a similar onset of therapeutic response , following a single dose administration under fasting conditions . the time taken to reach peak plasma concentration tmax of i m was little higher in case of microcid sr compared to formulation f3 , but no statistical significance differences between two products are observed ( table 4 ) . there was not much difference in the t1/2 for i m between both formulations , and no statistical significance differences were observed between both products . mean rate of absorption ka for microcid sr was 0.3934 0.002 h and for formulation f30.3856 0.002 h and mean elimination rate constants kel for microcid sr and for formulation f3 were 0.2843 0.004 h and 0.2678 0.004 h , respectively . however a small difference between both products related to cmax , tmax , t1/2 and reduced fluctuations ( peak to trough ratios ) of the plasma concentrations . the systematic availability of i m can be determined by comparison of the area under the plasma concentration ( auc ) versus time curves . slow in vitro release of i m from the products microcid sr and f3 formulations may be responsible for the decreased auc values when compared to the reported conventional dosage forms . the average value of the individual and mean auc024 ratio at 95% confidence limit ( 0.81.25 ) was within acceptable limits for bioequivalent products . in order to obtain in vitro - in vivo correlation , drug absorption profiles were compared for microcid sr and formulation f3 using the cumulative fraction of the drug absorbed in vivo against cumulative fraction of the drug dissolved in vitro up to 8 h. from the study it was noticed that both products showed an adequate correlation . currently accepted criteria in the us for bioequivalence for most dosage forms require that the mean pharmacokinetic parameters of the test dosage forms should be within 80120% of the reference dosage form using 90% confidence interval . pharmacokinetic parameters clearly indicate that the parameters of f3 are in good agreement with microcid sr . on the basis of fda recommendation , the two products , microcid sr and formulation f3 , no untoward effects were observed by any of the subjects after the administration of either product . the products microcid sr and formulation f3 investigated in the present study were found to be bioequivalent . the objective of the study was to prepare and evaluate wax microspheres loaded with i m by optimized meltable dispersion emulsified cooling - induced solidification method for controlled release . the results of the drug entrapment and micromeritic properties exhibited fairly good spherical nature as evidenced by sem photograph . the compatible state of the drug - loaded wax microspheres was evaluated by ftir and dsc . both formulations were found to be bioequivalent and both formulations showed an adequate correlation between cumulative fractions dissolved in vitro and cumulative fractions absorbed in vivo . optimized formulation f3 and marketed product microcid sr showed similarity in drug release profiles and in vivo bioequivalent behavior . from the present work , it can be concluded that the prepared wax microspheres demonstrate the potential use of cetyl alcohol for the development of controlled drug delivery systems for water insoluble or lipophilic drug .

mid - urethral sling procedures have been found to be a safe and effective treatment of choice in stress urinary incontinence ( sui ) . although the tension - free vaginal tape ( tvt ) procedure is considered to be the gold standard with excellent long - term efficacy , serious complications have been reported with this technique , including vascular and bowel injuries . to minimize these complications , an alternative procedure called the tension - free obturator tape ( tot ) procedure was developed in which the tape is introduced through the obturator foramen . the tot procedure has been shown in several randomized comparative studies to be as safe and effective in the surgical treatment of stress urinary incontinence as the tvt technique . however , in a minority of women with stress incontinence , stress incontinence surgery may result in failure owing to the presence of hidden intrinsic sphincter deficiency ( isd ) , mixed incontinence , hormonal changes , or age - related collagen alteration . the success rates of the tension - free sling in women with sui with isd range from 24 to 84% , although the results were reported differently in research on the success of treatment of anatomical urinary incontinence . some articles on tvt for intrinsic sphincter deficiency show satisfactory outcome with rates of 73 to 91% [ 8 - 10 ] . tot has been used for the treatment of sui patients along with tvt , but there have been only a few reports on its outcomes for sui with isd . reported that the cure rate of the tot procedure was 77.3% in women with isd . the most common problem in sling surgery has been excess tension , and concern over postoperative bladder outlet obstruction has led to the development of a number of methods for determining the proper tension . thus , the transobturator adjustable tape ( toa ) was developed to reduce these complications . the adjustable transobturator tape has been shown to allow for adjustment of tension for several days after surgical intervention , thus permitting correction of postoperative symptoms . therefore , it has the advantages that urinary retention can be achieved and incontinence can be minimized . in this study , we present a retrospective comparison after short - term follow - up of the effectiveness for sui with isd of the toa and tot procedures performed by one urologist . patients underwent toa ( n=33 with isd ) or tot ( n=47 with isd ) performed by one experienced surgeon between january 2007 and december 2009 . each patient underwent one of two techniques ( toa ; a.m.i toa sling , agency for medical innovations gmbh , austria ; tot , dowmedics co. , wonju , korea ) in accordance with the scheduling order . subjects were considered to have isd identified by a valsalva leak point pressure ( vlpp ) measurement < 60 cmh2o in the sitting position with a volume of 150 ml in the bladder or by a maximum urethral closure pressure ( mucp ) measurement < 20 cmh2o in the sitting position with a volume of 200 ml in the bladder . additional minor exclusion factors were chronic degenerative diseases that would affect muscular and nerve tissues , advanced genital prolapses , and active or recurrent urinary tract infections . we excluded patients who had pelvic prolapse greater than stage i on the international continence society grading system . all patients were given a routine workup for incontinence , including history , physical examination , stress cough test , standard 1-hour pad test , uroflowmetry , post - void residual ( pvr ) urine measurement , and complete multi - channel urodynamic study . all medical charts were retrospectively reviewed for certain data , including age , body weight , height , urodynamic study , and type of suburethral tape . we reviewed complications , postoperative urinary symptoms , and outcomes . during the pelvic examination , the severity of the vaginal wall defect was determined by using the pelvic organ prolapse quantification system . the stress cough test was performed with the patient in the standing position with 300 ml bladder filling . urodynamic study was performed with the patient in a birthing chair at a 45-degree angle . after catheterization , cystometry was performed by using a laborie 8 fr double - lumen urodynamic catheter at a fill rate of 50 ml / min . the vlpp , a measurement of the lowest abdominal pressure required to produce urine leakage , was also recorded . the vlpp was obtained with the subject seated when the total infused volume of sterile water reached 300 ml . routine postoperative follow - up for all patients included office visits at postoperative 7 days and at 3 and 6 months . at postoperative 7 days , patients underwent a stress cough test and uroflowmetry and residual urine volume measurement . at the 3- and 6-month follow - up sui cure was defined as no leakage of urine during cough stress testing and a pad weight gain of less than 2 g on a 1-hour pad test during the follow - up visit . improvement was defined as a more than 50% reduction of urine weight on a 1-hour pad test and a positive result on the stress cough test . failure was defined as less than a 50% reduction on a 1-hour pad test and a positive result on the cough stress test . patients who checked ' very satisfied ' or ' satisfied ' were placed in the satisfaction group . urinary obstruction was defined as a flow < 10 ml / s and/or residual urine > 50 ml . severe pain was defined as the presence of pain still requiring analgesic therapy 1 week after surgery . the toa tape is situated below the mid - urethra via a small incision in the anterior vaginal wall . two strings on either side are situated 1.5 cm from the midline of the tape , which is externalized via the anterior vaginal wall . when it is pulled down to reduce tension , the toa group is formed of three strings in each branch of the tape situated at the same distance from both thighs . these are externalized via the same orifice through which the mesh is , when pulled up , to increase the tension . the tension is adjusted with minimal tension by placing scissors between the tape and the urethra . the plastic envelope is removed , and the redundant portion of the mesh is cut . depending on the distance from the urethra to the skin , one or two of the lateral superior strings are also cut . the foley catheter was removed the next day , and the patients underwent studies that included measurements of flow rate and pvr before being discharged home . comparison of baseline patient characteristics as well as baseline uroflowmetric parameters was performed by using the independent t - test and chi - square test . the repeated - measures analysis of variance ( anova ) test was used to compare changes in clinical outcomes between the two groups . patients underwent toa ( n=33 with isd ) or tot ( n=47 with isd ) performed by one experienced surgeon between january 2007 and december 2009 . each patient underwent one of two techniques ( toa ; a.m.i toa sling , agency for medical innovations gmbh , austria ; tot , dowmedics co. , wonju , korea ) in accordance with the scheduling order . subjects were considered to have isd identified by a valsalva leak point pressure ( vlpp ) measurement < 60 cmh2o in the sitting position with a volume of 150 ml in the bladder or by a maximum urethral closure pressure ( mucp ) measurement < 20 cmh2o in the sitting position with a volume of 200 ml in the bladder . additional minor exclusion factors were chronic degenerative diseases that would affect muscular and nerve tissues , advanced genital prolapses , and active or recurrent urinary tract infections . we excluded patients who had pelvic prolapse greater than stage i on the international continence society grading system . all patients were given a routine workup for incontinence , including history , physical examination , stress cough test , standard 1-hour pad test , uroflowmetry , post - void residual ( pvr ) urine measurement , and complete multi - channel urodynamic study . all medical charts were retrospectively reviewed for certain data , including age , body weight , height , urodynamic study , and type of suburethral tape . we reviewed complications , postoperative urinary symptoms , and outcomes . during the pelvic examination , the severity of the vaginal wall defect was determined by using the pelvic organ prolapse quantification system . the stress cough test was performed with the patient in the standing position with 300 ml bladder filling . urodynamic study was performed with the patient in a birthing chair at a 45-degree angle . after catheterization , cystometry was performed by using a laborie 8 fr double - lumen urodynamic catheter at a fill rate of 50 ml / min . the vlpp , a measurement of the lowest abdominal pressure required to produce urine leakage , was also recorded . the vlpp was obtained with the subject seated when the total infused volume of sterile water reached 300 ml . routine postoperative follow - up for all patients included office visits at postoperative 7 days and at 3 and 6 months . at postoperative 7 days , patients underwent a stress cough test and uroflowmetry and residual urine volume measurement . at the 3- and 6-month follow - up sui cure was defined as no leakage of urine during cough stress testing and a pad weight gain of less than 2 g on a 1-hour pad test during the follow - up visit . improvement was defined as a more than 50% reduction of urine weight on a 1-hour pad test and a positive result on the stress cough test . failure was defined as less than a 50% reduction on a 1-hour pad test and a positive result on the cough stress test . patients who checked ' very satisfied ' or ' satisfied ' were placed in the satisfaction group . urinary obstruction was defined as a flow < 10 ml / s and/or residual urine > 50 ml . severe pain was defined as the presence of pain still requiring analgesic therapy 1 week after surgery . the toa tape is situated below the mid - urethra via a small incision in the anterior vaginal wall . two strings on either side are situated 1.5 cm from the midline of the tape , which is externalized via the anterior vaginal wall . when it is pulled down to reduce tension , the toa group is formed of three strings in each branch of the tape situated at the same distance from both thighs . these are externalized via the same orifice through which the mesh is , when pulled up , to increase the tension . the tension is adjusted with minimal tension by placing scissors between the tape and the urethra . the plastic envelope is removed , and the redundant portion of the mesh is cut . depending on the distance from the urethra to the skin , one or two of the lateral superior strings are also cut . the foley catheter was removed the next day , and the patients underwent studies that included measurements of flow rate and pvr before being discharged home . comparison of baseline patient characteristics as well as baseline uroflowmetric parameters was performed by using the independent t - test and chi - square test . the repeated - measures analysis of variance ( anova ) test was used to compare changes in clinical outcomes between the two groups . a total of 80 participants underwent the toa procedure ( n=33 ) or the tot procedure ( n=47 ) . in the tot group , 22 of the 47 patients had low vlpp ( < 60 cmh2o ) , and 29 of the 47 patients had low mucp ( < 20 cmh2o ) . in the toa group , 14 of the 33 patients had low vlpp ( < 60 cmh2o ) , and 20 of the 33 patients had low mucp ( < 20 cmh2o ) . there were no significant differences in preoperative characteristics between patients in the toa and tot groups ( table 1 ) . the operating time was 22.72.3 minutes in the tot group and 23.52.4 minutes in the toa group ( meansd ) . the mean hospitalization duration was 3.1 days in the tot group and 3.3 days in the toa group . uroflowmetry tests performed before and after surgery showed that flow in the tot group ranged from 23.07.2 ml / sec to 20.07.4 ml / sec and that in the toa group ranged from 22.57.7 ml / sec to 21.39.2 ml / sec ( table 2 ) . no statistically significant differences were found in the change in maximal urine flow rate between the two groups . however , the change in residual urine volume was significantly lower in the toa group than in the tot group ( 19.5 ml vs. 41 ml , p=0.016 , repeated - measures anova test ) . there was no definite difference in voiding volume or maximal urine flow between the two groups . at postoperative 6 months , the change in maximal urine flow rate , voiding volume , and residual urine volume was similar at postoperative 7 days . vaginal wall injury occurred in 2 patients ( 4.3% ) in the tot group and urethral perforation occurred in 1 patient ( 3.0% ) in the toa group . we promptly repaired the vaginal wall injury , and the patient with urethral perforation was catheterized for 3 days . there were no cases of wound infection , tape erosion , or urinary tract infection ( white blood cell count > 5 in urine analysis ) in either group . six patients ( 18.2% ) in the toa group tended to still have continuing groin pain 1 day after surgery . the incidence of such pain was 10.6% of the tot group compared with 6.1% of the toa group 7 days after surgery . the cause of these results may have been that with the toa procedure the strings remained until the end of the adjustment period . four patients in the toa group had improved pain 1 week after surgery , and two patients ( 6.1% ) had improved pain 4 weeks after surgery . postoperative urinary obstruction was seen in 10 patients ( 21.3% ) in the tot group at the 1-week follow - up visit . four patients ( 8.5% ) in the tot group had persistent obstructive voiding symptoms ( flow rate < 10 ml / sec and/or residual urine > 50 ml ) at the 6-month follow - up visit . these patients still showed no improvement , so we performed urethrolysis at 6 to 12 months after surgery . four patients in the toa group ( 12.1% ) required a reduction in tension due to urinary obstruction ( flow < 10 ml / sec and/or residual urine > 50 ml ) after postoperative day 1 . in 5 patients ( 15.2% ) in the toa group , the tension of the mesh was tightened because of a certain degree of continuing incontinence after postoperative day 1 . one patient ( 3.0% ) in the toa group had a complication of urethral obstruction at 5 months after surgery ; this case required urethrolysis at 6 months after surgery . table 4 shows the objective and subjective outcomes of both groups 6 months after surgery . the overall cure rate was 75.6% at 6 months in the toa group vs. 72.3% in the tot group . the satisfaction rate was higher in the toa group than in the tot group ( 84.8% vs. 78.7% ) . surgical failure rates increase 4 to 6-fold when there is evidence of isd ( mucp < 20 cmh2o or vlpp < 60 cmh2o at capacity ) . in one study , hsiou et al . retrospectively analyzed 61 cases of tvt and 60 cases of tot and found that low mucp ( < 40 cmh2o ) was an independent risk factor for sling failure for the tot group but not the tvt group . on the contrary , in ancillary studies from two separate rcts comparing tvt with tot , no significant differences in objective stress incontinence cure rates in isd cases based on a vlpp of 60 cmh2o or less were reported . surgical failure occurred in 8 of 25 patients ( 32% ) in the tot group compared with 6 ( 24% ) of 25 patients in the tvt group ( p>0.05 ) . in another report , barber found no significant association between low vlpp and recurrent stress incontinence in a multivariate analysis . however , it must be pointed out that both studies contained only between 14 and 25 isd patients in each arm and consisted of only one patient who had both isd and fixed urethra . on ultrasound assessment , compared tvt and the transobturator tape ( tvt - obturator ) and showed that , at rest or during valsalva , the middle of the tvt - obturator tape localized more distally than the tvt ( p=0.01 ) . a higher rate of urethral kinking during straining was observed in the tvt group than in the tvt - obturator group after surgery ( 86.9% compared with 23.9% , p=0.01 ) . also , the subjective cure rate was significantly lower for the tvt - obturator group than for the tvt group ( 82.4 vs. 93.4% , p=0.042 ) . the difference was because the mesh tape of the tot , which was more horizontally placed , lacked support because it wrapped a smaller part of the urethra in comparison with that wrapped by the tvt . the current literature would thus suggest that isd - related sui increases the surgical failure rate and that mid - urethral slings placed retropubically have a higher success rate than do those placed with a transobturator approach . in the present study , the overall cure rate was 75.6% at 6 months in the toa group vs. 72.3% in the tot group . the high cure rate of the toa procedure resulted from the adjustable system after the immediate postoperative period . the satisfaction rate was higher in the toa group than in the tot group ( 84.8% vs. 78.7% ) . in a study by rezapour et al . in an observational , retrospective , multicenter study , lee et al . recently evaluated pre- and intraoperative factors that might affect long - term ( 5-year ) success rates of the tvt procedure in 155 consecutive patients with sui . they found that 64 patients with abdominal leak point pressure 60 cmh2o had significantly higher cure rates than did 31 patients with abdominal leak point pressure < 60 cmh2o ( 82.8 vs. 51.6% , respectively ; p=0.003 ) . the success cure rate of the tvt procedure with isd varies from 52 to 86% ; we noted a similar success rate in both the toa group with isd in our present study and in the other article 's tvt group with isd . the overall goal of the sling operation is to produce adequate urethral resistance to prevent stress incontinence , allowing voluntary and complete bladder emptying . the two most frequent problems after stress incontinence surgery are persistence of incontinence and voiding dysfunction , both of which are related to how loose or how tight the tape is implanted . slings that are too tight are associated with voiding dysfunction and de novo urge incontinence . although most urinary retention resolves with conservative treatment , including medication , urethral dilation , or intermittent catheterization , refractory urethral obstruction ultimately requires midline or lateral excision of the tape . if sui reappears or the patient suffers persistence of urinary leakage after surgery , we will need to perform a new intervention to correct it . however , the success rate of incontinence reoperations is between 20% and 40% lower than that of first - time operations , and these procedures present a greater number of complications . in the present study , 13 patients ( 27.7% ) in the tot group had undergone urethral pull - down after postoperative 1 day . in urethral pull - down , a hegar dilator is inserted in the urethra and pulled down with the aim of moving downward and loosening the tape in the tot group . in four patients in the toa group ( 12.1% ) , it was necessary to reduce tension due to urinary obstruction ( flow < 10 ml / sec and/or residual urine > 50 ml ) after postoperative day 1 . tension is released from the mesh by pulling down on one side only of the vaginal strings , approximately 1 cm . the patients were checked again by uroflowmetry and residual urine volume after 3 hours . if the patients showed sui in the cough stress test , then we immediately pulled up the strings on each side approximately 0.5 cm . continence was tested , and the procedure was repeated until the patient was continent with a maximum flow rate equal to or greater than 10 ml / s and when there was less than 50 ml of residual urine . the strings were cut and extracted , and the patient was discharged . in the present study , the change in residual urine volume was significantly lower in the toa group than in the tot group at postoperative 1 week ( 19.5 ml vs. 41 ml , p=0.016 , repeated - measures anova test ) . four patients ( 8.5% ) in the tot group had persistent obstructive voiding symptoms ( flow rate < 10 ml / sec and/or residual urine > 50 ml ) at the 3-month follow - up visit . one patient ( 3.0% ) in the toa group had a complication of urethral obstruction at 5 months after surgery . these patients still showed no improvement , so we performed urethrolysis at 6 to 12 months after surgery . we think that the adjustable transobturator tape allowed for adjustment of tension for several days after surgical intervention , thus significantly reducing urethrolysis . the reported complication rates range from 4.3% to 75.1% for retropubic and 10.5 to 31.3% for transobturator mid - urethral slings . complications include bladder perforation , hemorrhage , bowel injury , vaginal extrusion , urinary tract infections , and voiding dysfunction . retropubic mid - urethral slings lead to a higher occurrence of complications such as bladder perforation ( 0.7 to 24% vs. 0.5% ) and hematoma ( 0.7 to 8% vs. 0% to 2% ) . in addition , the retropubic approach results in serious complications such as bowel injury , major vascular injury , and death . groin pain was more common after the transobturator approach . a randomized controlled study from finland revealed that 16% of women in the transobturator ( inside - to - outside ) arm had groin pain compared with 1.5% of those in the tvt arm , which led to an increased need for analgesia in the transobturator group . in the present study , groin pain occurred in 2 ( 6.1% ) patients . although rare , erosion of mesh into the urethra can occur after the transobturator approach . urethral erosions are believed to occur because of poor surgical technique that could damage the integrity of the urethral tissue or its blood supply , excessive tension placed on the tape , or local infection . however , the recommendation for cystoscopy after passage of the trocars can not be overemphasized , because these complications can be treated promptly in the operation field . vaginal erosion occurred in one patient , and it was located in the lateral fornix . the results of the present study suggest that toa is an effective and simple procedure for treating female stress urinary incontinence with isd , because the procedure had minimal complications and a high success rate . this sling system allows the surgeon to leave the sling very loose at the time of surgery with the ability to tighten or loosen the sling easily in the postoperative period to achieve continence and still maintain adequate voiding function . the present study was limited in that the study targeted a small number of subjects and the follow - up period was short . this study suggests that more subjects and a longer follow - up period are needed for more specific evaluation . the toa procedure allows postoperative adjustment of the suburethral sling pressure for a number of days after surgical intervention , which allows the achievement of good short - term results . these data suggest that better lower obstructive voiding symptoms than those achieved with the traditional nonadjustable mesh can be obtained . reassessment with a larger population and a randomized controlled trial is required to draw a definite conclusion .

purposethe transobturator adjustable tape ( toa ) allows adjustment of tension after surgical intervention , thus permitting correction of postoperative incontinence or obstruction . the aim of this study was to compare the efficacy and safety of toa versus transobturator tape ( tot ) for the treatment of stress urinary incontinence with intrinsic sphincter deficiency ( isd).materials and methodspatients underwent toa ( n=33 with isd ) or tot ( n=47 with isd ) insertion by one experienced surgeon . the patients were considered to have isd on the basis of a valsalva leak point pressure < 60 cmh2o or a maximum urethral closure pressure < 20 cmh2o . the preoperative evaluation included history taking , physical examination , voiding diary , stress and 1-hour pad tests , and a comprehensive urodynamic examination . postoperative evaluation included a stress test , 1-hour pad test , questionnaire , and uroflowmetry with postvoid residuals.resultsafter 6 months of follow - up , the rate of cure ( toa , 75.6% vs. tot , 72.3% ) was similar between the two groups . the rate of satisfaction was not significantly higher in the toa group than in the tot group ( 84.8% vs. 78.7% ; p=0.05 ) . four patients in the toa group ( 12.1% ) needed a reduction in tension because of urinary obstruction ( flow < 10 ml / sec and/or residual urine > 50 ml ) . the tension of the mesh was tightened in 5 patients ( 15.2% ) because of the persistence of a certain degree of incontinence . the residual urine volume at postoperative 7 days was significantly lower in the toa group than in the tot group ( 19.5 ml vs. 41 ml ; p=0.016 , repeated - measures analysis of variance test).conclusionsthe toa allows postoperative readjustment of the suburethral sling pressure for a number of days after surgical intervention , which allows for the achievement of good short - term results . these data suggest that better lower obstructive voiding symptoms than those achieved with the traditional nonadjustable mesh can be obtained with the toa .

INTRODUCTION MATERIALS AND METHODS 1. Patient group 2. Study design 3. Operation technique 4. Statistical analyses RESULTS DISCUSSION CONCLUSIONS

mid - urethral sling procedures have been found to be a safe and effective treatment of choice in stress urinary incontinence ( sui ) . although the tension - free vaginal tape ( tvt ) procedure is considered to be the gold standard with excellent long - term efficacy , serious complications have been reported with this technique , including vascular and bowel injuries . to minimize these complications , an alternative procedure called the tension - free obturator tape ( tot ) procedure was developed in which the tape is introduced through the obturator foramen . the tot procedure has been shown in several randomized comparative studies to be as safe and effective in the surgical treatment of stress urinary incontinence as the tvt technique . however , in a minority of women with stress incontinence , stress incontinence surgery may result in failure owing to the presence of hidden intrinsic sphincter deficiency ( isd ) , mixed incontinence , hormonal changes , or age - related collagen alteration . the success rates of the tension - free sling in women with sui with isd range from 24 to 84% , although the results were reported differently in research on the success of treatment of anatomical urinary incontinence . tot has been used for the treatment of sui patients along with tvt , but there have been only a few reports on its outcomes for sui with isd . reported that the cure rate of the tot procedure was 77.3% in women with isd . the most common problem in sling surgery has been excess tension , and concern over postoperative bladder outlet obstruction has led to the development of a number of methods for determining the proper tension . thus , the transobturator adjustable tape ( toa ) was developed to reduce these complications . the adjustable transobturator tape has been shown to allow for adjustment of tension for several days after surgical intervention , thus permitting correction of postoperative symptoms . in this study , we present a retrospective comparison after short - term follow - up of the effectiveness for sui with isd of the toa and tot procedures performed by one urologist . patients underwent toa ( n=33 with isd ) or tot ( n=47 with isd ) performed by one experienced surgeon between january 2007 and december 2009 . each patient underwent one of two techniques ( toa ; a.m.i toa sling , agency for medical innovations gmbh , austria ; tot , dowmedics co. , wonju , korea ) in accordance with the scheduling order . subjects were considered to have isd identified by a valsalva leak point pressure ( vlpp ) measurement < 60 cmh2o in the sitting position with a volume of 150 ml in the bladder or by a maximum urethral closure pressure ( mucp ) measurement < 20 cmh2o in the sitting position with a volume of 200 ml in the bladder . all patients were given a routine workup for incontinence , including history , physical examination , stress cough test , standard 1-hour pad test , uroflowmetry , post - void residual ( pvr ) urine measurement , and complete multi - channel urodynamic study . during the pelvic examination , the severity of the vaginal wall defect was determined by using the pelvic organ prolapse quantification system . after catheterization , cystometry was performed by using a laborie 8 fr double - lumen urodynamic catheter at a fill rate of 50 ml / min . the vlpp was obtained with the subject seated when the total infused volume of sterile water reached 300 ml . routine postoperative follow - up for all patients included office visits at postoperative 7 days and at 3 and 6 months . at postoperative 7 days , patients underwent a stress cough test and uroflowmetry and residual urine volume measurement . at the 3- and 6-month follow - up sui cure was defined as no leakage of urine during cough stress testing and a pad weight gain of less than 2 g on a 1-hour pad test during the follow - up visit . improvement was defined as a more than 50% reduction of urine weight on a 1-hour pad test and a positive result on the stress cough test . failure was defined as less than a 50% reduction on a 1-hour pad test and a positive result on the cough stress test . urinary obstruction was defined as a flow < 10 ml / s and/or residual urine > 50 ml . the toa tape is situated below the mid - urethra via a small incision in the anterior vaginal wall . when it is pulled down to reduce tension , the toa group is formed of three strings in each branch of the tape situated at the same distance from both thighs . these are externalized via the same orifice through which the mesh is , when pulled up , to increase the tension . the tension is adjusted with minimal tension by placing scissors between the tape and the urethra . the plastic envelope is removed , and the redundant portion of the mesh is cut . depending on the distance from the urethra to the skin , one or two of the lateral superior strings are also cut . the foley catheter was removed the next day , and the patients underwent studies that included measurements of flow rate and pvr before being discharged home . the repeated - measures analysis of variance ( anova ) test was used to compare changes in clinical outcomes between the two groups . patients underwent toa ( n=33 with isd ) or tot ( n=47 with isd ) performed by one experienced surgeon between january 2007 and december 2009 . each patient underwent one of two techniques ( toa ; a.m.i toa sling , agency for medical innovations gmbh , austria ; tot , dowmedics co. , wonju , korea ) in accordance with the scheduling order . subjects were considered to have isd identified by a valsalva leak point pressure ( vlpp ) measurement < 60 cmh2o in the sitting position with a volume of 150 ml in the bladder or by a maximum urethral closure pressure ( mucp ) measurement < 20 cmh2o in the sitting position with a volume of 200 ml in the bladder . all patients were given a routine workup for incontinence , including history , physical examination , stress cough test , standard 1-hour pad test , uroflowmetry , post - void residual ( pvr ) urine measurement , and complete multi - channel urodynamic study . during the pelvic examination , the severity of the vaginal wall defect was determined by using the pelvic organ prolapse quantification system . the stress cough test was performed with the patient in the standing position with 300 ml bladder filling . urodynamic study was performed with the patient in a birthing chair at a 45-degree angle . after catheterization , cystometry was performed by using a laborie 8 fr double - lumen urodynamic catheter at a fill rate of 50 ml / min . the vlpp was obtained with the subject seated when the total infused volume of sterile water reached 300 ml . routine postoperative follow - up for all patients included office visits at postoperative 7 days and at 3 and 6 months . at postoperative 7 days , patients underwent a stress cough test and uroflowmetry and residual urine volume measurement . at the 3- and 6-month follow - up sui cure was defined as no leakage of urine during cough stress testing and a pad weight gain of less than 2 g on a 1-hour pad test during the follow - up visit . improvement was defined as a more than 50% reduction of urine weight on a 1-hour pad test and a positive result on the stress cough test . failure was defined as less than a 50% reduction on a 1-hour pad test and a positive result on the cough stress test . urinary obstruction was defined as a flow < 10 ml / s and/or residual urine > 50 ml . the toa tape is situated below the mid - urethra via a small incision in the anterior vaginal wall . two strings on either side are situated 1.5 cm from the midline of the tape , which is externalized via the anterior vaginal wall . when it is pulled down to reduce tension , the toa group is formed of three strings in each branch of the tape situated at the same distance from both thighs . these are externalized via the same orifice through which the mesh is , when pulled up , to increase the tension . the tension is adjusted with minimal tension by placing scissors between the tape and the urethra . the plastic envelope is removed , and the redundant portion of the mesh is cut . depending on the distance from the urethra to the skin , one or two of the lateral superior strings are also cut . the repeated - measures analysis of variance ( anova ) test was used to compare changes in clinical outcomes between the two groups . a total of 80 participants underwent the toa procedure ( n=33 ) or the tot procedure ( n=47 ) . in the tot group , 22 of the 47 patients had low vlpp ( < 60 cmh2o ) , and 29 of the 47 patients had low mucp ( < 20 cmh2o ) . in the toa group , 14 of the 33 patients had low vlpp ( < 60 cmh2o ) , and 20 of the 33 patients had low mucp ( < 20 cmh2o ) . there were no significant differences in preoperative characteristics between patients in the toa and tot groups ( table 1 ) . the operating time was 22.72.3 minutes in the tot group and 23.52.4 minutes in the toa group ( meansd ) . the mean hospitalization duration was 3.1 days in the tot group and 3.3 days in the toa group . uroflowmetry tests performed before and after surgery showed that flow in the tot group ranged from 23.07.2 ml / sec to 20.07.4 ml / sec and that in the toa group ranged from 22.57.7 ml / sec to 21.39.2 ml / sec ( table 2 ) . no statistically significant differences were found in the change in maximal urine flow rate between the two groups . however , the change in residual urine volume was significantly lower in the toa group than in the tot group ( 19.5 ml vs. 41 ml , p=0.016 , repeated - measures anova test ) . there was no definite difference in voiding volume or maximal urine flow between the two groups . at postoperative 6 months , the change in maximal urine flow rate , voiding volume , and residual urine volume was similar at postoperative 7 days . vaginal wall injury occurred in 2 patients ( 4.3% ) in the tot group and urethral perforation occurred in 1 patient ( 3.0% ) in the toa group . six patients ( 18.2% ) in the toa group tended to still have continuing groin pain 1 day after surgery . the incidence of such pain was 10.6% of the tot group compared with 6.1% of the toa group 7 days after surgery . the cause of these results may have been that with the toa procedure the strings remained until the end of the adjustment period . four patients in the toa group had improved pain 1 week after surgery , and two patients ( 6.1% ) had improved pain 4 weeks after surgery . postoperative urinary obstruction was seen in 10 patients ( 21.3% ) in the tot group at the 1-week follow - up visit . four patients ( 8.5% ) in the tot group had persistent obstructive voiding symptoms ( flow rate < 10 ml / sec and/or residual urine > 50 ml ) at the 6-month follow - up visit . four patients in the toa group ( 12.1% ) required a reduction in tension due to urinary obstruction ( flow < 10 ml / sec and/or residual urine > 50 ml ) after postoperative day 1 . in 5 patients ( 15.2% ) in the toa group , the tension of the mesh was tightened because of a certain degree of continuing incontinence after postoperative day 1 . one patient ( 3.0% ) in the toa group had a complication of urethral obstruction at 5 months after surgery ; this case required urethrolysis at 6 months after surgery . the overall cure rate was 75.6% at 6 months in the toa group vs. 72.3% in the tot group . the satisfaction rate was higher in the toa group than in the tot group ( 84.8% vs. 78.7% ) . surgical failure rates increase 4 to 6-fold when there is evidence of isd ( mucp < 20 cmh2o or vlpp < 60 cmh2o at capacity ) . retrospectively analyzed 61 cases of tvt and 60 cases of tot and found that low mucp ( < 40 cmh2o ) was an independent risk factor for sling failure for the tot group but not the tvt group . on the contrary , in ancillary studies from two separate rcts comparing tvt with tot , no significant differences in objective stress incontinence cure rates in isd cases based on a vlpp of 60 cmh2o or less were reported . surgical failure occurred in 8 of 25 patients ( 32% ) in the tot group compared with 6 ( 24% ) of 25 patients in the tvt group ( p>0.05 ) . on ultrasound assessment , compared tvt and the transobturator tape ( tvt - obturator ) and showed that , at rest or during valsalva , the middle of the tvt - obturator tape localized more distally than the tvt ( p=0.01 ) . a higher rate of urethral kinking during straining was observed in the tvt group than in the tvt - obturator group after surgery ( 86.9% compared with 23.9% , p=0.01 ) . also , the subjective cure rate was significantly lower for the tvt - obturator group than for the tvt group ( 82.4 vs. 93.4% , p=0.042 ) . the difference was because the mesh tape of the tot , which was more horizontally placed , lacked support because it wrapped a smaller part of the urethra in comparison with that wrapped by the tvt . in the present study , the overall cure rate was 75.6% at 6 months in the toa group vs. 72.3% in the tot group . the high cure rate of the toa procedure resulted from the adjustable system after the immediate postoperative period . the satisfaction rate was higher in the toa group than in the tot group ( 84.8% vs. 78.7% ) . recently evaluated pre- and intraoperative factors that might affect long - term ( 5-year ) success rates of the tvt procedure in 155 consecutive patients with sui . they found that 64 patients with abdominal leak point pressure 60 cmh2o had significantly higher cure rates than did 31 patients with abdominal leak point pressure < 60 cmh2o ( 82.8 vs. 51.6% , respectively ; p=0.003 ) . the success cure rate of the tvt procedure with isd varies from 52 to 86% ; we noted a similar success rate in both the toa group with isd in our present study and in the other article 's tvt group with isd . the two most frequent problems after stress incontinence surgery are persistence of incontinence and voiding dysfunction , both of which are related to how loose or how tight the tape is implanted . if sui reappears or the patient suffers persistence of urinary leakage after surgery , we will need to perform a new intervention to correct it . however , the success rate of incontinence reoperations is between 20% and 40% lower than that of first - time operations , and these procedures present a greater number of complications . in the present study , 13 patients ( 27.7% ) in the tot group had undergone urethral pull - down after postoperative 1 day . in urethral pull - down , a hegar dilator is inserted in the urethra and pulled down with the aim of moving downward and loosening the tape in the tot group . in four patients in the toa group ( 12.1% ) , it was necessary to reduce tension due to urinary obstruction ( flow < 10 ml / sec and/or residual urine > 50 ml ) after postoperative day 1 . tension is released from the mesh by pulling down on one side only of the vaginal strings , approximately 1 cm . the patients were checked again by uroflowmetry and residual urine volume after 3 hours . if the patients showed sui in the cough stress test , then we immediately pulled up the strings on each side approximately 0.5 cm . continence was tested , and the procedure was repeated until the patient was continent with a maximum flow rate equal to or greater than 10 ml / s and when there was less than 50 ml of residual urine . in the present study , the change in residual urine volume was significantly lower in the toa group than in the tot group at postoperative 1 week ( 19.5 ml vs. 41 ml , p=0.016 , repeated - measures anova test ) . four patients ( 8.5% ) in the tot group had persistent obstructive voiding symptoms ( flow rate < 10 ml / sec and/or residual urine > 50 ml ) at the 3-month follow - up visit . one patient ( 3.0% ) in the toa group had a complication of urethral obstruction at 5 months after surgery . we think that the adjustable transobturator tape allowed for adjustment of tension for several days after surgical intervention , thus significantly reducing urethrolysis . retropubic mid - urethral slings lead to a higher occurrence of complications such as bladder perforation ( 0.7 to 24% vs. 0.5% ) and hematoma ( 0.7 to 8% vs. 0% to 2% ) . a randomized controlled study from finland revealed that 16% of women in the transobturator ( inside - to - outside ) arm had groin pain compared with 1.5% of those in the tvt arm , which led to an increased need for analgesia in the transobturator group . urethral erosions are believed to occur because of poor surgical technique that could damage the integrity of the urethral tissue or its blood supply , excessive tension placed on the tape , or local infection . however , the recommendation for cystoscopy after passage of the trocars can not be overemphasized , because these complications can be treated promptly in the operation field . vaginal erosion occurred in one patient , and it was located in the lateral fornix . the results of the present study suggest that toa is an effective and simple procedure for treating female stress urinary incontinence with isd , because the procedure had minimal complications and a high success rate . this sling system allows the surgeon to leave the sling very loose at the time of surgery with the ability to tighten or loosen the sling easily in the postoperative period to achieve continence and still maintain adequate voiding function . the present study was limited in that the study targeted a small number of subjects and the follow - up period was short . this study suggests that more subjects and a longer follow - up period are needed for more specific evaluation . the toa procedure allows postoperative adjustment of the suburethral sling pressure for a number of days after surgical intervention , which allows the achievement of good short - term results . these data suggest that better lower obstructive voiding symptoms than those achieved with the traditional nonadjustable mesh can be obtained .

fungi of the genus aspergillus can cause a wide variety of respiratory disorders , depending on the immune status of the patient . such disorders can range from simple allergic reactions and saprophytic colonization to invasive destruction of lung tissue with systemic dissemination , designated invasive pulmonary aspergillosis ( ipa ) , which can be fatal . it is now also being seen in critically ill patients , without apparent immunosuppression , in intensive care units . other predisposing risk factors include chemotherapy ( in patients with acute hematological malignancy ) , alcoholism , diabetes mellitus , immunosuppression therapy ( in recipients of organ transplants ) , and high doses of systemic corticosteroids . 1 2 there have been few reports of acute ipa caused by brief but massive exposure to aspergillus sp . a search of the literature revealed four reports of six cases occurring in immunocompetent subjects . 3 6 there have also been a few reports of acute ipa in immunocompetent subjects after near - drowning in contaminated water . 7 the rarity of such scenarios prompted us to report the case of a previously healthy 50-year - old male who developed acute ipa after working for 2 h in a pit containing polluted muddy water . a 50-year - old , hiv - negative male former smoker who was a water pipeline maintenance worker with the water supply organization of the state of delhi , india , was referred to our institute for evaluation of dyspnea and fever that had developed acutely and had peaked over a period of three days . he presented to us one month later , and his clinical course was characterized by progressive exertional dyspnea , together with cough and scant mucoid sputum , accompanied by low grade , intermittent fever with chills and rigors . there was no wheezing , chest pain , palpitations , or hemoptysis . when questioned , the patient reported having been exposed to polluted muddy water for 2 h while working in a deep pit , to repair a water pipeline , one day prior to the onset of the symptoms . however , the patient emphatically denied having aspirated any of the water . the patient was completely healthy prior to this event and had no history of using corticosteroids or any other medication . he had a 10 pack - year smoking history : before quitting smoking 10 years prior , he had smoked 10 cigarettes per day for 20 years . the type of cigarette he smoked is known as a bidi ( or beedi ) , which is manufactured in india and consists of finely ground , sun - dried tobacco rolled in a brown leaf of one of two broadleaf plants native to india - diospyros melanoxylon or diospyros ebenum . 8 physical examination revealed that the patient was a middle - aged man in respiratory distress . he was tachypneic , with a respiratory rate of 25 breaths / min , and febrile . breath sounds of equal intensity were audible bilaterally , together with coarse bibasilar crackles . while breathing room air , the patient had an spo2 of 90% , with a ph of 7.37 , paco2 of 41.2 mmhg , and a pao2 of 46 mmhg . the results of an electrocardiogram , urinalysis , and blood glucose testing , as well as of tests of renal and hepatic function , were all within normal limits . a chest x - ray obtained after the onset of symptoms showed bilateral multifocal consolidations with cavitation in some of the lesions ( figure 1a ) . a contrast - enhanced hrct scan of the chest obtained one week after the onset of symptoms revealed multiple areas of consolidation as well as nodular lesions of varying sizes with ill - defined margins . some of those lesions showed cavitation , whereas others were surrounded by ground - glass opacity , suggestive of a halo sign ( figure 1b ) . sputum smear microscopy and cultures revealed no growth of mycobacterium tuberculosis or other aerobic organisms . figure 1.a ) chest x - ray , obtained after the onset of symptoms , showing bilateral multifocal consolidations with cavitation in some of the lesions ( arrows ) ; b ) hrct scan of the chest , obtained one week after the onset of symptoms , showed multiple areas of consolidation as well as nodular lesions of varying sizes , some showing cavitation and others surrounded by ground - glass opacity consistent with a halo sign ( arrow ) . spirometry showed that the patient had an fvc of 3.01 l ( 82% of predicted ) , an fev1 of 1.88 l ( 62% of predicted ) , and an fev1/fvc ratio of 0.62 . fiberoptic bronchoscopy revealed hyperemic mucosa with whitish patches and foci of mucosal bleeding in the right upper lobe . cultures of post - bronchoscopy sputum and bronchial aspirate showed heavy growth of a. fumigatus . on skin prick testing total serum ige was 114 kilo units of allergen per liter ( kua / l ; reference : < 64 kua / l ) , and a fluorescence enzyme immunoassay ( immunocap 100e ; phadia , uppsala , sweden ) identified specific ige and igg antibodies against a. fumigatus . the diagnosis was substantiated by the heavy growth of a. fumigatus seen in the post - bronchoscopy sputum cultures , as well as in those of the bronchial aspirate . the patient was started on itraconazole ( 200 mg twice daily for two months ) , together with treatment for obstructive lung disease in the form of an inhaled long - acting 2 agonist and an inhaled muscarinic antagonist . the patient showed remarkable clinical improvement , and chest imaging showed marked resolution of the lesions , with residual fibrosis ( figures 2a and 2b ) . at this writing , the patient is on regular therapy for obstructive lung disease , the severity of which has been reduced considerably , and is largely asymptomatic . figure 2.chest x - ray ( a ) and hrct scan of the chest ( b ) , both obtained after two months of treatment , showing marked resolution of the lesions , with residual fibrosis . seen predominantly in immunocompromised subjects , ipa is a life - threatening form of pneumonia caused by aspergillus spp . , most commonly a. fumigatus . it is increasingly being seen in critically ill patients admitted to intensive care units and in recipients of organ transplants . 1 2 the occurrence of acute ipa in a previously healthy subject is rather uncommon , and ipa due to massive but brief environmental exposure to aspergillus sp . is quite rare ; in the literature , we identified four reports describing a collective total of six cases . 3 6 the clinical descriptions of those six patients and of our patient are summarized in table 1 . 3 6 all six patients had a history of exposure to damp material likely to harbor aspergillus sp . , potentially resulting in massive inhalation of conidia . of the six patients , five died , 3 6 and the diagnosis was established only at autopsy in four . 3 5 6 cultures of sputum or bronchial aspirate were positive for a. fumigatus in three of the six patients . 4 6 in our patient chest x - rays showed patchy / homogeneous infiltrate in five patients , 3 6 with right upper lobe cavitation in one . 6 our patient also showed bilateral diffuse cavitary nodules with ill - defined margins . an hrct scan of the chest revealed bilateral interstitial infiltrates in only one of the six patients , 6 none of the other five patients having undergone hrct . in our patient , hrct demonstrated multiple nodules of varying sizes with ill - defined margins , some of which showed cavitation , whereas others were surrounded by ground - glass opacity suggestive of a halo sign , which is a hallmark of ipa and therefore facilitated the diagnosis . four of the six patients underwent biopsy 3 4 6 : needle biopsy in one ; open lung biopsy in one ; and bronchial biopsy in two . aspergillus hyphae were identified in only two of the six patients 4 6 : from an open lung biopsy sample in one and from a bronchial biopsy sample in the other . table 1.clinical descriptions of cases of acute invasive pulmonary aspergillosis , after massive exposure to aspergillus conidia , in immunocompetent individuals . reference case history of acute exposure smoking status other comorbidities respiratory symptoms imaging cultures diagnostic confirmation serological markers treatment outcome strelling et al.,conducted in the uk in 19661sisters who played long hours in a barn during the months preceding the disease , thus being exposed to natural and artificial manure ; linseed oil ; cattle cake ; and grain.not statednonedyspnea , fever , and coughchest x - ray showed bilateral patchy infiltrates , more in the middle lobessputum , negative for funguslung needle biopsy , no organism identifiednot statednot stateddeath ( in both cases ) ; positive staining for fungus was observed at autopsy2dyspnea and feverchest x - ray showed bilateral homogeneous infiltratesnot statedmeeker et al . , conducted in the usa in 19913exposure to old damp hay two weeks prior to presentationnot statednonelow - grade fever , chills , and dry coughchest x - ray showed diffuse nodular and interstitial infiltratessputum and bal , negative for fungus ; lung tissue , positive for aspergillus fumigatus open lung biopsy , hyphae of aspergillus sp.total ige , 6,630 kua / l ( reference : < 260 kua / l ) ; immunodiffusion , positive for aspergillus antibodiesamphotericin bdeath ; no autopsy performedbatard e et al.,conducted in france in 20034exposed to vegetal dust one day prior to presentationcurrent ( 10 cigarette - a - day ) smoker fever , headache , myalgia , and dry coughchest x - ray showed bilateral interstitial miliary infiltratesbronchial aspirate , positive for a. fumigatus not statedserology , negative for aspergillus amphotericin bdeath ; postmortem lung biopsy culture positive for a. fumigatus arendrup et al . , conducted in denmark in 20065both exposed to bark chippings on the day symptoms startednot statedheart diseasefever and dry coughchest x - ray and hrct scan showed bilateral interstitial infiltratesbal , positive for a. fumigatus bronchial biopsy , hyphaepositive aspergillus antibody titeramphotericin bdeath ; postmortem histopathology suggestive of aspergillus pneumonia6current ( 20-cigarette - a - day ) smokermild copd and barrett 's esophagusdry cough , pleuritic chest pain , and sweatingchest x - ray showed cavitating pneumonia in the right upper lobebal , negative for fungusbronchial biopsy , not suggestive of aspergillus positive aspergillosis precipitin testitraconazoleimprovementpresent study7worked for 2 h in a deep pit containing polluted muddy water , 1 day prior to symptom onsetformer smoker who quit 10 years prior ( smoking history , 10 pack - years)undiagnosed moderate obstructive lung diseasedyspnea , cough , and feverchest x - ray showed bilateral diffuse patchy infiltrates with bilateral cavitation ; chest hrct showed multiple ill - defined nodular lesions affecting both lungs , with cavitation , some surrounded by ground - glass opacity ( halo sign)sputum and bronchial aspirate , positive for a. fumigatus chronic nonspecific inflammationtotal ige , 114 kua / l ( reference : < 64 kua / l ) ; skin prick test reactivity to a. fumigatus and a. niger ; positivity for specific ige and igg antibodies against a. fumigatus itraconazole ( 200 mg twice a day for 2 months)clinical and radiological improvementkua : kilo units of allergen . fungi are known to occur in polluted water . a study conducted in malaysia identified fungi in wastewater from a sewage treatment plant , 9 aspergillus spp . another study , conducted in denmark , highlighted the dangers of the inhalation of aerosols , including the conidia of aspergillus spp . 10 putrid sludge from wastewater provides warm , humid conditions , which are ideal for the growth of molds . 10 our patient was exposed to polluted muddy water for 2 h while working in a deep pit . given that his symptoms commenced the very next day , that water was most probably the source of his exposure to aspergillus conidia . near - drowning episodes , especially in polluted water , can also result in acute ipa in immunocompetent individuals are often recovered from water bodies and are a potential cause of invasive disease in near - drowning victims , because the lung tissue can be damaged due to immersion and the large inoculum of aspergillus that can be deposited therein under such circumstances . 7 our patient showed immediate skin reactivity to aspergillus antigens , as well as testing positive for specific ige and igg antibodies against a. fumigatus , which suggests prior sensitization . because his profession entailed working in such environments on a regular basis , although the disease typically occurs in immunocompromised subjects , igg antibodies against aspergillus antigens are detected in 29 - 100% of ipa patients , such sensitization being proportionally highest in non - neutropenic patients . however , the fact that it takes a mean of 10.8 days for antibodies to appear during the acute phase of the disease reduces their diagnostic utility . 11 a halo sign is defined as a nodule or mass surrounded by ground - glass opacity on hrct and is the earliest manifestation of ipa . this sign is transient , mainly seen during the early stages , and tends to disappear with time . 12 it is thus imperative to obtain hrct scans in the early stages of ipa , because this key radiological marker might not be visible thereafter . in our patient , the halo sign raised the suspicion of ipa and enabled rapid introduction of an antifungal agent , which likely helped our patient survive . although most commonly seen in ipa , halo signs can also be seen in other fungal , viral , or bacterial infections and even in some systemic or neoplastic diseases . 12 because ipa usually occurs in immunocompromised hosts , it rarely comes to mind in the diagnosis of those who are immunocompetent . our report highlights the importance of suspecting ipa in immunocompetent patients who , in comparable scenarios , might have suffered brief but massive exposure to aspergillus conidia , leading to acute ipa . os fungos do gnero aspergillus podem causar uma grande variedade de distrbios respiratrios , dependendo do estado imunolgico do paciente . tais distrbios podem variar de simples reaes alrgicas e colonizao saproftica at a destruio do tecido pulmonar com disseminao sistmica , designada aspergilose pulmonar invasiva ( api ) , que pode ser fatal . atualmente , vem sendo observada tambm em pacientes crticos , sem imunossupresso aparente , em unidades de terapia intensiva . outros fatores de risco predisponentes incluem quimioterapia ( em pacientes com malignidade hematolgica aguda ) , alcoolismo , diabetes mellitus , terapia de imunossupresso ( em receptores de transplantes de rgos ) e altas doses de corticosteroides sistmicos . 1 2 h poucos relatos de api aguda causada por exposio breve , mas macia , a aspergillus sp . a busca na literatura revelou quatro relatos de seis casos que ocorreram em indivduos imunocompetentes . 3 6 tambm h alguns relatos de api aguda em indivduos imunocompetentes aps quase afogamento em gua contaminada . 7 a raridade de tais cenrios nos levou a relatar o caso de um homem previamente saudvel de 50 anos de idade que desenvolveu api aps trabalhar 2 h em uma vala contendo gua poluda barrenta homem de 50 anos de idade , hiv negativo , ex - fumante , trabalhador de manuteno de tubulao de gua junto ao rgo desabastecimento de gua do estado de delhi , ndia , foi encaminhado ao nosso instituto para avaliao de dispneia e febre que haviam evoludo de forma aguda e atingiu um pico num perodo de trs dias . apresentou - se novamente a ns aps um ms , e sua evoluo clnica se caracterizava por dispneia progressiva aos esforos , juntamente com tosse e escarro escasso e mucoide , acompanhados de febre baixa intermitente com calafrios e rigores . ausncia de sibilncia , dor torcica , palpitaes e hemoptise . quando questionado , relatou ter sido exposto a gua poluda barrenta por 2 h enquanto trabalhava em uma vala funda , para consertar uma tubulao de gua , um dia antes do incio dos sintomas . o paciente era totalmente saudvel antes desse evento e no tinha histria de uso de corticosteroides ou qualquer outra medicao . apresentava carga tabgica de 10 anos - mao : antes de parar de fumar h 10 anos , havia fumado 10 cigarros por dia durante 20 anos . o tipo de cigarro que ele fumou conhecido como bidi ( ou beedi ) , que fabricado na ndia e consiste em tabaco seco ao sol , modo muito fino , enrolado em uma folha marrom de diospyros melanoxylon ou diospyros ebenum - duas plantas de folha larga nativas da ndia . 8 ao exame fsico , o paciente de meia idade apresentava dificuldade respiratria . respirando ar ambiente , o paciente apresentava spo2 de 90% , com ph de 7,37 , paco2 de 41,2 mmhg e pao2 de 46 mmhg . a contagem total de leuccitos foi de 28,6 10 clulas/l , sendo que os neutrfilos representaram 89% . os resultados do eletrocardiograma , da anlise da urina e do teste de glicose no sangue , bem como dos testes de funo renal e heptica , estavam todos dentro dos limites normais . a radiografia de trax realizada aps o incio dos sintomas mostrou consolidaes multifocais bilaterais com cavitao em algumas das leses ( figura 1a ) . a tcar de trax com contraste realizada uma semana aps o incio dos sintomas revelou mltiplas reas de consolidao bem como leses nodulares de tamanhos variados com limites mal definidos . algumas dessas leses apresentavam cavitao , enquanto outras eram circundadas por opacidade em vidro fosco , sugestiva de sinal do halo ( figura 1b ) . a ultrassonografia abdominal revelou hepatomegalia com infiltrao gordurosa grau 2 . a baciloscopia e as culturas de escarro revelaram ausncia de crescimento de mycobacterium tuberculosis e outros organismos aerbicos . figura 1.a ) radiografia de trax , realizada aps o incio dos sintomas , mostrando consolidaes multifocais bilaterais com cavitao em algumas das leses ( setas ) ; b ) tcar de trax , realizada uma semana aps o incio dos sintomas , mostrando mltiplas reas de consolidao bem como leses nodulares de tamanhos variados , algumas apresentando cavitao e outras circundadas por opacidade em vidro fosco compatvel com sinal do halo ( seta ) . a espirometria mostrou que o paciente apresentava cvf de 3,01 l ( 82% do previsto ) , vef1 de 1,88 l ( 62% do previsto ) e relao vef1/cvf de 0,62 . a fibrobroncoscopia revelou mucosa hipermica com manchas esbranquiadas e focos de sangramento de mucosa no lobo superior direito . as culturas de escarro ps - broncoscopia e de aspirado brnquico apresentaram crescimento significativo de a. fumigatus no teste cutneo , o paciente tambm apresentou reatividade imediata a a. fumigatus e a. niger . a ige srica total foi de 114 quilo unidades de alrgeno por litro ( kua / l ; referncia < 64 kua / l ) , e o imunoensaio enzimtico com fluorescncia ( immunocap 100e ; phadia , uppsala , sucia ) identificou anticorpos ige e igg especficos contra a. fumigatus . o aspirado brnquico revelou - se negativo para m. tuberculosis e qualquer outro organismo aerbico . o diagnstico foi fundamentado pelo crescimento significativo de a. fumigatus observado nas culturas de escarro aps a broncoscopia , bem como nas de aspirado brnquico . o paciente iniciou tratamento com itraconazol ( 200 mg duas vezes ao dia por dois meses ) , juntamente com tratamento para doena pulmonar obstrutiva na forma de um 2-agonista inalatrio de longa durao e um antagonista muscarnico inalatrio . o paciente apresentou notvel melhora clnica , e os exames de imagem do trax mostraram resoluo importante das leses , com fibrose residual ( figuras 2a e 2b ) . encontra - se atualmente em tratamento regular para doena pulmonar obstrutiva , cuja gravidade diminuiu consideravelmente , e amplamente assintomtico . figura 2.radiografia de trax ( a ) e tcar de trax ( b ) , ambas realizadas aps dois meses de tratamento , mostrando resoluo importante das leses , com fibrose residual . a api , observada predominantemente em indivduos imunocomprometidos , uma forma de pneumonia causada por aspergillus spp . vem sendo observada cada vez mais em pacientes crticos internados em unidades de terapia intensiva e em receptores de transplantes de rgos . 1 2 a ocorrncia de api em um indivduo previamente saudvel muito incomum , e a api causada por exposio ambiental macia , mas breve , ao aspergillus sp . bastante rara ; na literatura , identificamos quatro relatos descrevendo um total coletivo de seis casos . 3 6 as descries clnicas desses seis pacientes e do nosso paciente encontram - se resumidas na tabela 1 . 3 6 todos os seis pacientes apresentavam histria de exposio a material mido que provavelmente abrigava aspergillus sp . dos seis pacientes , cinco foram a bito , 3 6 e o diagnstico foi estabelecido apenas na autpsia em quatro . 3 5 6 as culturas de escarro ou aspirado brnquico foram positivas para a. fumigatus em trs dos seis pacientes . 4 6 em nosso paciente , o a. fumigatus tambm foi cultivado no escarro coletado na admisso . as radiografias de trax mostraram infiltrados irregulares / homogneos em cinco pacientes , 3 6 com cavitao no lobo superior direito em um . a tcar de trax , realizada em apenas um dos seis pacientes , 6 revelou infiltrados intersticiais bilaterais . em nosso paciente , a tcar demonstrou mltiplos ndulos de tamanhos variados com limites mal definidos , alguns dos quais apresentavam cavitao , enquanto outros eram circundados por opacidade em vidro fosco sugestiva de sinal do halo , que uma caracterstica da api e , portanto , facilitou o diagnstico . quatro dos seis pacientes foram submetidos a bipsia 3 4 6 : bipsia com agulha em um ; bipsia pulmonar a cu aberto em um ; e bipsia brnquica em dois . hifas de aspergillus foram identificadas em apenas dois dos seis pacientes 4 6 : a partir de fragmento de bipsia pulmonar a cu aberto em um e a partir de fragmento de bipsia brnquica no outro . tabela 1.descries clnicas de casos de aspergilose pulmonar invasiva aguda , aps exposio macia a condios de aspergillus , em indivduos imunocompetentes . referncia caso histria de exposio aguda status tabgico outras comorbidades sintomas respiratrios exames de imagem culturas confirmao diagnstica marcadores sorolgicos tratamento desfecho strelling et al.,realizado no reino unido em 19661irms que brincaram muitas horas em um celeiro durante os meses que antecederam a doena , expondo - se assim a esterco natural e artificial ; leo de linhaa ; torta para gado ; e gros.no referidonenhumadispneia , febre e tossea radiografia de trax mostrou infiltrados irregulares bilaterais , mais nos lobos mdios.escarro , negativas para fungosbipsia pulmonar com agulha , nenhum organismo foi identificadono referidosno referidobito ( em ambos os casos ) ; colorao positiva para fungos foi observada na autpsia2dispneia e febrea radiografia de trax mostrou infiltrados homogneos bilaterais.no referidameeker et al . , realizado nos eua em 19913exposio a feno velho e mido duas semanas antes da apresentaono referidonenhumafebre baixa , calafrios e tosse secaa radiografia de trax mostrou infiltrados intersticiais e nodulares difusos.escarro e lba , negativas para fungos ; tecido pulmonar , positiva para aspergillus fumigatus bipsia pulmonar a cu aberto , hifas de aspergillus sp.ige total , 6.630 kua / l ( referncia < 260 kua / l ) ; imunodifuso , positiva para anticorpos contra aspergillus anfotericina bbito ; nenhuma autpsia foi realizadabatard e et al.,realizado na frana em 20034exposio a poeira vegetal um dia antes da apresentaofumante atual ( de 10 cigarros por ) febre , cefaleia , mialgia e tosse secaa radiografia de trax mostrou infiltrados intersticiais miliares bilaterais.aspirado brnquico , positiva para a. fumigatus no referidasorologia , negativa para aspergillus anfotericina bbito ; cultura de bipsia pulmonar post - mortem positiva para a. fumigatus arendrup et al . , realizado na dinamarca em 20065ambos expostos a lascas de casca de rvore no dia em que os sintomas comearamno referidodoena cardacafebre e tosse seca a radiografia de trax e a tcar mostraram infiltrados intersticiais bilaterais.lba , positiva para a. fumigatus bipsia brnquica , hifasttulo positivo de anticorpos contra aspergillus anfotericina bbito ; histopatologia post - mortem sugestiva de pneumonia por aspergillus 6fumante atual ( de 20 cigarros por dia)dpoc leve e esfago de barretttosse seca , dor torcica pleurtica e sudoresea radiografia de trax mostrou pneumonia cavitria no lobo superior direito.lba , negativa para fungosbipsia brnquica , no sugestiva de aspergillus resultado positivo no teste de precipitina para aspergiloseitraconazolmelhorapresente estudo7trabalhou 2 h em uma vala funda contendo gua barrenta poluda , 1 dia antes do incio dos sintomasex - fumante que parou de fumar h 10 anos ( carga tabgica , 10 maos - ano)doena pulmonar obstrutiva moderada no diagnosticadadispneia , tosse e febrea radiografia de trax mostrou infiltrados irregulares difusos bilaterais com cavitao bilateral ; a tcar de trax mostrou mltiplas leses nodulares mal definidas acometendo ambos os pulmes , com cavitao , algumas circundadas por opacidade em vidro fosco ( sinal do halo).escarro e aspirado brnquico , positivas para a. fumigatus inflamao crnica inespecficaige total , 114 kua / l ( referncia < 64 kua / l ) ; reatividade no teste cutneo para a. fumigatus e a. niger ; positividade para anticorpos ige e igg especficos contra a. fumigatus itraconazol ( 200 mg duas vezes ao dia por 2 meses)melhora clnica e radiolgicakua : quilo unidades de alrgeno . um estudo realizado na malsia identificou fungos em gua residuria proveniente de uma estao de tratamento de esgoto , 9 sendo que os isolados de aspergillus spp . outro estudo , realizado na dinamarca , destacou os perigos da inalao de aerossis , inclusive os condios de aspergillus spp . 10 o lodo ptrido de guas residurias propicia condies aquecidas e midas , que so ideais para o crescimento de mofos . 10 nosso paciente foi exposto a gua barrenta poluda por 2 h enquanto trabalhava em uma vala funda . uma vez que os sintomas comearam j no dia seguinte , essa gua foi muito provavelmente a fonte de sua exposio a condios de aspergillus . episdios de quase afogamento , especialmente em guas poludas , podem tambm resultar em api aguda em indivduos imunocompetentes . costumam ser recuperados em corpos d'gua e so uma possvel causa de doena invasiva em vtimas de quase afogamento , pois o tecido pulmonar pode ser danificado devido imerso e a um grande inoculo de aspergillus que pode ser depositado no mesmo sob tais circunstncias . 7 nosso paciente apresentou reatividade cutnea imediata a antgenos de aspergillus , e tambm testou positivo para anticorpos ige e igg especficos contra a. fumigatus , o que sugere sensibilizao prvia . como sua profisso implicava trabalhar em tais ambientes regularmente , provvel que ele tenha sido previamente exposto a antgenos de aspergillus . embora a doena tipicamente ocorra em indivduos imunocomprometidos , anticorpos igg contra antgenos de aspergillus so detectados em 29 - 100% dos pacientes com api , sendo tal sensibilizao proporcionalmente maior em pacientes no neutropnicos . porm , o fato de os anticorpos levarem uma mdia de 10,8 dias para aparecer durante a fase aguda da doena reduz sua utilidade diagnstica . 11 o sinal do halo definido como um ndulo ou massa circundada por opacidade em vidro fosco na tcar e a manifestao mais precoce da api . esse sinal transitrio , observado principalmente durante os estgios iniciais , e tende a desaparecer com o tempo . 12 , portanto , imperativo realizar tcar nos estgios iniciais da api , pois esse marcador radiolgico fundamental pode no ser visvel posteriormente . em nosso paciente , o sinal do halo levantou a suspeita de api e possibilitou a rpida introduo de um antifngico , o que provavelmente ajudou nosso paciente a sobreviver . embora sejam mais comuns na api , os sinais do halo tambm podem ser observados em outras infeces fngicas , virais ou bacterianas e at mesmo em algumas doenas sistmicas ou neoplsicas . 12 como a api geralmente ocorre em hospedeiros imunocomprometidos , a mesma raramente lembrada no diagnstico daqueles que so imunocompetentes . isso frequentemente leva a atrasos considerveis no diagnstico e , consequentemente , a desfechos ruins . nosso relato destaca a importncia da suspeio de api em pacientes imunocompetentes que , em cenrios comparveis , possam ter sofrido exposio breve , mas macia , a condios de aspergillus , levando a api aguda .

invasive pulmonary aspergillosis ( ipa ) predominantly occurs in severely neutropenic immunocompromised subjects . the occurrence of acute ipa after brief but massive exposure to aspergillus conidia in previously healthy subjects has been documented , although only six such cases have been reported . the diagnosis was delayed in all six of the affected patients , five of whom died . we report the case of a 50-year - old hiv - negative male , a water pipeline maintenance worker , who presented with acute - onset dyspnea and fever one day after working for 2 h in a deep pit containing polluted , muddy water . over a one - month period , his general condition deteriorated markedly , despite antibiotic therapy . imaging showed bilateral diffuse nodules with cavitation , some of which were surrounded by ground - glass opacity suggestive of a halo sign ( a hallmark of ipa ) . cultures ( of sputum / bronchial aspirate samples ) and serology were positive for aspergillus fumigatus . after being started on itraconazole , the patient improved . we conclude that massive exposure to aspergillus conidia can lead to acute ipa in immunocompetent subjects .

INTRODUCTION CASE REPORT DISCUSSION INTRODUO RELATO DE CASO DISCUSSO

fungi of the genus aspergillus can cause a wide variety of respiratory disorders , depending on the immune status of the patient . such disorders can range from simple allergic reactions and saprophytic colonization to invasive destruction of lung tissue with systemic dissemination , designated invasive pulmonary aspergillosis ( ipa ) , which can be fatal . other predisposing risk factors include chemotherapy ( in patients with acute hematological malignancy ) , alcoholism , diabetes mellitus , immunosuppression therapy ( in recipients of organ transplants ) , and high doses of systemic corticosteroids . 1 2 there have been few reports of acute ipa caused by brief but massive exposure to aspergillus sp . a search of the literature revealed four reports of six cases occurring in immunocompetent subjects . 3 6 there have also been a few reports of acute ipa in immunocompetent subjects after near - drowning in contaminated water . 7 the rarity of such scenarios prompted us to report the case of a previously healthy 50-year - old male who developed acute ipa after working for 2 h in a pit containing polluted muddy water . a 50-year - old , hiv - negative male former smoker who was a water pipeline maintenance worker with the water supply organization of the state of delhi , india , was referred to our institute for evaluation of dyspnea and fever that had developed acutely and had peaked over a period of three days . when questioned , the patient reported having been exposed to polluted muddy water for 2 h while working in a deep pit , to repair a water pipeline , one day prior to the onset of the symptoms . however , the patient emphatically denied having aspirated any of the water . the type of cigarette he smoked is known as a bidi ( or beedi ) , which is manufactured in india and consists of finely ground , sun - dried tobacco rolled in a brown leaf of one of two broadleaf plants native to india - diospyros melanoxylon or diospyros ebenum . 8 physical examination revealed that the patient was a middle - aged man in respiratory distress . while breathing room air , the patient had an spo2 of 90% , with a ph of 7.37 , paco2 of 41.2 mmhg , and a pao2 of 46 mmhg . a chest x - ray obtained after the onset of symptoms showed bilateral multifocal consolidations with cavitation in some of the lesions ( figure 1a ) . a contrast - enhanced hrct scan of the chest obtained one week after the onset of symptoms revealed multiple areas of consolidation as well as nodular lesions of varying sizes with ill - defined margins . some of those lesions showed cavitation , whereas others were surrounded by ground - glass opacity , suggestive of a halo sign ( figure 1b ) . figure 1.a ) chest x - ray , obtained after the onset of symptoms , showing bilateral multifocal consolidations with cavitation in some of the lesions ( arrows ) ; b ) hrct scan of the chest , obtained one week after the onset of symptoms , showed multiple areas of consolidation as well as nodular lesions of varying sizes , some showing cavitation and others surrounded by ground - glass opacity consistent with a halo sign ( arrow ) . spirometry showed that the patient had an fvc of 3.01 l ( 82% of predicted ) , an fev1 of 1.88 l ( 62% of predicted ) , and an fev1/fvc ratio of 0.62 . cultures of post - bronchoscopy sputum and bronchial aspirate showed heavy growth of a. fumigatus . the diagnosis was substantiated by the heavy growth of a. fumigatus seen in the post - bronchoscopy sputum cultures , as well as in those of the bronchial aspirate . the patient was started on itraconazole ( 200 mg twice daily for two months ) , together with treatment for obstructive lung disease in the form of an inhaled long - acting 2 agonist and an inhaled muscarinic antagonist . the patient showed remarkable clinical improvement , and chest imaging showed marked resolution of the lesions , with residual fibrosis ( figures 2a and 2b ) . at this writing , the patient is on regular therapy for obstructive lung disease , the severity of which has been reduced considerably , and is largely asymptomatic . figure 2.chest x - ray ( a ) and hrct scan of the chest ( b ) , both obtained after two months of treatment , showing marked resolution of the lesions , with residual fibrosis . 1 2 the occurrence of acute ipa in a previously healthy subject is rather uncommon , and ipa due to massive but brief environmental exposure to aspergillus sp . 3 6 all six patients had a history of exposure to damp material likely to harbor aspergillus sp . of the six patients , five died , 3 6 and the diagnosis was established only at autopsy in four . 3 5 6 cultures of sputum or bronchial aspirate were positive for a. fumigatus in three of the six patients . 4 6 in our patient chest x - rays showed patchy / homogeneous infiltrate in five patients , 3 6 with right upper lobe cavitation in one . 6 our patient also showed bilateral diffuse cavitary nodules with ill - defined margins . an hrct scan of the chest revealed bilateral interstitial infiltrates in only one of the six patients , 6 none of the other five patients having undergone hrct . in our patient , hrct demonstrated multiple nodules of varying sizes with ill - defined margins , some of which showed cavitation , whereas others were surrounded by ground - glass opacity suggestive of a halo sign , which is a hallmark of ipa and therefore facilitated the diagnosis . four of the six patients underwent biopsy 3 4 6 : needle biopsy in one ; open lung biopsy in one ; and bronchial biopsy in two . table 1.clinical descriptions of cases of acute invasive pulmonary aspergillosis , after massive exposure to aspergillus conidia , in immunocompetent individuals . reference case history of acute exposure smoking status other comorbidities respiratory symptoms imaging cultures diagnostic confirmation serological markers treatment outcome strelling et al.,conducted in the uk in 19661sisters who played long hours in a barn during the months preceding the disease , thus being exposed to natural and artificial manure ; linseed oil ; cattle cake ; and grain.not statednonedyspnea , fever , and coughchest x - ray showed bilateral patchy infiltrates , more in the middle lobessputum , negative for funguslung needle biopsy , no organism identifiednot statednot stateddeath ( in both cases ) ; positive staining for fungus was observed at autopsy2dyspnea and feverchest x - ray showed bilateral homogeneous infiltratesnot statedmeeker et al . , conducted in the usa in 19913exposure to old damp hay two weeks prior to presentationnot statednonelow - grade fever , chills , and dry coughchest x - ray showed diffuse nodular and interstitial infiltratessputum and bal , negative for fungus ; lung tissue , positive for aspergillus fumigatus open lung biopsy , hyphae of aspergillus sp.total ige , 6,630 kua / l ( reference : < 260 kua / l ) ; immunodiffusion , positive for aspergillus antibodiesamphotericin bdeath ; no autopsy performedbatard e et al.,conducted in france in 20034exposed to vegetal dust one day prior to presentationcurrent ( 10 cigarette - a - day ) smoker fever , headache , myalgia , and dry coughchest x - ray showed bilateral interstitial miliary infiltratesbronchial aspirate , positive for a. fumigatus not statedserology , negative for aspergillus amphotericin bdeath ; postmortem lung biopsy culture positive for a. fumigatus arendrup et al . , conducted in denmark in 20065both exposed to bark chippings on the day symptoms startednot statedheart diseasefever and dry coughchest x - ray and hrct scan showed bilateral interstitial infiltratesbal , positive for a. fumigatus bronchial biopsy , hyphaepositive aspergillus antibody titeramphotericin bdeath ; postmortem histopathology suggestive of aspergillus pneumonia6current ( 20-cigarette - a - day ) smokermild copd and barrett 's esophagusdry cough , pleuritic chest pain , and sweatingchest x - ray showed cavitating pneumonia in the right upper lobebal , negative for fungusbronchial biopsy , not suggestive of aspergillus positive aspergillosis precipitin testitraconazoleimprovementpresent study7worked for 2 h in a deep pit containing polluted muddy water , 1 day prior to symptom onsetformer smoker who quit 10 years prior ( smoking history , 10 pack - years)undiagnosed moderate obstructive lung diseasedyspnea , cough , and feverchest x - ray showed bilateral diffuse patchy infiltrates with bilateral cavitation ; chest hrct showed multiple ill - defined nodular lesions affecting both lungs , with cavitation , some surrounded by ground - glass opacity ( halo sign)sputum and bronchial aspirate , positive for a. fumigatus chronic nonspecific inflammationtotal ige , 114 kua / l ( reference : < 64 kua / l ) ; skin prick test reactivity to a. fumigatus and a. niger ; positivity for specific ige and igg antibodies against a. fumigatus itraconazole ( 200 mg twice a day for 2 months)clinical and radiological improvementkua : kilo units of allergen . another study , conducted in denmark , highlighted the dangers of the inhalation of aerosols , including the conidia of aspergillus spp . 10 our patient was exposed to polluted muddy water for 2 h while working in a deep pit . given that his symptoms commenced the very next day , that water was most probably the source of his exposure to aspergillus conidia . near - drowning episodes , especially in polluted water , can also result in acute ipa in immunocompetent individuals are often recovered from water bodies and are a potential cause of invasive disease in near - drowning victims , because the lung tissue can be damaged due to immersion and the large inoculum of aspergillus that can be deposited therein under such circumstances . 7 our patient showed immediate skin reactivity to aspergillus antigens , as well as testing positive for specific ige and igg antibodies against a. fumigatus , which suggests prior sensitization . because his profession entailed working in such environments on a regular basis , although the disease typically occurs in immunocompromised subjects , igg antibodies against aspergillus antigens are detected in 29 - 100% of ipa patients , such sensitization being proportionally highest in non - neutropenic patients . however , the fact that it takes a mean of 10.8 days for antibodies to appear during the acute phase of the disease reduces their diagnostic utility . 11 a halo sign is defined as a nodule or mass surrounded by ground - glass opacity on hrct and is the earliest manifestation of ipa . 12 it is thus imperative to obtain hrct scans in the early stages of ipa , because this key radiological marker might not be visible thereafter . in our patient , the halo sign raised the suspicion of ipa and enabled rapid introduction of an antifungal agent , which likely helped our patient survive . 12 because ipa usually occurs in immunocompromised hosts , it rarely comes to mind in the diagnosis of those who are immunocompetent . our report highlights the importance of suspecting ipa in immunocompetent patients who , in comparable scenarios , might have suffered brief but massive exposure to aspergillus conidia , leading to acute ipa . 1 2 h poucos relatos de api aguda causada por exposio breve , mas macia , a aspergillus sp . 7 a raridade de tais cenrios nos levou a relatar o caso de um homem previamente saudvel de 50 anos de idade que desenvolveu api aps trabalhar 2 h em uma vala contendo gua poluda barrenta homem de 50 anos de idade , hiv negativo , ex - fumante , trabalhador de manuteno de tubulao de gua junto ao rgo desabastecimento de gua do estado de delhi , ndia , foi encaminhado ao nosso instituto para avaliao de dispneia e febre que haviam evoludo de forma aguda e atingiu um pico num perodo de trs dias . figura 2.radiografia de trax ( a ) e tcar de trax ( b ) , ambas realizadas aps dois meses de tratamento , mostrando resoluo importante das leses , com fibrose residual . em nosso paciente , a tcar demonstrou mltiplos ndulos de tamanhos variados com limites mal definidos , alguns dos quais apresentavam cavitao , enquanto outros eram circundados por opacidade em vidro fosco sugestiva de sinal do halo , que uma caracterstica da api e , portanto , facilitou o diagnstico . , realizado nos eua em 19913exposio a feno velho e mido duas semanas antes da apresentaono referidonenhumafebre baixa , calafrios e tosse secaa radiografia de trax mostrou infiltrados intersticiais e nodulares difusos.escarro e lba , negativas para fungos ; tecido pulmonar , positiva para aspergillus fumigatus bipsia pulmonar a cu aberto , hifas de aspergillus sp.ige total , 6.630 kua / l ( referncia < 260 kua / l ) ; imunodifuso , positiva para anticorpos contra aspergillus anfotericina bbito ; nenhuma autpsia foi realizadabatard e et al.,realizado na frana em 20034exposio a poeira vegetal um dia antes da apresentaofumante atual ( de 10 cigarros por ) febre , cefaleia , mialgia e tosse secaa radiografia de trax mostrou infiltrados intersticiais miliares bilaterais.aspirado brnquico , positiva para a. fumigatus no referidasorologia , negativa para aspergillus anfotericina bbito ; cultura de bipsia pulmonar post - mortem positiva para a. fumigatus arendrup et al . , realizado na dinamarca em 20065ambos expostos a lascas de casca de rvore no dia em que os sintomas comearamno referidodoena cardacafebre e tosse seca a radiografia de trax e a tcar mostraram infiltrados intersticiais bilaterais.lba , positiva para a. fumigatus bipsia brnquica , hifasttulo positivo de anticorpos contra aspergillus anfotericina bbito ; histopatologia post - mortem sugestiva de pneumonia por aspergillus 6fumante atual ( de 20 cigarros por dia)dpoc leve e esfago de barretttosse seca , dor torcica pleurtica e sudoresea radiografia de trax mostrou pneumonia cavitria no lobo superior direito.lba , negativa para fungosbipsia brnquica , no sugestiva de aspergillus resultado positivo no teste de precipitina para aspergiloseitraconazolmelhorapresente estudo7trabalhou 2 h em uma vala funda contendo gua barrenta poluda , 1 dia antes do incio dos sintomasex - fumante que parou de fumar h 10 anos ( carga tabgica , 10 maos - ano)doena pulmonar obstrutiva moderada no diagnosticadadispneia , tosse e febrea radiografia de trax mostrou infiltrados irregulares difusos bilaterais com cavitao bilateral ; a tcar de trax mostrou mltiplas leses nodulares mal definidas acometendo ambos os pulmes , com cavitao , algumas circundadas por opacidade em vidro fosco ( sinal do halo).escarro e aspirado brnquico , positivas para a. fumigatus inflamao crnica inespecficaige total , 114 kua / l ( referncia < 64 kua / l ) ; reatividade no teste cutneo para a. fumigatus e a. niger ; positividade para anticorpos ige e igg especficos contra a. fumigatus itraconazol ( 200 mg duas vezes ao dia por 2 meses)melhora clnica e radiolgicakua : quilo unidades de alrgeno . 10 nosso paciente foi exposto a gua barrenta poluda por 2 h enquanto trabalhava em uma vala funda . isso frequentemente leva a atrasos considerveis no diagnstico e , consequentemente , a desfechos ruins . nosso relato destaca a importncia da suspeio de api em pacientes imunocompetentes que , em cenrios comparveis , possam ter sofrido exposio breve , mas macia , a condios de aspergillus , levando a api aguda .

the development of controllable and scalable growth , processing , and interfacing techniques remains the largest challenge in realizing the application potential of two - dimensional ( 2d ) materials . chemical vapor deposition ( cvd ) has emerged as the most viable route for high - quality graphene synthesis , and a lot of progress has also been made in developing cvd processes for other 2d materials , such as hexagonal boron nitride ( h - bn ) and related 2d heterostructures . essential to the development of process rationales for 2d material cvd is an understanding of the underlying growth mechanisms , in particular the role of typically used process catalysts . while graphene cvd relies on the interactions of carbon with the surface , sub - surface , and bulk of the catalyst , the growth mechanisms for compound 2d materials such as h - bn are inherently more complex , as two elements need to be fed and incorporated into the growing nanostructure and bond polarity can affect the structural formation . there is typically a range of poly - types for compound materials , and hence selective growth becomes increasingly challenging . monolayer and few - layer h - bn has received considerable attention recently , reflecting an ever increasing demand for ultrathin dielectric , support , or barrier layers in electronics , photonics , and many other applications . while progress has been made in synthesizing h - bn on several catalyst metals , the focus has remained limited to catalyst surface pre - treatment and tuning of precursors and/or exposure conditions . further progress and , for instance , control of layer texture , grain structure , and number of layers require a more detailed understanding , in particular of the basic interactions of b and n with the catalyst . the interaction of h - bn with the catalyst also affects h - bn transfer and an increasing number of applications , such as oxidation barriers and corrosion protection , that utilize h - bn as - grown on top of the catalyst metal . unlike bulk h - bn which is typically thermally and chemically very stable , the use of few - layer h - bn as a high - temperature oxidation barrier on ni , cu , and stainless steel has recently highlighted that the stability and maximum possible temperatures of these ultrathin films are critically linked to their support and environment . here , using a combination of in situ x - ray photoelectron spectroscopy ( xps ) and x - ray diffraction ( xrd ) during actual synthesis conditions , we fingerprint the entire h - bn cvd growth process on polycrystalline cu using borazine , ( hbnh)3 , as the precursor . we focus on the important questions of how the precursor and constituent b and n as well as the as - grown h - bn interact with the catalyst during and after growth . polycrystalline cu foil catalysts are currently most widely used for graphene cvd , motivated by the rationale that the low carbon solubility of cu offers a relatively error - tolerant growth window for monolayer graphene formation via a mainly cu surface - based mechanism . based on the ease of processing , polycrystalline cu foils are now also used for h - bn cvd . our data show that , at the typically used growth temperatures , the borazine molecule decomposes and b incorporates into the bulk of the cu catalyst . during h - bn growth the cu is in the metallic face - centered - cubic ( fcc ) phase ; whereby , we can measure a lattice dilation due to the dissolved b. upon post - growth cooling we observe precipitation of b from the fcc cu . compared to graphene cvd , the role of the surface , sub - surface , and bulk of the catalyst is more complex , considering that n has a considerably lower solubility in cu and its incorporation into the cu bulk is not detected . we invoke a comparison to catalytically mediated iii v nanowire growth , i.e. , the cvd of 1d nanomaterials , where the growth kinetics crucially depend on the different solubilities of the group iii and v constituents in the catalyst and where atomically sharp and controlled heterostructures can be grown despite a comparably high bulk solubility of the group iii element . we further show that after h - bn cvd and during commonly used further processing of h - bn on the cu catalyst in ambient air , oxygen intercalation readily occurs under as - grown h - bn and negatively affects the stability of h - bn on the catalyst . for extended air exposure cu oxidation is observed , and upon reheating in vacuum an oxygen - mediated disintegration of the h - bn film via volatile boron oxides occurs . of key importance is that this disintegration is catalyst mediated , i.e. , occurs at the catalyst / h - bn interface and depends on the level of oxygen present at this interface . in turn , however , we demonstrate that pre- and co - exposure with oxygen species can be used to influence h - bn growth morphologies . we discuss the implications of these observations in the context of corrosion protection and relate them to challenges for process integration and heterostructure growth . we adopt a simple cvd process , and figure 1 outlines the nomenclature for the process steps that will be used throughout this study . cvd of h - bn was performed at 9501000 c in customized cold - wall cvd reactors using borazine , ( hbnh)3 , as precursor on commercially available cold - rolled polycrystalline cu foils as catalyst ( see methods ) . process diagram illustrating the salient stages of h - bn cvd on polycrystalline cu . figure 2 highlights via various ex situ characterization techniques , the representative h - bn film quality for the cvd recipe used throughout this study . transmission electron microscopy images at high magnification show a hexagonal lattice structure ( figure 2a ) , and selected area diffraction ( figure 2b ) shows the typical hexagonal spot pattern of h - bn , confirming the high crystalline quality at the microscopic level . figure 2c shows an optical microscopy image of a continuous h - bn film transferred to a sio2(300 nm)/si wafer . the image shows large areas of uniform contrast indicating the macroscopic uniformity of the h - bn films along with specs of residual polymethylmethacrylate ( pmma ) typically seen for such transferred films . the pmma contamination toward the edge of the layer helps to guide the eye to distinguish the film from the substrate in spite of the low optical contrast of h - bn placed on sio2(300 nm)/si wafer support . raman spectra ( figure 2d ) taken on the transferred h - bn film on sio2 ( 300 nm)/si ( see figure 2c ) show a single peak at 1368 cm consistent with the signature for h - bn . the corresponding ex situ xps core - level signatures [ i.e. , post ambient air exposure ( step 7 , figure 1 ) ] acquired on the h - bn films on cu show in the b1s region a main component at 190.6 ev ( figure 2e , inset additionally shows the bond shake up satellite at 200 ev ) and in the n1s region a main component at 398.1 ev ( figure 2f ) . from the intensities we estimate a b : n ratio of 1 , again consistent with h - bn . we also note that our measured binding energies ( bes ) are consistent with previous xps characterization studies of h - bn . further additional fitted components are seen in the ex situ xps spectra at b1s 191.1 , 191.9 ev and in the n1s at 398.6 , 399.3 ev , respectively , and we will discuss the assignment of these peaks in the sections below . ( a ) high - magnification tem image showing the hexagonal lattice structure of h - bn . ( b ) selected area diffraction pattern for the h - bn film . ( c ) optical microscopy image for h - bn film transferred onto a sio2(300 nm)/si wafer . the h - bn layer can be clearly distinguished in spite of the low contrast due to the edge where pmma residue accumulates . ( d ) raman spectrum for the transferred h - bn film in ( c ) . ( e , f ) xps b1s ( e , inset shows the bond shake - up satellite at 200 ev ) and n1s ( f ) core - level signatures for h - bn on the cu foil . figure 3a and figure s1 in the supporting information show post - growth ( step 7 , figure 1 ) scanning electron microscopy ( sem ) images of as - grown h - bn on polycrystalline cu foil for 5 , 15 , and 30 min of borazine exposure . while the sem contrast mechanisms of such 2d materials supported on metallic foils / films is still an area of active research , in figures 3a and s1 the h - bn appears darker than the uncovered cu . figure 3a and s1 highlight that a typical h - bn cvd film grows via multiple nucleation points within each facet of the polycrystalline cu foil ( see figure s1 ) which then expand as isolated h - bn domains and eventually merge to form a continuous polycrystalline h - bn film . in contrast to graphene , the isolated h - bn domains often adopt more strict geometrical shapes ( figure 3a , 300 s and figure s1a ) , such as triangles or other simple polygons , due to the bond polarity and related larger energetic differences between different edge terminations . longer exposures lead here to the formation of more irregular - shaped domains ( figure 3a , 900 s ) , which eventually cover the entire cu surface , as indirectly confirmed by the wrinkles in the resulting h - bn film ( figure 3a , 1800 s , and figure s1c ) . analogous to graphene , extended exposure times also lead here to the nucleation of further h - bn layers , i.e. , multilayer formation via secondary nucleation , as highlighted by the darker contrast regions ( figure 3a , 1800 s ) . atomic force microscopy ( afm ) with step heights measured on the edges of such a h - bn film transferred to sio2 ( 300 nm)/si wafer ( see supporting information , figure s2 ) indeed confirms the growth of few - layer h - bn films for extended ( 30 min ) exposure times under our experimental conditions . having confirmed the growth of atomically thin h - bn ( monolayer islands for short exposures ; full - coverage few - layer films for longer exposure times ) under the given cvd conditions we now turn to study the growth process using in situ experiments . ( a ) ex situ high - magnification sem images for experiments with 300 , 900 , and 1800 s of borazine exposure . corresponding low - magnification images are presented in the supporting information , figure s1 . ( b , c ) in situ xps confirming isothermal h - bn growth on cu : in situ cvd process - resolved xps b1s ( b ) and n1s ( c ) core - level scans at 975 c before ( step 3 ) , during ( step 4 ) , and after borazine exposure ( step 5 ) , after cooling ( step 6 ) , and after air exposure ( step7 ) . figure 3b , c shows cvd process - resolved xps b1s and n1s core - level signatures measured in situ at steps 27 ( figure 1 ) , respectively . to simplify assignments , we refer to pairs of b1s / n1s components for b n compound species while giving separate respective b1s and n1s bes for other species . during annealing in h2 ( step 2 ) the flat lines in the respective b1s and n1s spectra ( figure 3b , c ) indicate a clean cu surface as baseline for the subsequent borazine exposure . the time - resolved b1s spectra during borazine exposure ( inset in figure 3b , step 4 ) show that upon borazine introduction a broad peak in the b1s spectra emerges after 290 s. this is consistent with the time scale of the nucleation and growth of h - bn domains observed ex situ by sem ( figure 3a ) . at 880 s the broad b1s peak can be resolved into two components , which are also reflected in the matching n1s scans with be pairings of 191.1/398.6 ev and 190.6/398.1 ev . around this exposure time the islands have grown in size but have not coalesced yet ( figure 3a ) . for continued exposures ( up to 2270 s ) the component at 190.6/398.1 ev starts to dominate the spectrum , while the 191.1/398.6 ev peak strongly decreases in relative intensity . concurrently , the ex situ sem now shows coalescence of the islands into a closed few - layer h - bn film . the 191.1/398.6 ev component could be attributed to cubic boron nitride ( c - bn ) , but is also consistent with a slightly higher be of monolayer h - bn ( as compared to few - layer h - bn at 190.6/398.1 ev ) . we exclude significant homogeneous c - bn formation based on the ex situ characterization of our films ( figure 2 ) and also based on the observation of a bond shake - up satellite at 200 ev in the b1s spectra ( see inset of figure 2e ) . therefore , we interpret the observed initial emergence of the two peaks at 190.6/398.1 and 191.1/398.6 ev at equal intensities followed by domination of the spectrum by the 190.6/398.1 ev component for extended exposure times as evidence for isothermal growth of monolayer h - bn that interacts with the cu substrate ( 191.1/398.6 ev ) and some fraction of multilayer h - bn nuclei ( 190.6/398.1 ev ) , where the initial monolayer domains further evolve to multilayers with extended exposure time ( hence the decrease of 191.1/398.6 ev contribution relative to the 190.6/398.1 ev peaks ) . we suggest that the observed peak shift between 191.1/398.6 and 190.6/398.1 ev is caused by interactions of h - bn with the cu substrate , similar to monolayer graphene i.e. , charge transfer between the substrate and the growing 2d material . in turn this interaction is not present for multilayer h - bn , where the top layer ( which contributes most significantly to the xps signal ) is supported by the other h - bn layers and thus shows the be at 190.6/398.1 ev . further evidence for this interpretation of h - bn / cu interactions for monolayer h - bn is provided below . in addition to the assignment of the monolayer h - bn ( 191.1/398.6 ev ) and multilayer h - bn ( 190.6/398.1 ev ) xps components , we note that , due to the overlap of be positions for multiple species , part of the signal at 191.1/398.6 ev may also be assigned to local cubic - like sp bonding arrangements at h - bn island edges or defects . also such a cubic - like sp contribution from nuclei edges would similarly decrease in relative intensity with exposure time as the islands increase in area and therefore exhibit a decreasing fraction of signal from edges . our ex situ characterization however excludes significant cubic - like bn contributions . besides the 191.1/398.6 and 190.6/398.1 ev components , we also observe small xps fitted components at 191.9/399.3 and 189.8/397.5 ev . we note that borazine is known to dissociate on metal catalyst surfaces at the given growth temperature , meaning that these remaining unassigned b n - related components during growth can not be ascribed to adsorption of intact borazine molecules on the catalyst surface . instead we find that the 191.9/399.3 ev component also emerges when we sputter ex situ grown h - bn on cu post air exposure ( see supporting information , figure s3 ) . as sputtering is known to induce defects and dangling bonds , we assign the very small components at b1s / n1s 191.9/399.3 ev to h - bn defect species saturated with residual oxygen . similarly , we also assign the 189.8/397.5 ev components to defects in the h - bn . our observations so far indicate isothermal growth of h - bn on cu with initial monolayer nucleation ( which then evolve toward few - layer h - bn ) with a small amount of defects incorporated in the h - bn films ( lattice , edges , grain boundaries ) . such an isothermal growth scenario is consistent with ultra - high vacuum ( uhv)/single crystal studies , where the precursor pressure however remained below the threshold for multilayer nucleation . the in situ xps b1s and n1s fingerprints of growing h - bn are preserved upon removal of borazine at temperature ( step 5 , figure 1 ) , but interestingly one small additional component emerges upon cooling of the sample in vacuum in the b1s at 188.1 ev ( step 6 , figure 1 ) . this be is known to correspond to atomic b diffused to the catalyst surface or sub - surface . this assignment of atomic b ( non - nitrogen bonded ) is also corroborated by the lack of a concurrently emerging n1s component upon cooling , as well as by the disappearance of the 188.1 ev signal upon subsequent air exposure , since atomic boron is known to quickly oxidize in ambient air and desorb as highly volatile boron oxides . the emergence of atomic b on the catalyst surface upon cooling strongly implies b precipitation from the catalyst bulk and in turn suggests dissolution of b in the polycrystalline cu subsurface / bulk during the high - temperature borazine exposure . to confirm this b dissolution , we employ in situ xrd to characterize the crystalline structure of the catalyst bulk during cvd . figure 4a shows xrd patterns recorded during cvd , starting with as loaded cu catalyst ( before step 1 , figure 1 ) , after h2 anneal ( step 3 , figure 1 ) , during borazine exposure ( step 4 , figure 1 ) , and after ambient air exposure ( step 7 , figure 1 ) . the growing h - bn layers are not seen in the xrd patterns due to the setup geometry / sensitivity used here ( see methods ) . from the xrd patterns we observe metallic fcc cu as the state of the catalyst bulk during the entire growth process . importantly , no ( non - equilibrium ) boride or borate phases are observed . rietveld refinement of the patterns in figure 4a reveals that when borazine is introduced the cu lattice constant expands by 0.0006 ( figure 4b ) compared to a vacuum baseline . this lattice expansion is indicative of interstitial uptake of a borazine - derived component into the cu lattice . we note that this lattice constant change remains after borazine exposure with the sample still at temperature ( step 5 , figure 1 ) . in order to get some indication of the nature of the dissolving species , we characterized by xrd the cu catalyst exposed to ammonia ( nh3 , i.e. , a nitrogen and hydrogen source without b ) instead of borazine under similar pressures . for this ammonia exposure , no expansion in the cu lattice constant is found . as ammonia is known to dissociate on cu in this temperature regime , our data imply that n and h uptake is not responsible for the increase in the cu lattice constant , and instead b is dissolved into the cu during growth . therefore , our combined xps and xrd data suggest that the h - bn growth mechanism on cu is not limited to a pure surface mechanism as previously suggested . rather our data indicate element - specific feeding mechanisms including the bulk of the catalyst . we suggest that b is taken up in the cu catalyst while n is not ( by relative amounts ) , which is also in agreement with thermodynamic phase diagrams . bulk crystallography of the cu catalyst during isothermal growth of h - bn by cvd measured using in situ xrd . ( a ) xrd patterns during salient stages of cvd as loaded ( before step 1 ) , after h2 anneal ( step 3 ) , during borazine exposure ( step 4 ) , and after ambient air exposure ( step 7 ) . ( b ) rietveld refinement derived change in fcc cu lattice parameter during vacuum and borazine and nh3 exposure ( corrected for thermal expansion via measuring a baseline temperature series in vacuum ) . returning to the xps data in figure 3b , c , we find that after extended air exposure the n1s spectra show an additional small component at 400.5 ev that does not have a b1s counterpart and is attributed to the formation of n o bonds . we note that the final in situ xps fingerprint after air exposure ( step 7 ) in figure 3b , c resembles our x - ray photoelectron spectra of the ex situ grown few - layer h - bn in figure 2e , f which highlights the validity and relevance of our in situ measurements for scalable high - quality h - bn growth under realistic process conditions . in addition to following the evolution of b and n species during h - bn cvd , in situ xps also allows us ( within its detection limits ) to observe the surface chemistry of the cu catalyst by simultaneously monitoring the cu2p , culmm , o1s ( figure 5 ) , and valence band ( supporting information , figure s4 ) regions . we find that , before cvd , the as - loaded cu foil surface is heavily oxidized due to storage and transportation in ambient air ( before step 1 ) . following an anneal ( step 2 ) in h2 the cu surface is reduced to metallic cu ( step 3 ) and remains metallic during growth ( step 4 ) and cooling post - growth ( step 6 ) . this is in good agreement with the xrd data in figure 4a that suggested the cu bulk to be metallic during h - bn growth . upon air exposure ( step 7 ) the h - bn covered cu surface oxidizes again . the oxidation of the cu upon air exposure implies that the inherent polycrystallinity of cvd grown h - bn film provides diffusion pathways for species to reach the cu / h - bn interface . this suggests that h - bn films as - grown on cu per se are not as perfect oxidation barriers as recently reported . we emphasize here that the o1s and culmm are more sensitive to measure the oxidation of the cu than the cu2p . the observation of oxidation upon air exposure is also consistent with the disappearance of the elemental b peak ( desorbed as volatile boron - oxides , figure 3b ) and the formation of n o bonds ( figure 3c ) upon air exposure . surface chemistry of the cu catalyst during isothermal growth of h - bn by cvd : in situ xps ( a ) cu2p3/2 , ( b ) culmm auger , and ( c ) o1s spectra after h2 anneal in vacuum ( step 3 ) , during borazine exposure ( step 4 ) , after exposure ( step 5 ) , after cooling in vacuum ( step 6 ) , and after ambient air exposure ( step 7 ) . to further probe this evolution of h - bn on cu catalysts during ambient air exposure ( step 7 , figure 1 ) , we undertake complementary experiments with ex situ grown h - bn islands on cu , for which the borazine exposure was limited to 5 min to obtain islands of monolayer h - bn . this significantly increases the direct contribution of the cu / h - bn interface . we measure xps on these ex situ grown h - bn islands on cu exposed to ambient air for 4 days after cvd ( figure 6 ) . the as - loaded sample shows a b1s peak centered at 190.6 ev and a n1s peak centered at 398.1 ev , and the o1s and culmm peaks indicate an oxidized cu surface . the bes of the b1s and n1s peaks are surprising , as we established above that monolayer h - bn should exhibit a be of 191.1/398.6 ev . interestingly , however , upon annealing the sample in vacuum ( 10 mbar ) we observe a shift of the xps peak centers to the expected positions for monolayer h - bn , i.e. , the b1s to 191.1 ev and n1s to 398.6 ev . concurrently , we see a reduction of the cu in the o1s and culmm spectra during vacuum annealing and the appearance of a defect related component at 191.9/399.3 ev . in turn , after cooling and upon subsequent room temperature re - exposure to air for 2 days , the initial as - loaded positions of the b1s and n1s are recovered ( 190.6/398.1 ev ) along with a re - oxidation of cu . re - heating of triangular h - bn nuclei on cu in 10 mbar vacuum , after sample was stored in ambient air for 4 days . in situ x - ray photoelectron spectra of ( a ) b1s , ( b ) n1s , ( c ) cu auger ( lmm ) , and ( d ) o1s region of as - loaded sample , during subsequent heating in vacuum up to 650 c , post - cooling , and after re - exposure to air ( from bottom to top ) . this behavior , where changes in the xps bes of h - bn are linked to reversible support oxidation / reduction cycles , closely resembles previous observations of oxygen intercalation phenomena under graphene on metals . to the best of our knowledge oxygen intercalation has not been reported yet for h - bn on metals , but previous studies of au intercalated under h - bn on ru , ni , and hydrogen intercalated under h - bn on rh showed xps peak shifts consistent with our interpretation . therefore , our data strongly suggest that air exposure of h - bn on cu leads to oxygen intercalation under the h - bn , i.e. , at the h - bn / cu interface . in this intercalated state monolayer h - bn has a xps fingerprint of 190.6/398.1 ev , resembling the be of few - layer h - bn but being different to the 191.1/398.6 ev measured in figure 3b , c for monolayer h - bn during growth directly on cu . upon vacuum annealing the intercalated oxygen is de - intercalated and desorbed , and thus direct contact between h - bn and cu is ( re-)established albeit with a small number of defects introduced into the h - bn layer . the weak but nevertheless present electronic interaction of cu and monolayer h - bn leads to a small shift in the b1s / n1s bes to 191.1/398.6 ev ( same as during growth ) . thus , our data in figure 6 evidence the decoupling ( upon air exposure ) and recoupling ( upon vacuum annealing ) of monolayer h - bn and the cu support . we emphasize here that coupling does not necessarily imply a strong interaction with the substrate . in the case of few - layer h - bn only the decoupled bes 190.6/398.1 ev are observed , as for the top h - bn layers ( which mainly contribute to the xps signal ) the interaction with the cu support is screened by the h - bn layers below . therefore , in our in situ growth experiments in figure 3b , c where few - layer h - bn was eventually grown , no b1s / n1s peak shifts were observed upon air exposure . while our data strongly suggest intercalation of oxygen , we however note that a second possible scenario could also explain the b1s / n1s peak shifts , where oxygen species decorate edges ( which are present in large number for h - bn islands ) or defect sites on top of the h - bn . oxygen in this scenario is not intercalated under the h - bn but rather adsorbed onto it and then removed by heating . we note that the already slightly back shifting b1s / n1s bes during cooling in vacuum in figure 6 could point to fast oxygen decoration of edge / defects sites from residual oxygen groups in the vacuum chamber accompanied by a small amount of degradation / defect generation in h - bn due to atmospheric contaminants . thus , a combination of fast oxygen decoration of defects on top of h - bn and oxygen intercalation under h - bn at higher oxygen pressures is also conceivable as a combined source of the observed b1s / n1s be shifts upon air exposure / vacuum annealing cycles . irrespective of the detailed configuration of the oxygen causing the b1s / n1s shifts , we find that the evolution upon vacuum - annealing of ex situ grown h - bn samples on cu is highly dependent on their length of exposure to ambient air . in contrast to the short - term air - exposed monolayer sample in figure 6 ( 4 days , termed fresh sample for the remainder of the text ) , figure 7 shows b1s , n1s , and o1s xps core - level signatures for an ex situ grown , full - coverage few - layer h - bn film on cu foil stored in ambient air for 10 months ( termed aged sample ) . the as - loaded b1s and n1s spectra show peaks corresponding to h - bn , and the o1s spectrum shows the presence of copper oxide species ( cu2o ) . for this aged sample , upon heating in vacuum ( 10 mbar ) to 500 c , the intensity of the b1s and n1s increases which is attributed to desorption of adsorbed atmospheric dirt . the b1s and n1s bes remain consistent with few - layer h - bn as expected . concurrently , the o1s spectrum shows the onset of reduction of the cu2o and reaches a state that resembles a sub - stoichiometric oxide or adsorbed o. when the sample is further heated to 700 c all three spectra change drastically . the b1s and n1s peaks reduce dramatically in intensity , and the b1s shows predominantly features of boron oxide ( bxoy ) species at > 191.5 ev . this is accompanied by a corresponding increase in the o1s intensity , also indicating an increase in boron oxide species . this points toward oxygen - mediated disintegration of the h - bn film on the cu foil upon vacuum annealing , where the oxygen was fed from the cu oxide underneath the h - bn . the formed boron oxides are largely volatile resulting in loss of b and n into the gas phase . this is in stark contrast to the higher stability of the fresh h - bn islands in figure 6 which under similar treatment only showed signs of minor degradation . we suggest that the higher degree of oxidation of the aged cu foil provided a larger oxygen reservoir where instead of simple de - intercalation under the h - bn , catalytic oxygen - mediated etching of h - bn occurs ( which in the case of the fresh h - bn on cu is much milder due to the smaller oxygen reservoir in the less oxidized cu ) . reheating of full - coverage , few - layer h - bn films on cu in vacuum ( 10 mbar ) , after sample was stored in ambient air for 10 months : ( a ) b1s , ( b ) n1s , and ( c ) o1s . from top to bottom : as loaded , at 500 c , at 700 c , and post - cooling . to cross - check this hypothesis , we return to annealing similarly fresh h - bn islands as in figure 6 but now anneal in the presence of 1 10 mbar oxygen ( figure 8) . these experimental conditions can be considered as heating the fresh h - bn sample in an infinite oxygen reservoir . figure 8 shows that in the presence of oxygen the islands quickly disintegrate on heating ( confirmed by sem in the inset ) , similar to the aged h - bn film in figure 7 . this confirms our assertion that when sufficient amounts of oxygen are provided ( either from a heavily oxidized cu as in the aged sample or via the gas phase as in figure 8) h - bn on cu is etched via a reaction with oxygen . we emphasize that control experiments with h - bn that was transferred to sio2 , i.e. , removed from the cu catalyst , did not lead to such a drastic disintegration during annealing in air up to 700 c . this highlights the role of the cu catalyst in the proposed oxygen - mediated etching of h - bn . such catalytically mediated oxygen - based etching of h - bn is consistent with recent uhv - based reports of etching of h - bn on ir and ru . re - heating of triangular h - bn nuclei on cu in 1 10 mbar o2 , after sample was stored in ambient air for 15 days : ( a ) b1s , ( b ) n1s , ( c ) o1s , and ( d ) culmm as loaded and during subsequent heating in 1 10 mbar o2 ( from top to bottom ) . the insets show low- and high - magnification sem images before ( left ) and after ( right ) re - heating in o2 . heating in o2 causes the h - bn on cu to disintegrate in contrast to figure 6 but similar to the observations in figure 7 . having established that oxygen can have a drastic detrimental influence on the stability of h - bn on cu during ambient air processing after cvd , we now study whether this impact of oxygen can also be used advantageously during h - bn growth to controllably influence h - bn characteristics by controlled etching . in analogy to graphene cvd we expect that oxygen feeding prior / during borazine exposure will have an impact on nucleation and might change the balance between growth and etching reactions , and thus strongly modify the resulting h - bn film morphologies . confirming this hypothesis , figure 9a , b demonstrates a reduction in nucleation density of h - bn on cu by dosing 1 10 mbar of air after annealing in hydrogen and before borazine exposure , i.e. , between steps 2 and 4 . in the case of air - dosing ( figure 9b ) , the nucleation density is reduced by an order of magnitude compared to the standard oxygen - free growth conditions ( figure 9a ) . the short exposure to air / oxygen after h2 pre - treatment partly re - oxidized the cu surface , and subsequently , upon borazine exposure , boro - thermal reduction ( see figure 5c ) of these surface copper oxides to active metallic cu ( confirmed by xrd measurements ) takes place , which significantly reduces catalyst activity and thus the resulting nucleation density . in figure 9c we show the effect of co - exposure of air and borazine : co - dosing a small air partial pressure alongside the borazine changes the h - bn domain morphology toward an flower - like shape ( figure 9c ) . we suggest that this change of growth morphology is due to increased selective etching reactions occurring concurrent with the growth reactions which might also contribute to a less defined h - bn termination . these observations introduce oxygen pre - treatment and co - feeding as highly relevant parameters toward optimizing h - bn cvd . sem images of h - bn nuclei on cu after 5 min growth , ( a ) similar to figure 3a ( pborazine 1 10 mbar ) and ( b ) with 5 10 mbar air exposure prior to step 3 . air exposure caused an order of magnitude reduction in nucleation density of h - bn on cu for identical processing conditions . ( c ) co - exposure of air ( 1 10 mbar ) and pborazine 1 10 mbar for 30 min leads to nuclei that are irregular in shape . in order to put these results in context , we start by outlining the growth models introduced for graphene . without going into the details of the specific chemistry for each catalyst material , graphene nucleation requires a certain carbon surface concentration which is fed by the precursor dissociation and can be balanced by carbon diffusion in to and out from the catalyst subsurface and bulk . the growth kinetics thereby strongly depend on any possible phase changes of the catalyst surface / bulk , if the carbon incorporates from the catalyst surface or via the bulk , and how the graphene layer edges are anchored on the catalyst surface . in the case of h - bn the growth mechanism is expected to be more complex than for graphene since a balance of two elements , b and n , are needed to form h - bn on the catalyst . while a stoichiometric balance can be introduced into the cvd reactor by selecting a precursor with a pre - defined stoichiometry ( i.e. , borazine in this case with a b : n = 1 ) , the interaction of the constituent elements with the catalyst will dictate the actual supply of the elements during cvd . in this context , diffusion rates and solubility of b , n , or both into the catalyst sub - surface or bulk , metastable or stable boride / nitride formation , and the phase of the catalyst will critically affect the supply of the constituent elements . further , the rate of supply of the constituent elements based on dissociation kinetics , impingement flux of the precursor on the catalyst surface , sticking coefficients , and any related change in interaction between the growing h - bn and the catalyst need to be carefully considered . figure 10a schematically puts forward key points of a kinetic growth model that we propose for h - bn cvd on the basis of a consistent interpretation of the experimental data . our data show no signatures of adsorbed borazine molecules on cu , indicating that at the given conditions borazine dissociates on the reduced cu surface . we also observe a lattice expansion during borazine exposure which suggests uptake of boron into the cu bulk , in agreement with our conjectures from the xps b1s data , where upon post - growth cooling we observe b precipitation from cu . we further see no evidence of bulk nitrogen dissolution or surface nitrogen species in bonding configurations other than those related to bn compounds during exposure . further , the cu catalyst remains in its metallic state during the entire cvd process , and no borides or nitride phases are seen . n phase diagrams as well as with previous uhv measurements of h - bn cvd on rh and ir catalysts and the general metallurgy literature . the simplified schematic of figure 10a neglects a certain level of cu sublimation under low - pressure cvd conditions , but this can be minimized or indeed might be significantly suppressed with h - bn coverage , and hence this complexity is not further discussed here . ( a ) schematic diagram for h - bn growth mechanism on cu , where ji is the impingement flux of the precursor ( borazine ) , rd is the rate of dissociation of the precursor , jb is the diffusion flux of b into the cu subsurface / bulk , jn is the diffusion flux of n away from the catalyst surface , and jb = jn is the flux of b and n required for the formation of h - bn on the catalyst surface . we propose that multilayer h - bn grows beneath the first layer in contact with the catalyst surface as seen for graphene . the schematic neglects a certain level of cu sublimation under low - pressure cvd conditions . ( b ) schematic diagram for stability of h - bn on cu and the oxygen - mediated catalytic dissociation mechanism while heating in air or an oxygen reservoir . the nucleation and growth of h - bn layers is found to occur isothermally , i.e. , at a constant elevated temperature . although a stoichiometrically balanced precursor is used , our data show that the boron and nitrogen supply routes for h - bn growth can be different . the boron dissolution could leave a nitrogen excess at the cu surface which might be balanced by nitrogen desorption , as indicated in figure 10a . whereas boron can enter the growing h - bn either via the bulk or the catalyst surface , the nitrogen is fed only via the catalyst surface , and nitrogen desorption can lead to a situation where the h - bn growth is limited by the nitrogen supply . this is reminiscent of the situation for catalytic iii v nanowire growth , where the group v constituent typically has a low solubility in the conventionally used au catalyst , and despite a high bulk solubility of the group iii element atomically sharp heterostructures can be obtained . the focus of prior literature on h - bn cvd has been on the balance of gaseous precursors . our data here highlight that , for further process optimization , it is crucial to consider and understand the catalyst - mediated flux balance as highlighted for cu in figure 10a . considering the growth of 2d multilayer films and heterostructures , the complexity of the kinetics of catalyst mediated flux balances increases for h - bn . growth of h - bn multilayers can be seen analogous to graphene cvd , where additional layers nucleate and grow only in contact with the metal catalyst , i.e. , at the interface between the catalyst and an existing graphene layer . the feeding of such additional layer will depend on the leakage rate of both ( b , n ) constituent species through an existing 2d layer . further , for the growth of h - bn / graphene heterostructures via , e.g. , sequential exposure to carbon and boron / nitrogen precursors , complex interactions can be expected not only for the catalytic precursor dissociation on the catalyst surface but also for the catalyst - mediated flux balances . sutter et al . have studied potential smear - out effects during h - bn - graphene heterostructures synthesis and reported techniques such a pulsed oxygen etch to address reservoir effects and improve the sharpness of transitions , albeit this has been very specific so far to the exposure sequence and catalyst material ( ru ) . we see our insights here as being highly relevant as a framework for the understanding and further optimization of a range of compound 2d materials and more complex cvd grown heterostructures . the catalyst support is relevant not only to the cvd growth process but , as our data show , also to the post - growth stability of as - grown h - bn , which is crucial to post - growth processing and numerous applications . practically all current use and processing of cvd grown h - bn involves some stage of ambient air exposure when the material is still on the catalyst . figure 10b schematically summarizes the key points regarding catalyst- and oxygen - mediated thermal h - bn stability . we find that post growth ambient air exposure leads to intercalation of oxygen under h - bn on cu . while oxygen can reach the interface through inherent defects in the h - bn layer ( including point defects , vacancy defects , stone wales defects , or out - of - plane bonds ) and domain boundaries , even when the catalyst surface is completely covered with monolayer and few - layer h - bn , the rate of arrival at the interface is expected to reduce significantly with increasing number of h - bn layers . interacts with cu . for extended air exposure cu oxidation is observed , and upon re - heating in vacuum an oxygen - mediated disintegration of the h - bn film occurs via volatile boron oxides . i.e. , occurs at the catalyst / h - bn interface and depends on the catalyst and the level of oxygen fed to this interface ( figure 10b ) . this mechanism is in conceptual agreement with uhv - based reports of h - bn etching on ir and ru . it is important to note that annealing of h - bn films transferred to a 300 nm sio2 /si wafer support in air up to 700 c did not lead to such a drastic disintegration consistent with recent reports . hence our data highlight that key to the thermal stability are the catalyst interaction and the level of oxygen fed to the catalyst interface . for few - layer and multilayer h - bn this means that the layer most likely to be etched is the one in contact with the catalyst as long as an oxygen leakage pathway is provided . we note that for certain catalysts , in particular ni , which more strongly interact with the 2d layer on its surface , graphene has been shown to passivate the catalyst surface and prevent oxygen from reaching the catalyst / graphene interface . this further highlights the importance of the catalyst interaction , and the resultant different levels of oxygen intercalation can explain previous reports in literature for instance on the stability of few - layer h - bn on cu being limited to 500 c compared to 1100 c for ni . combining the discussion of the h - bn growth kinetics ( figure 10a ) and thermal stability ( figure 10b ) , it is obvious that the presence of oxygen during cvd can affect the h - bn formation in multiple ways . a more oxidized catalyst surface is likely to reduce the h - bn nucleation density , assuming a catalyst surface reduction is required . the presence of oxygen might also change the catalyst / h - bn interaction and change the b and n flux balance , hence affecting the shape of growing h - bn domains and their growth rate . similar to graphene cvd , we note oxygen ( co-)exposure should be considered an important aspect of the parameter space for h - bn cvd . although a detailed study of the effect of gaseous additions to the cvd atmosphere is beyond the scope of this paper , we emphasize that the insights provided here give a fundamental framework and highlight multiple avenues toward more advanced integrated growth and integration of h - bn and other related compound 2d materials . in summary , we have used a combination of complementary in situ xps and xrd during h - bn cvd to gain a fundamental understanding of the mechanisms underlying the growth of h - bn on polycrystalline cu under scalable cvd conditions . h - bn was shown to grow isothermally on cu during exposure to borazine at elevated temperature . the growth , however , is not limited to a surface - mediated growth mechanism as previously reported . expansion of the cu lattice during borazine exposure at high temperature and elemental b precipitation from cu upon cooling confirm b incorporation into the bulk of the catalyst . cu maintains its metallic state during h - bn cvd , and no additional boride or borate phases are seen . our in situ insights into the mechanisms of h - bn growth provide guidelines for future rational cvd process engineering towards controlled h - bn film characteristics such as film quality , domain size , or number of layers . the element - specific mechanistic insights obtained here are highly relevant as a framework for the understanding and further optimization of a range of compound 2d materials and more complex cvd - grown heterostructures . after cvd , we find that exposure to ambient air leads to intercalation of oxygen under h - bn on cu . the stability of h - bn following this air exposure is strongly dependent on the catalyst interaction and accumulated oxygen dose , where high oxygen levels lead to drastic catalytically mediated h - bn etching and h - bn disintegration on cu upon processing at elevated temperatures . in turn , however , these etching effects could potentially open new routes to control the growth morphology of h - bn when oxygen is pre - dosed or co - fed under controlled conditions during h - bn cvd . large - scale processing schemes for h - bn integration and processes involving growth of in - plane / out - of - plane h - bn/2d material heterostructures will have to carefully consider these findings in terms of allowable levels of oxygen exposure during ( re-)processing and suggested use of h - bn as high - temperature passivation layers . cvd of h - bn using borazine ( ( hbnh)3 , fluorochem , uk ) as precursor was performed in customized in situ compatible cold - wall cvd reactors on cold - rolled polycrystalline cu foils ( alfa aesar , 25 m thick , 99.999% purity ) at 9501000 c and at pborazine 1 105 10 mbar for 530 min . borazine vapor was introduced into the reaction chambers via leak valves from liquid borazine reservoirs . the cvd process commonly consisted of a h2 pre - treatment step ( 0.2 mbar ) , followed by pump - down to base pressure , and then the precursor was introduced to initiate h - bn growth . after a pre - defined growth time the borazine precursor was removed , followed by pump - down to base pressure and cooling in vacuum . figure 1 summaries the salient steps during the h - bn cvd process . for experiments with air pre - dosing , the dosing was performed after h2 annealing , i.e. , between steps 2 and 3 in figure 1 . the isiss end station of fhi mpg at the bessy ii synchrotron was used for in situ xps measurements . further details on the experimental setup / measurements can be found elsewhere . in situ xrd ( 2 geometry ) was performed at the bm20 beamline ( rossendorf beamline ) of the european synchrotron radiation facility ( esrf ) in a cold - wall stainless steel reactor mounted on a high - precision six - circle goniometer . the kapton windows of the reaction chamber allow transmission of x - rays at different scattering geometries . a monochromatic x - ray beam of 11.5 kev ( corresponding wavelength of 1.078 ) was used , and the diffracted x - rays were measured with a 1d line detector ( mythen ) . cu powder ( alfa aesar , < 5 m , 99.9% purity ) pressed into a thick granular film onto a sapphire wafer was used as the model system since the high degree of texture in cold rolled cu foils prevented reliable measurement of the powder diffraction geometry . a boron nitride - coated graphite resistive heating element ( boralectric ) was used to heat the sample inside the chamber . the sample was clamped down with alumina spacers , and the temperature was measured with a thermocouple in contact with the sapphire substrate . gases were fed via computer - controlled mass flow controllers and the borazine via a manual leak valve . quantitative lattice parameters were derived by rietveld refinement using xpert plus and file 64699 ( fcc cu ) from the inorganic crystal structure database ( icsd ) . scanning electron microscopy ( sem , carl zeiss sigma vp , 12 kv ) and raman spectroscopy ( renishaw in - via 532 nm laser ) after transfer , using a pmma support layer as described in detail elsewhere , were used to characterize h - bn growth . we note that the presented sem images are representative of measurements across several macroscopically separated points on the samples . an fei titan 80 - 300 etem equipped with a monochromator at the electron gun and a spherical aberration ( cs)-corrector for the objective lens was used for acquiring tem images . samples are heated for 1 h at 350 c in a vacuum prior to imaging . images were acquired at 80 kv , energy spread < 0.3 ev , and corrector alignment to minimize cs to obtain a resolution better than 0.12 nm .

using a combination of complementary in situ x - ray photoelectron spectroscopy and x - ray diffraction , we study the fundamental mechanisms underlying the chemical vapor deposition ( cvd ) of hexagonal boron nitride ( h - bn ) on polycrystalline cu . the nucleation and growth of h - bn layers is found to occur isothermally , i.e. , at constant elevated temperature , on the cu surface during exposure to borazine . a cu lattice expansion during borazine exposure and b precipitation from cu upon cooling highlight that b is incorporated into the cu bulk , i.e. , that growth is not just surface - mediated . on this basis we suggest that b is taken up in the cu catalyst while n is not ( by relative amounts ) , indicating element - specific feeding mechanisms including the bulk of the catalyst . we further show that oxygen intercalation readily occurs under as - grown h - bn during ambient air exposure , as is common in further processing , and that this negatively affects the stability of h - bn on the catalyst . for extended air exposure cu oxidation is observed , and upon re - heating in vacuum an oxygen - mediated disintegration of the h - bn film via volatile boron oxides occurs . importantly , this disintegration is catalyst mediated , i.e. , occurs at the catalyst / h - bn interface and depends on the level of oxygen fed to this interface . in turn , however , deliberate feeding of oxygen during h - bn deposition can positively affect control over film morphology . we discuss the implications of these observations in the context of corrosion protection and relate them to challenges in process integration and heterostructure cvd .

Introduction Results Discussion Conclusions Methods

chemical vapor deposition ( cvd ) has emerged as the most viable route for high - quality graphene synthesis , and a lot of progress has also been made in developing cvd processes for other 2d materials , such as hexagonal boron nitride ( h - bn ) and related 2d heterostructures . while graphene cvd relies on the interactions of carbon with the surface , sub - surface , and bulk of the catalyst , the growth mechanisms for compound 2d materials such as h - bn are inherently more complex , as two elements need to be fed and incorporated into the growing nanostructure and bond polarity can affect the structural formation . the interaction of h - bn with the catalyst also affects h - bn transfer and an increasing number of applications , such as oxidation barriers and corrosion protection , that utilize h - bn as - grown on top of the catalyst metal . here , using a combination of in situ x - ray photoelectron spectroscopy ( xps ) and x - ray diffraction ( xrd ) during actual synthesis conditions , we fingerprint the entire h - bn cvd growth process on polycrystalline cu using borazine , ( hbnh)3 , as the precursor . we focus on the important questions of how the precursor and constituent b and n as well as the as - grown h - bn interact with the catalyst during and after growth . our data show that , at the typically used growth temperatures , the borazine molecule decomposes and b incorporates into the bulk of the cu catalyst . compared to graphene cvd , the role of the surface , sub - surface , and bulk of the catalyst is more complex , considering that n has a considerably lower solubility in cu and its incorporation into the cu bulk is not detected . we further show that after h - bn cvd and during commonly used further processing of h - bn on the cu catalyst in ambient air , oxygen intercalation readily occurs under as - grown h - bn and negatively affects the stability of h - bn on the catalyst . for extended air exposure cu oxidation is observed , and upon reheating in vacuum an oxygen - mediated disintegration of the h - bn film via volatile boron oxides occurs . of key importance is that this disintegration is catalyst mediated , i.e. , occurs at the catalyst / h - bn interface and depends on the level of oxygen present at this interface . in turn , however , we demonstrate that pre- and co - exposure with oxygen species can be used to influence h - bn growth morphologies . we discuss the implications of these observations in the context of corrosion protection and relate them to challenges for process integration and heterostructure growth . , post ambient air exposure ( step 7 , figure 1 ) ] acquired on the h - bn films on cu show in the b1s region a main component at 190.6 ev ( figure 2e , inset additionally shows the bond shake up satellite at 200 ev ) and in the n1s region a main component at 398.1 ev ( figure 2f ) . figure 3a and figure s1 in the supporting information show post - growth ( step 7 , figure 1 ) scanning electron microscopy ( sem ) images of as - grown h - bn on polycrystalline cu foil for 5 , 15 , and 30 min of borazine exposure . longer exposures lead here to the formation of more irregular - shaped domains ( figure 3a , 900 s ) , which eventually cover the entire cu surface , as indirectly confirmed by the wrinkles in the resulting h - bn film ( figure 3a , 1800 s , and figure s1c ) . ( b , c ) in situ xps confirming isothermal h - bn growth on cu : in situ cvd process - resolved xps b1s ( b ) and n1s ( c ) core - level scans at 975 c before ( step 3 ) , during ( step 4 ) , and after borazine exposure ( step 5 ) , after cooling ( step 6 ) , and after air exposure ( step7 ) . the time - resolved b1s spectra during borazine exposure ( inset in figure 3b , step 4 ) show that upon borazine introduction a broad peak in the b1s spectra emerges after 290 s. this is consistent with the time scale of the nucleation and growth of h - bn domains observed ex situ by sem ( figure 3a ) . therefore , we interpret the observed initial emergence of the two peaks at 190.6/398.1 and 191.1/398.6 ev at equal intensities followed by domination of the spectrum by the 190.6/398.1 ev component for extended exposure times as evidence for isothermal growth of monolayer h - bn that interacts with the cu substrate ( 191.1/398.6 ev ) and some fraction of multilayer h - bn nuclei ( 190.6/398.1 ev ) , where the initial monolayer domains further evolve to multilayers with extended exposure time ( hence the decrease of 191.1/398.6 ev contribution relative to the 190.6/398.1 ev peaks ) . we suggest that the observed peak shift between 191.1/398.6 and 190.6/398.1 ev is caused by interactions of h - bn with the cu substrate , similar to monolayer graphene i.e. our observations so far indicate isothermal growth of h - bn on cu with initial monolayer nucleation ( which then evolve toward few - layer h - bn ) with a small amount of defects incorporated in the h - bn films ( lattice , edges , grain boundaries ) . the in situ xps b1s and n1s fingerprints of growing h - bn are preserved upon removal of borazine at temperature ( step 5 , figure 1 ) , but interestingly one small additional component emerges upon cooling of the sample in vacuum in the b1s at 188.1 ev ( step 6 , figure 1 ) . this assignment of atomic b ( non - nitrogen bonded ) is also corroborated by the lack of a concurrently emerging n1s component upon cooling , as well as by the disappearance of the 188.1 ev signal upon subsequent air exposure , since atomic boron is known to quickly oxidize in ambient air and desorb as highly volatile boron oxides . the emergence of atomic b on the catalyst surface upon cooling strongly implies b precipitation from the catalyst bulk and in turn suggests dissolution of b in the polycrystalline cu subsurface / bulk during the high - temperature borazine exposure . figure 4a shows xrd patterns recorded during cvd , starting with as loaded cu catalyst ( before step 1 , figure 1 ) , after h2 anneal ( step 3 , figure 1 ) , during borazine exposure ( step 4 , figure 1 ) , and after ambient air exposure ( step 7 , figure 1 ) . as ammonia is known to dissociate on cu in this temperature regime , our data imply that n and h uptake is not responsible for the increase in the cu lattice constant , and instead b is dissolved into the cu during growth . rather our data indicate element - specific feeding mechanisms including the bulk of the catalyst . we suggest that b is taken up in the cu catalyst while n is not ( by relative amounts ) , which is also in agreement with thermodynamic phase diagrams . bulk crystallography of the cu catalyst during isothermal growth of h - bn by cvd measured using in situ xrd . we note that the final in situ xps fingerprint after air exposure ( step 7 ) in figure 3b , c resembles our x - ray photoelectron spectra of the ex situ grown few - layer h - bn in figure 2e , f which highlights the validity and relevance of our in situ measurements for scalable high - quality h - bn growth under realistic process conditions . in addition to following the evolution of b and n species during h - bn cvd , in situ xps also allows us ( within its detection limits ) to observe the surface chemistry of the cu catalyst by simultaneously monitoring the cu2p , culmm , o1s ( figure 5 ) , and valence band ( supporting information , figure s4 ) regions . the oxidation of the cu upon air exposure implies that the inherent polycrystallinity of cvd grown h - bn film provides diffusion pathways for species to reach the cu / h - bn interface . surface chemistry of the cu catalyst during isothermal growth of h - bn by cvd : in situ xps ( a ) cu2p3/2 , ( b ) culmm auger , and ( c ) o1s spectra after h2 anneal in vacuum ( step 3 ) , during borazine exposure ( step 4 ) , after exposure ( step 5 ) , after cooling in vacuum ( step 6 ) , and after ambient air exposure ( step 7 ) . to further probe this evolution of h - bn on cu catalysts during ambient air exposure ( step 7 , figure 1 ) , we undertake complementary experiments with ex situ grown h - bn islands on cu , for which the borazine exposure was limited to 5 min to obtain islands of monolayer h - bn . interestingly , however , upon annealing the sample in vacuum ( 10 mbar ) we observe a shift of the xps peak centers to the expected positions for monolayer h - bn , i.e. in turn , after cooling and upon subsequent room temperature re - exposure to air for 2 days , the initial as - loaded positions of the b1s and n1s are recovered ( 190.6/398.1 ev ) along with a re - oxidation of cu . in situ x - ray photoelectron spectra of ( a ) b1s , ( b ) n1s , ( c ) cu auger ( lmm ) , and ( d ) o1s region of as - loaded sample , during subsequent heating in vacuum up to 650 c , post - cooling , and after re - exposure to air ( from bottom to top ) . therefore , our data strongly suggest that air exposure of h - bn on cu leads to oxygen intercalation under the h - bn , i.e. in the case of few - layer h - bn only the decoupled bes 190.6/398.1 ev are observed , as for the top h - bn layers ( which mainly contribute to the xps signal ) the interaction with the cu support is screened by the h - bn layers below . thus , a combination of fast oxygen decoration of defects on top of h - bn and oxygen intercalation under h - bn at higher oxygen pressures is also conceivable as a combined source of the observed b1s / n1s be shifts upon air exposure / vacuum annealing cycles . irrespective of the detailed configuration of the oxygen causing the b1s / n1s shifts , we find that the evolution upon vacuum - annealing of ex situ grown h - bn samples on cu is highly dependent on their length of exposure to ambient air . in contrast to the short - term air - exposed monolayer sample in figure 6 ( 4 days , termed fresh sample for the remainder of the text ) , figure 7 shows b1s , n1s , and o1s xps core - level signatures for an ex situ grown , full - coverage few - layer h - bn film on cu foil stored in ambient air for 10 months ( termed aged sample ) . this points toward oxygen - mediated disintegration of the h - bn film on the cu foil upon vacuum annealing , where the oxygen was fed from the cu oxide underneath the h - bn . we suggest that the higher degree of oxidation of the aged cu foil provided a larger oxygen reservoir where instead of simple de - intercalation under the h - bn , catalytic oxygen - mediated etching of h - bn occurs ( which in the case of the fresh h - bn on cu is much milder due to the smaller oxygen reservoir in the less oxidized cu ) . this highlights the role of the cu catalyst in the proposed oxygen - mediated etching of h - bn . re - heating of triangular h - bn nuclei on cu in 1 10 mbar o2 , after sample was stored in ambient air for 15 days : ( a ) b1s , ( b ) n1s , ( c ) o1s , and ( d ) culmm as loaded and during subsequent heating in 1 10 mbar o2 ( from top to bottom ) . having established that oxygen can have a drastic detrimental influence on the stability of h - bn on cu during ambient air processing after cvd , we now study whether this impact of oxygen can also be used advantageously during h - bn growth to controllably influence h - bn characteristics by controlled etching . in analogy to graphene cvd we expect that oxygen feeding prior / during borazine exposure will have an impact on nucleation and might change the balance between growth and etching reactions , and thus strongly modify the resulting h - bn film morphologies . confirming this hypothesis , figure 9a , b demonstrates a reduction in nucleation density of h - bn on cu by dosing 1 10 mbar of air after annealing in hydrogen and before borazine exposure , i.e. the short exposure to air / oxygen after h2 pre - treatment partly re - oxidized the cu surface , and subsequently , upon borazine exposure , boro - thermal reduction ( see figure 5c ) of these surface copper oxides to active metallic cu ( confirmed by xrd measurements ) takes place , which significantly reduces catalyst activity and thus the resulting nucleation density . in the case of h - bn the growth mechanism is expected to be more complex than for graphene since a balance of two elements , b and n , are needed to form h - bn on the catalyst . we also observe a lattice expansion during borazine exposure which suggests uptake of boron into the cu bulk , in agreement with our conjectures from the xps b1s data , where upon post - growth cooling we observe b precipitation from cu . ( a ) schematic diagram for h - bn growth mechanism on cu , where ji is the impingement flux of the precursor ( borazine ) , rd is the rate of dissociation of the precursor , jb is the diffusion flux of b into the cu subsurface / bulk , jn is the diffusion flux of n away from the catalyst surface , and jb = jn is the flux of b and n required for the formation of h - bn on the catalyst surface . ( b ) schematic diagram for stability of h - bn on cu and the oxygen - mediated catalytic dissociation mechanism while heating in air or an oxygen reservoir . the nucleation and growth of h - bn layers is found to occur isothermally , i.e. whereas boron can enter the growing h - bn either via the bulk or the catalyst surface , the nitrogen is fed only via the catalyst surface , and nitrogen desorption can lead to a situation where the h - bn growth is limited by the nitrogen supply . the catalyst support is relevant not only to the cvd growth process but , as our data show , also to the post - growth stability of as - grown h - bn , which is crucial to post - growth processing and numerous applications . practically all current use and processing of cvd grown h - bn involves some stage of ambient air exposure when the material is still on the catalyst . while oxygen can reach the interface through inherent defects in the h - bn layer ( including point defects , vacancy defects , stone wales defects , or out - of - plane bonds ) and domain boundaries , even when the catalyst surface is completely covered with monolayer and few - layer h - bn , the rate of arrival at the interface is expected to reduce significantly with increasing number of h - bn layers . for extended air exposure cu oxidation is observed , and upon re - heating in vacuum an oxygen - mediated disintegration of the h - bn film occurs via volatile boron oxides . , occurs at the catalyst / h - bn interface and depends on the catalyst and the level of oxygen fed to this interface ( figure 10b ) . hence our data highlight that key to the thermal stability are the catalyst interaction and the level of oxygen fed to the catalyst interface . this further highlights the importance of the catalyst interaction , and the resultant different levels of oxygen intercalation can explain previous reports in literature for instance on the stability of few - layer h - bn on cu being limited to 500 c compared to 1100 c for ni . in summary , we have used a combination of complementary in situ xps and xrd during h - bn cvd to gain a fundamental understanding of the mechanisms underlying the growth of h - bn on polycrystalline cu under scalable cvd conditions . expansion of the cu lattice during borazine exposure at high temperature and elemental b precipitation from cu upon cooling confirm b incorporation into the bulk of the catalyst . after cvd , we find that exposure to ambient air leads to intercalation of oxygen under h - bn on cu . the stability of h - bn following this air exposure is strongly dependent on the catalyst interaction and accumulated oxygen dose , where high oxygen levels lead to drastic catalytically mediated h - bn etching and h - bn disintegration on cu upon processing at elevated temperatures . in turn , however , these etching effects could potentially open new routes to control the growth morphology of h - bn when oxygen is pre - dosed or co - fed under controlled conditions during h - bn cvd .

as the demand and usage of polymers increases , so do issues associated with the sustainability of their source materials , their end of life fates , and costs . economic and environmental concerns have driven the quest to replace some petroleum - derived materials with alternative polymers sourced from renewable raw materials and with improved end of life options . polylactic acid ( pla ) is one such material : it is efficiently prepared from starch and after use can be either composted or recycled . although current production is around 100 000 t / a , growth in manufacture is predicted , and its properties make it suitable for applications spanning packaging , fibers , composites , and medical devices . pla is manufactured by the ring - opening polymerization ( rop ) of lactide ( la ) ( figure 1 ) , a process that is initiated by lewis acidic metal alkoxide complexes . quite a range of metals have precedent for initiating rop , and for further details on the scope of this catalysis , the reader is referred to several comprehensive reviews . there has been a long - standing academic interest in aluminum - based initiators , due to the excellent control conferred by this metal and the potential for some al complexes to enable stereocontrolled rop of rac - lactide . from the materials property perspective , the production of stereoblock or stereocomplex pla , via the iso - selective rop of rac - la , remains a high priority target . such polymers exhibit significantly improved thermal - mechanical properties as compared to isotactic pla ; for example , the tm for isotactic pla is 170 c , whereas that for stereoblock / stereocomplex pla has been reported from 170 to 230 c . aluminum initiators have a strong track record in the production of stereoblock / complex pla from rac - la . so far , the most iso - selective catalysts reported have been aluminum salen complexes . these are important academic targets , but the very slow rate of polymerization and high catalyst loadings required are significant drawbacks to these systems . recently , several groups have reported improved activity and good iso - selectivities using indium catalysts . for example , mehrkhodavandi and co - workers reported the dinuclear complex , a ( figure 2 ) , which polymerized 200 equiv of rac - la in 30 min ( 298 k , methylene chloride , 0.5 m in [ la ] , 90% conversion , pi = 0.62 ) . this is a marked increase in rate as compared to the al salen systems , which typically take hours to achieve high conversions and require increased temperatures . recently , the same group has extended the study of dinuclear and mononuclear indium complexes , resulting in efficient catalysis with a moderate - good isotactic bias ( pi = 0.77 ) in the best cases . during the past 6 years , various other researchers have also reported promising indium - based catalysts , showing good rates and control . one attractive system was reported by tolman and hillmyer , where incl3 , in the presence of an equivalent each of an alcohol and triethylamine , polymerized rac - la quite rapidly and with high heteroselectivity ( ps = 0.97 , 1:100 [ i]:[la ] , 5 h ) . another example of an interesting indium complex was a racemic homoleptic species , b ( figure 2 ) , reported by okuda and arnold , which was found to induce a slight isotactic bias ( pi = 0.63 ) . given the significant potential for al and in initiators , it was somewhat surprising to discover that ga complexes have been far less investigated in the rop of la . the first report of a dialkyl gallium alkoxide complex , c ( figure 2 ) , demonstrated the potential of this group 13 congener for fast , controlled polymerization catalysis . a recent comparative study of aluminum and gallium complexes , with the structures represented by compound d ( figure 2 ) , showed that the ga catalysts were faster and equally well controlled as the al counterparts ( ga > 99% conversion , 100 equiv of la , 353 k , toluene 1 h , versus al 45% conversion under the same conditions ) . despite the difference in rate , the change in metal center did not affect the polymerization stereoselectivity , with moderate iso - selectivity being observed for both the al and the ga initiators ( pi = 0.70 ) . examples of the structures of some stereoselective in and ga initiators for rac - la rop . we have previously reported a series of bis(8-quinolinolato ) aluminum ethyl complexes , which , in the presence of iso - propyl alcohol , were efficient , iso - selective initiators ( pi = 0.76 ) , for the rop of rac - la . in this article , the preparation and application of a series of gallium complexes , with some of the same 8-quinolinato ligands , is presented . two 8-hydroxyquinoline ligands , 1 and 2 , were selected for investigation as ancillary ligands for gallium polymerization initiators ( figure 3 ) . the ligands were selected as both are commercially available compounds , an attractive feature if polymerization activity of the initiators can be increased . furthermore , the aluminum complexes of 1 and 2 showed distinct differences in polymerization activity with the halide substituents of ligand 2 enhancing activity and iso - selectivity . the structures of the pro - ligands , 1 and 2 . pro - ligands 1 and 2 were reacted with gallium(iii)chloride , in toluene at 298 k , to form bis(8-quinolinolato ) gallium chloride compounds , 1a and 2a ( figure 4 ) . the completion of the complexation reaction was confirmed by h nmr spectroscopy where the resonances assigned to the quinolinolato protons were at higher chemical shift as compared to the pro - ligands , due to the lewis acidic ga center deshielding the nuclei . for each complex , the stoichiometry was further confirmed by elemental analysis and mass spectrometry . for compound 2a , single crystals suitable for x - ray diffraction experiments were obtained from a saturated toluene solution . reagents and conditions : ( i ) gacl3 , toluene , 298 k , 12 h , 1a ( 51% ) , 2a ( 53% ) ; ( ii ) kotbu , benzene - d6 , 298 k , 24 h , product not isolated . compounds 1a and 2a were tested as initiators for the rop of rac - la ( 1 m solution of la in thf , 298 k , 20 h ) but showed no activity . this is in line with the findings of other researchers that metal halide complexes are rarely effective initiators , presumably due to the high m cl bond strength ( 481 kj mol versus 285 kj mol for ga to prepare active initiators , the gallium chloride complexes were reacted further to generate new alkoxide and amide complexes . thus , various salt metathesis reactions , with the relevant potassium salts ( figure 4 ) , were attempted . the reactions of either compound 1a or 2a with potassium tert - butoxide , potassium ethoxide , or potassium bis(trimethylsilyl)amide all resulted in a mixture of products . as an illustrative example , the reaction between 2a and potassium tert - butoxide yielded a yellow powder whose h nmr spectrum indicated the presence of the desired alkoxide complex 2b ( vide infra ) together with another complex , showing only resonances for the quinolinate ligands . the product was determined to be the oxo - bridged dimeric complex [ ( l2ga)2(-o ) ] , which formed due to undesired oxidation of the target alkoxide complex ( see the supporting information ) . the oxygen / moisture necessary for such a degradation reaction is presumed to have arisen from residual oxygen / water in the reaction / nmr solvents . it should be noted that the same solvents were applied successfully for the isolation of aluminum and gallium alkyl complexes , without any such degradation , illustrating the high sensitivity of the alkoxide derivatives . the pure alkoxide complex 2b was prepared by an nmr scale reaction , in a young s tap nmr tube with centrifugation to remove the kcl byproduct ( supporting information figure s1 ) . the h nmr spectrum shows that all of the quinolinate resonances are shifted to lower field , and furthermore the singlet at 1.17 ppm , with a relative integral of 9 , confirms the presence of the tert - butyl alkoxide . the low intensity signal at 1.05 ppm corresponds to residual tert - butyl alcohol . crystals suitable for x - ray diffraction analyses ( vide infra ) were grown by addition of hexane to this solution . as an alternative route to gallium alkoxide complexes , gallium alkyl complexes [ l2gar ] ( r = alkyl ) were targeted as suitable precursors for further reaction , via protonolysis , with various alcohols . the reaction of 2 equiv . of the pro - ligands , 1 and 2 , with trimethylgallium resulted only in the formation of the monoligated complexes 1c and 2c [ lgame2 ] together with the residual pro - ligands . it is relevant that the reaction was conducted under the same conditions as had previously enabled the isolation of the bis - ligated aluminum ethyl complexes [ l2alet ] . changing the conditions , for example , by using more polar solvents such as thf or pyridine , resulted solely in the formation of the same monoligated product . it was only possible to form some of the desired bis - ligated gallium complexes , by refluxing the solutions in toluene for 24 h. even under such forcing conditions , complex 2c was only partly converted , in low yield ( 25% ) , to the desired bis - ligated product 2d . given the preferential formation of monoligated complexes , these species were themselves directly targeted . complexes 1c and 2c were prepared in high yields ( 74% and 87% , respectively ) by reaction between equimolar quantities of trimethylgallium and the appropriate pro - ligands ( figure 5 ) . the complex formation was confirmed by h nmr spectroscopy , where characteristic shifts in the quinolinolate resonances were observed . a singlet peak was observed between 0 and 0.1 ppm , with a total relative integral of 6 , due to the two gallium coordinated methyl groups . synthetic routes attempted for the preparation of bis(8-quinolinolato ) gallium methyl compounds , and the synthesis of compounds 1c , 2c , and 2d . reagents and conditions : ( i ) 0.5 equiv of game3 , toluene , 298 k ; ( ii ) game3 , toluene , 298 k ( 1c 74% , 2c 87% ) ; ( iii ) 0.5 equiv of game3 , toluene , 393 k ( 25% ) . crystals suitable for x - ray crystallographic analysis were isolated for compounds 2a and 2b from toluene and benzene - d6/hexane solutions , respectively . both compounds crystallized in centrosymmetric ( i.e. , racemic ) space groups . the structure of 2a has c2 symmetry about an axis that passes through the gallium and chlorine atoms ( figure 6 ) , while the structure of 2b contains two crystallographically independent complexes , 2b - a ( shown in figure 7 ) and 2b - b ( shown in supporting information figure s4 ) . all three molecules have pentacoordinate gallium centers with similar distorted trigonal bipyramidal coordination geometries , the values being 0.69 , 0.57 , and 0.57 for 2a , 2b - a , and 2b - b , respectively . the greater deviation of 2b - a and 2b - b from the trigonal bipyramidal geometry is due to the steric bulk of the tert - butoxide coligand . as with the aluminum complexes , in every case the only geometric isomer observed has the nitrogen atoms of the ligand coordinated trans to one another in the axial positions . the o - donors , from the quinolinolate rings , are coordinated at equatorial sites , with the remaining site being occupied by a chloride or alkoxide coligand , for 2a and 2b , respectively . n bond lengths to the quinolinolato ligand differ between 2a and 2b ( a and b ) significantly ; in the case of 2a , all of the ga o and ga n bonds are shorter than those in 2b - a and 2b - b . again , this can be attributed to the larger tert - butoxide group versus chloride ( figure 6 and table 1 ) . in both cases , the ga n and al o bonds previously observed for the aluminum quinolinolate complexes . the molecular structure of the c2-symmetric complex 2a . selected bond lengths ( ) : ga the structure of one ( 2b - a ) of the two independent complexes present in the crystals of 2b . compounds 1c , 2b d were tested as initiators in the rop of rac - la ( table 2 ) . polymerizations were conducted under a standard set of conditions : using a 1 m solution of lactide in toluene , at 348 k with an initiator concentration of 10 mm . in the case of compounds 1c and 2c , an equivalent ( vs initiator ) of isopropyl alcohol was added so as to enable generation of the active gallium alkoxide species in situ . the polymerizations were monitored by taking aliquots at regular time intervals , which were analyzed using h nmr spectroscopy , to determine the la conversion , and by gpc - malls ( gel permeation chromatography - multiangle laser light scattering ) , to determine the number average molecular weight , mn , and the polydispersity index , pdi . in each case , the polymerization rate was compared by determination of the pseudo - first - order rate constant , kobs , which is the gradient of the linear plots of ln([la]0/[la]t ) versus time . the tacticity of the pla obtained using compound 2b was determined by integration of the methyne region of the homonuclear decoupled nmr spectrum . the normalized integrals were compared against the values predicted using bernoullian statistics to give the probability of isotactic linkages , pi . polymerization conditions : toluene , 348 k , 1:1:100 [ i]:[iproh]:[la ] , 1 m [ la ] . determined by integration of the methine region of the h nmr spectrum ( la 4.985.04 ppm ; pla 5.085.22 ppm ) . determined from the gradients of the plots of ln{[la]0/[la]t } versus time , where the average errors = 910% ( determined using initiator 1c ) . determined by gpc in thf , using multiangle laser light scattering ( gpc - malls ) . determined by analysis of all of the tetrad signals in the methine region of the homonuclear decoupled h nmr spectrum . because of the previously discussed problems with the isolation of compound 2b , the compound was synthesized in situ at a known concentration in toluene . the potassium chloride salt was removed by centrifugation , and the stock solution was added to the polymerization mixture . at this point , it is worth highlighting that the stoichiometric salt metathesis reaction proceeds to full conversion , as observed in the h nmr experiments . we can therefore eliminate the possibility of any excess potassium tert - butoxide salt , a known active initiator for the rop reaction , initiating the polymerization . the initiator , 2b , was moderately active in the rop of rac - lactide , with the polymerization proceeding to high conversion in excess of 50 h. such rates are typical for discrete group 13 initiators , and indeed the polymerization occurs at a rate similar to that of the previously reported bis(8-quinolinolato ) aluminum ethyl initiators . the initiator gives rise to pla with a moderate degree of isotactic enchainment , with a pi value of 0.70 consistent with a bias toward a stereoblock microstructure ( supporting information figure s8 ) . there are only two other examples of iso - selective gallium initiators : compound d ( figure 2 , pi = 0.70 ) and a dimethyl gallium n - heterocyclic carbene complex , reported by horeglad and co - workers , where a pi value of up to 0.78 was claimed . the initiator bis(5,7-dichloro-2-methyl-8-quinolinolato ) gallium tert - butoxide , 2b , is analogous to the bis(5,7-dichloro-2-methyl-8-quinolinolato ) aluminum ethyl initiator , where the active alkoxide initiator is formed in situ by reaction with isopropyl alcohol during the course of the rop reaction . initiator 2b is significantly faster than its aluminum analogue , with around a 3 times higher kobs value ( 1.3 10 s for 2b vs 5.0 10 s for the al analogue ) ( figure 8) . this finding is in line with the only other comparison between ga and al complexes , in that case using -n , o , n-{(c6f5n c6h4)2o}mnme2-type complexes . the increased activity of the gallium compounds is tentatively attributed to the reduced lewis acidity of ga resulting in a weaker or more labile gallium alkoxide bond . in addition to the beneficial increase in rates , the ga complex also shows high iso - selectivity and good polymerization control ( equaling those observed with the al analogue ) . this is particularly relevant as studies comparing al and in catalysts have been hampered by a reduction in stereocontrol for the indium congeners . the proposed active alkoxide complex is formed in situ during the polymerizations by reaction between 2d and the equivalent of alcohol . it was therefore somewhat surprising that complex 2d completely failed to polymerize rac - la , even in the presence of an equivalent of isopropyl alcohol . used a -n , o , n-{(c6f5n c6h4)2o}game initiator , in the presence of an equivalent of alcohol ; this system was also completely inactive toward the rop of rac - la . this was in contrast to the analogous aluminum complex , which showed moderate activity under the same conditions . the lack of reactivity of compound 2d , in the presence of iso - propyl alcohol , can be attributed to the failure to form the active alkoxide initiating species . an nmr scale experiment between 2d and a slight excess of iso - propyl alcohol , heated at 348 k for 24 h in toluene - d8 , showed no conversion to the desired alkoxide product . in contrast , monoligated compounds , 1c and 2c , were active initiators for the rop of la , in the presence of 1 equiv of isopropyl alcohol . the polymerizations using 1c and 2c are marginally faster than those using initiator 2b , with kobs values of 1.8 and 2.6 10 s , respectively ( figure 9 ) . thus , the halide ligand substituents increase the rate of polymerization , in line with previous structure activity studies using al complexes . in both systems , an initiation period of approximately 23 h was observed ( note such initiation periods are not observed using the gallium alkoxide complex 2b ) . once again , the alcoholysis reaction was studied using compound 2c , which was heated at 348 k in toluene with 110 equiv of iso - propyl alcohol for 72 h. this experiment failed to result in any formation of a gallium alkoxide complex , leaving only compound 2c unreacted . the lack of reactivity of compound 2c toward alcoholysis indicates that the ring - opening polymerization may be likely to proceed via a different mechanism to the usual coordination insertion route . a possible reaction pathway could be via an activated monomer mechanism ; such a mechanism does not require any formation of a gallium alkoxide species . instead , the lewis acidic gallium metal center acts as a nucleophile , activating the lactide monomer unit to attack by uncoordinated iso - propyl alcohol . such a mechanism has been previously proposed for a series of ( phenoxy - imine)indium complexes , which were also shown to be inert to alcoholysis reactions . furthermore , invoking this mechanistic pathway could rationalize the lack of polymerization activity observed with compound 2d , which would be expected to have a lower lewis acidity than 1c/2c due to its higher coordination number ( penta- vs tetracoordinate ga ) . thus , 2c might be insufficiently lewis acidic to activate the lactide , a key step in the activated monomer polymerization pathway . analysis of the homonuclear decoupled h{h } nmr spectrum showed the formation of atactic pla for 1c and 2c , consistent with the reduced steric influence of the hydroxyl - quinoline ligands in monoligated complexes . all of the initiators display a high degree of polymerization control , showing a linear evolution of the mn with % conversion ( supporting information figures s9 and s10 ) and mn values that are close to those predicted on the basis of the initiator concentration ( table 2 ) . the monomodal molecular weight distribution , observed with all initiators , is especially relevant for 2b as it provides further support for the lack of any additional initiating species , such as kotbu , in the polymerization system . all of the pdis are also narrow throughout the course of the polymerizations , below 1.25 in all cases , indicative of limited transesterification side reactions . the end groups of the polymers formed by initiators 1c and 2c were analyzed by maldi - tof mass spectrometry , which confirmed that the chains were end - capped with iso - propyl ester groups ( supporting information figure s11 ) . the peaks were separated by 144 amu . however , there is also a series of low molecular weight peaks that correspond to the formation of cyclic pla ( supporting information figure s12 ) . conditions : [ la]0 1 m , 1:100 [ i]:[la ] , toluene , 348 k. plot of ln([la]0/[la]t ) versus time of initiator 1c and 2c . conditions : [ la]0 1 m , 1:1:100 [ i]:[iproh]:[la ] , toluene , 348 k. a series of gallium complexes featuring one or two 8-quinolinolato ligands have been synthesized with either chloride , tert - butyl alkoxide , or methyl coligands . the new complexes were fully characterized , and in two cases x - ray crystallographic data were obtained . some of the complexes were tested as initiators ( 2b ) or as part of initiating systems , with exogenous alcohol ( 1c , 2c ) for the polymerization of rac - la . both the initiator and the initiating systems displayed moderate rates and excellent polymerization control . it is tentatively proposed that compounds 1c and 2c operate by an activated monomer mechanism , due to their lack of reactivity with exogenous alcohol . initiator 2b yielded stereoblock pla , with a pi value of 0.70 , from rac - la . this is an unusual example of a gallium complex capable of exerting iso - selectivity and yielding the higher value stereoblock pla . of particular note is that the complex exhibits equivalent good stereocontrol to an aluminum analogue but operates at approximately 3 times the rate . it is clear that further improvements to the rate and stereocontrol are desirable and could be achieved by rational ligand design . all reactions were conducted under an inert nitrogen atmosphere , using a nitrogen - filled glovebox or standard schlenk techniques . all solvents and reagents were obtained from commercial sources , and triethyl aluminum was obtained from strem . isopropyl alcohol was heated to reflux over cah2 , distilled onto fresh cah2 and further refluxed , then distilled , degassed , and stored under nitrogen . benzene - d6 was distilled from sodium , and toluene - d8 and cdcl3 were dried over cah2 ; all of the nmr solvents were degassed and stored under nitrogen . rac - lactide was obtained from purac plc . and was purified by recrystallization ( dry toluene ) and sublimation ( three times ) . nuclear magnetic resonance ( nmr ) spectra were recorded on a bruker av400 spectrometer operating at 400 mhz for h , and 100 mhz for c{h } spectra . solvent peaks were used as internal references for h and c chemical shifts ( ppm ) . nmr ( homodecoupled spectroscopy ) experiments were performed on a bruker av500 spectrometer and also a drx 400 spectrometer . stephen boyer at london metropolitan university , science centre , 29 hornsey road , london n7 7dd , uk . gpc - malls measurements were conducted on a polymer laboratories pl gpc-50 instrument at 35 c , using two polymer laboratories mixed d columns in series and thf as the eluent , at a flow rate of 1 ml min . the light scattering detector was a dawn 8 , wyatt technology , and data were analyzed using astra v version 5.3.4.18 . the refractive angle increment for polylactide ( dn / dc ) in thf was 0.042 ml g. 8-hydroxy-2-methylquinoline ( 0.45 g , 2.8 mmol ) in toluene ( 10 ml ) was added dropwise , with stirring , to a solution of gallium(iii)chloride , in toluene ( 0.25 g , 4.4 mmol ) at 273 k. the reaction was stirred for 12 h , after which time a pale green solid precipitated from the reaction . the solid was then crystallized from thf , at low temperature , to yield a pale yellow , microcrystalline , air - stable solid ( 0.30 mg , 0.71 mmol , 51% ) . h nmr ( 400 mhz , cdcl3 ) ( ppm ) : 3.24 ( s , 6h , ch3 ) , 7.15 ( dd , 2h , ch , jhh = 8.0 hz , jhh = 1.0 hz ) , 7.22 ( dd , 2h , ch , jhh = 8.0 hz , jhh = 1.0 hz ) , 7.50 ( m , 4h , ch ) , 8.31 ( d , 2h , ch , jhh = 8.0 hz ) . c{h } nmr ( 100 mhz , cdcl3 ) ( ppm ) : 23.3 ( ch3 ) , 112.8 ( ch ) , 114.2 ( ch ) , 124.3 ( c ) , 127.0 ( ch ) , 129.0 ( ch ) , 136.9 ( c ) , 139.8 ( ch ) , 155.6 ( c ) , 156.9 ( c ) calcd ( gac10h16n2o2cl ) : c , 56.99 ; h , 3.83 ; n , 6.65 . m / z ( lsims ) : 422 ( m , 15% ) , 385 ( m cl , 45% ) . 5,7-dichloro-8-hydroxy-2-methylquinoline ( 0.65 g , 2.8 mmol ) in toluene ( 15 ml ) was added dropwise with stirring to a solution of gallium(iii)chloride ( 0.25 g , 1.4 mmol ) in toluene ( 10 ml ) at 273 k. the reaction was left to stir for 12 h at 298 k , after which time the solvent was removed in vacuo . the yellow solid was then recrystallized from toluene to yield yellow crystals ( 391 mg , 0.70 mmol , 53% ) . h nmr ( 400 mhz , cdcl3 ) ( ppm ) : 3.25 ( s , 6h ) , 7.59 ( s , 2h ) , 7.60 ( d , 2h , jhh = 8.4 hz ) , 8.59 ( d , 2h , jhh = 8.4 hz ) . c{h } nmr ( 100 mhz , cdcl3 ) ( ppm ) : 23.3 ( ch3 ) , 117.1 ( c ) , 118.0 ( c ) , 123.6 ( c ) , 125.1 ( ch ) , 128.2 ( c ) , 129.2 ( ch ) , 137.5 ( ch ) , 150.7 ( c ) , 159.4 ( c ) n , 5.01 . found : c , 43.09 ; h , 2.08 ; n , 4.92 . m / z ( lsims ) : 560 ( m , 40% ) , 523 ( m cl , 25% ) . bis(5,7-dichloro-2-methylquinolinato)gallium chloride ( 0.10 g , 0.18 mmol ) was added portion - wise over 30 min to a suspension of potassium tert - butoxide ( 20 mg , 0.18 mmol ) in benzene - d6 ( 4 ml ) . the reaction was allowed to stir at 298 k for 24 h. the orange precipitate was removed by centrifugation , and h nmr spectroscopy of the solution revealed complete conversion to the product . h nmr ( 400 mhz , benzene - d6 ) ( ppm ) : 1.17 ( s , 9h , oc(ch3)3 ) , 3.16 ( s , 6h , ch3 ) , 6.50 ( d , 2h , ch , jhh = 8.8 hz ) , 7.41 ( s , 2h , ch ) , 7.79 ( d , 2h , ch , jhh = 8.8 hz ) . h nmr ( 400 mhz , toluene - d8 ) ( ppm ) : 3.23 ( s , 6h , ch3 ) , 6.10 ( d , 2h , ch , jhh = 8.6 hz ) , 7.26 ( s , 2h , ch ) , 7.49 ( d , 2h , ch , jhh = 8.6 hz ) . trimethylgallium ( 108 mg , 0.94 mmol ) in toluene ( 3 ml ) was added dropwise to a solution of 5,7-dichloro-8-hydroxy-2-methylquinoline ( 0.15 g , 0.94 mmol ) in toluene ( 10 ml ) . the solution was stirred at 298 k for 16 h , after which time a yellow precipitate had formed . the solid was isolated by filtration and dried in vacuo to yield a yellow solid . the volume of the filtrate was reduced , and further product was isolated ( 0.18 g , 0.70 mmol , 74% ) . h nmr ( 400 mhz , benzene - d6 ) ( ppm ) : 0.10 ( s , 6h , ch3 ) , 1.97 ( s , 3h , ch3 ) , 6.27 ( d , 1h , ch , jhh = 8.4 hz ) , 6.73 ( dd , 1h , ch , jhh = 8.0 hz , jhh = 1.2 hz ) , 7.29 ( t , 1h , ch , jhh = 7.8 hz ) , 7.34 ( dd , 1h , ch , jhh = 7.8 hz , jhh = 1.2 hz ) , 7.38 ( d , 1h , ch , jhh = 8.4 hz ) . c{h } nmr ( 100 mhz , cdcl3 ) ( ppm ) : 6.0 ( ch3 ) , 22.4 ( ch3 ) , 110.3 ( c ) , 111.6 ( ch ) , 114.7 ( ch ) , 122.2 ( ch ) , 130.6 ( ch ) , 139.0 ( c ) , 139.8 ( ch ) , 153.6 ( c ) , 162.0 ( c ) . anal . n , 5.43 . found : c , 55.96 ; h , 5.51 ; n , 5.57 . trimethylgallium ( 0.20 g , 1.8 mmol ) in toluene ( 3 ml ) was added dropwise to a solution of 5,7-dichloro-8-hydroxy-2-methylquinoline ( 0.40 g , 1.8 mmol ) in toluene ( 15 ml ) . the solution was stirred at 298 k for 16 h , after which time the solvent was removed in vacuo to yield a yellow solid ( 0.50 g , 1.53 mmol , 87% ) . h nmr ( 400 mhz , benzene - d6 ) ( ppm ) : 0.00 ( s , 6h , ch3 ) , 1.82 ( s , 3h , ch3 ) , 6.15 ( d , 1h , ch , jhh = 8.4 hz ) , 7.47 ( s , 1h , ch ) , 7.82 ( d , 1h , ch , jhh = 8.4 hz ) . c{h } nmr ( 100 mhz , cdcl3 ) ( ppm ) : 6.1 ( ch3 ) , 22.3 ( ch3 ) , 113.9 ( c ) , 119.1 ( c ) , 122.7 ( ch ) , 124.4 ( c ) , 130.6 ( ch ) , 137.1 ( ch ) , 138.9 ( c ) , 155.3 ( c ) , 156.9 ( c ) . calcd ( gac12h12nocl2 ) : c , 44.10 ; h , 3.70 ; n , 4.29 . trimethylgallium ( 50.8 mg , 0.44 mmol ) in toluene ( 3 ml ) was added dropwise to a solution of 5,7-dichloro-8-hydroxy-2-methylquinoline ( 0.20 g , 0.88 mmol ) in toluene ( 15 ml ) . the solution was heated at reflux for 24 h , after which time the solvent was removed in vacuo to yield a yellow solid . the product was isolated by washing with hot hexane to remove the side products , yielding a pale yellow solid ( 60 mg , 0.11 mmol , 25% ) . h nmr ( 400 mhz , benzene - d6 ) ( ppm ) : 0.16 ( s , 3h , ga ch3 ) , 2.83 ( s , 6h , ch3 ) , 6.45 ( d , 2h , ch , jhh = 8.8 hz ) , 7.44 ( s , 2h , ch ) , 7.82 ( s , 2h , ch , jhh = 8.8 hz ) . c{h } nmr ( 100 mhz , cdcl3 ) ( ppm ) : 4.9 ( ga ch3 ) , 22.9 ( ch3 ) , 115.5 ( c ) , 117.5 ( c ) , 124.0 ( ch ) , 124.0 ( c ) , 129.2 ( ch ) , 135.9 ( ch ) , 138.8 ( c),153.9 ( c ) , 157.5 ( c ) . calcd ( gac21h15n2o2cl4 ) : c , 46.80 ; h , 2.81 ; n , 5.20 . found : c , 46.72 ; h , 2.87 ; n , 5.15 . in a glovebox , a young s tap ampule was loaded with rac - lactide ( 432 mg , 3 mmol ) and ( 2-methyl-8-quinolinolato ) 1c ( 7.7 mg , 0.03 mmol ) . toluene ( 2.9 ml ) and iso - propyl alcohol ( 0.03 mmol ) were injected into the reaction , such that the overall concentration of lactide was 1 m and of initiator was 10 mm . the ampule was removed from the glovebox and placed in an oil bath at 348 k. aliquots were taken from the reaction , under an nitrogen atmosphere . aliquots were quenched with hexane ( 12 ml ) , and the solvent was allowed to evaporate . the crude product was analyzed by h nmr and homonuclear decoupled h nmr spectroscopy and gpc - malls . the conversion of la to pla was determined by integration of the methine proton peaks of the h nmr spectra , 5.005.30 . the pi value was determined by integration of the methine region of the homonuclear decoupled h nmr spectrum , 5.15.24 . the pla number - averaged molecular weight , mn , and polydispersity index ( mw / mn ; pdi ) were determined using gel permeation chromatography equipped with multiangle laser light scattering ( gpc - malls ) .

the synthesis and characterization of a series of 8-quinolinolato gallium complexes is presented , and the complexes are analogous to a series of aluminum complexes previously reported . the complexes have been shown to be active initiators for the ring - opening polymerization of rac - lactide . high degrees of polymerization control are demonstrated , as exemplified by the linear evolution of molecular weight as the polymerization progresses , narrow polydispersity indices , and molecular weights corresponding to those predicted on the basis of initiator concentration . some of the initiators show iso - selective polymerization of rac - lactide , with pi = 0.70 . the polymerization rates have been monitored , and the pseudo first - order rate constants are compared to those of analogous aluminum compounds . the 8-quinolinolato gallium initiators show rates approximately 3 times higher than those of the series of aluminum compounds , while maintaining equivalently high iso - selectivity ( pi = 0.70 ) and polymerization control .

Introduction Results and Discussion Conclusions Experimental Section

as the demand and usage of polymers increases , so do issues associated with the sustainability of their source materials , their end of life fates , and costs . pla is manufactured by the ring - opening polymerization ( rop ) of lactide ( la ) ( figure 1 ) , a process that is initiated by lewis acidic metal alkoxide complexes . quite a range of metals have precedent for initiating rop , and for further details on the scope of this catalysis , the reader is referred to several comprehensive reviews . there has been a long - standing academic interest in aluminum - based initiators , due to the excellent control conferred by this metal and the potential for some al complexes to enable stereocontrolled rop of rac - lactide . from the materials property perspective , the production of stereoblock or stereocomplex pla , via the iso - selective rop of rac - la , remains a high priority target . so far , the most iso - selective catalysts reported have been aluminum salen complexes . for example , mehrkhodavandi and co - workers reported the dinuclear complex , a ( figure 2 ) , which polymerized 200 equiv of rac - la in 30 min ( 298 k , methylene chloride , 0.5 m in [ la ] , 90% conversion , pi = 0.62 ) . recently , the same group has extended the study of dinuclear and mononuclear indium complexes , resulting in efficient catalysis with a moderate - good isotactic bias ( pi = 0.77 ) in the best cases . one attractive system was reported by tolman and hillmyer , where incl3 , in the presence of an equivalent each of an alcohol and triethylamine , polymerized rac - la quite rapidly and with high heteroselectivity ( ps = 0.97 , 1:100 [ i]:[la ] , 5 h ) . another example of an interesting indium complex was a racemic homoleptic species , b ( figure 2 ) , reported by okuda and arnold , which was found to induce a slight isotactic bias ( pi = 0.63 ) . given the significant potential for al and in initiators , it was somewhat surprising to discover that ga complexes have been far less investigated in the rop of la . a recent comparative study of aluminum and gallium complexes , with the structures represented by compound d ( figure 2 ) , showed that the ga catalysts were faster and equally well controlled as the al counterparts ( ga > 99% conversion , 100 equiv of la , 353 k , toluene 1 h , versus al 45% conversion under the same conditions ) . despite the difference in rate , the change in metal center did not affect the polymerization stereoselectivity , with moderate iso - selectivity being observed for both the al and the ga initiators ( pi = 0.70 ) . examples of the structures of some stereoselective in and ga initiators for rac - la rop . we have previously reported a series of bis(8-quinolinolato ) aluminum ethyl complexes , which , in the presence of iso - propyl alcohol , were efficient , iso - selective initiators ( pi = 0.76 ) , for the rop of rac - la . in this article , the preparation and application of a series of gallium complexes , with some of the same 8-quinolinato ligands , is presented . the ligands were selected as both are commercially available compounds , an attractive feature if polymerization activity of the initiators can be increased . furthermore , the aluminum complexes of 1 and 2 showed distinct differences in polymerization activity with the halide substituents of ligand 2 enhancing activity and iso - selectivity . the structures of the pro - ligands , 1 and 2 . the completion of the complexation reaction was confirmed by h nmr spectroscopy where the resonances assigned to the quinolinolato protons were at higher chemical shift as compared to the pro - ligands , due to the lewis acidic ga center deshielding the nuclei . compounds 1a and 2a were tested as initiators for the rop of rac - la ( 1 m solution of la in thf , 298 k , 20 h ) but showed no activity . this is in line with the findings of other researchers that metal halide complexes are rarely effective initiators , presumably due to the high m cl bond strength ( 481 kj mol versus 285 kj mol for ga to prepare active initiators , the gallium chloride complexes were reacted further to generate new alkoxide and amide complexes . as an illustrative example , the reaction between 2a and potassium tert - butoxide yielded a yellow powder whose h nmr spectrum indicated the presence of the desired alkoxide complex 2b ( vide infra ) together with another complex , showing only resonances for the quinolinate ligands . the product was determined to be the oxo - bridged dimeric complex [ ( l2ga)2(-o ) ] , which formed due to undesired oxidation of the target alkoxide complex ( see the supporting information ) . it should be noted that the same solvents were applied successfully for the isolation of aluminum and gallium alkyl complexes , without any such degradation , illustrating the high sensitivity of the alkoxide derivatives . the h nmr spectrum shows that all of the quinolinate resonances are shifted to lower field , and furthermore the singlet at 1.17 ppm , with a relative integral of 9 , confirms the presence of the tert - butyl alkoxide . as an alternative route to gallium alkoxide complexes , gallium alkyl complexes [ l2gar ] ( r = alkyl ) were targeted as suitable precursors for further reaction , via protonolysis , with various alcohols . of the pro - ligands , 1 and 2 , with trimethylgallium resulted only in the formation of the monoligated complexes 1c and 2c [ lgame2 ] together with the residual pro - ligands . changing the conditions , for example , by using more polar solvents such as thf or pyridine , resulted solely in the formation of the same monoligated product . it was only possible to form some of the desired bis - ligated gallium complexes , by refluxing the solutions in toluene for 24 h. even under such forcing conditions , complex 2c was only partly converted , in low yield ( 25% ) , to the desired bis - ligated product 2d . complexes 1c and 2c were prepared in high yields ( 74% and 87% , respectively ) by reaction between equimolar quantities of trimethylgallium and the appropriate pro - ligands ( figure 5 ) . a singlet peak was observed between 0 and 0.1 ppm , with a total relative integral of 6 , due to the two gallium coordinated methyl groups . synthetic routes attempted for the preparation of bis(8-quinolinolato ) gallium methyl compounds , and the synthesis of compounds 1c , 2c , and 2d . the structure of 2a has c2 symmetry about an axis that passes through the gallium and chlorine atoms ( figure 6 ) , while the structure of 2b contains two crystallographically independent complexes , 2b - a ( shown in figure 7 ) and 2b - b ( shown in supporting information figure s4 ) . all three molecules have pentacoordinate gallium centers with similar distorted trigonal bipyramidal coordination geometries , the values being 0.69 , 0.57 , and 0.57 for 2a , 2b - a , and 2b - b , respectively . as with the aluminum complexes , in every case the only geometric isomer observed has the nitrogen atoms of the ligand coordinated trans to one another in the axial positions . n bond lengths to the quinolinolato ligand differ between 2a and 2b ( a and b ) significantly ; in the case of 2a , all of the ga o and ga n bonds are shorter than those in 2b - a and 2b - b . compounds 1c , 2b d were tested as initiators in the rop of rac - la ( table 2 ) . the polymerizations were monitored by taking aliquots at regular time intervals , which were analyzed using h nmr spectroscopy , to determine the la conversion , and by gpc - malls ( gel permeation chromatography - multiangle laser light scattering ) , to determine the number average molecular weight , mn , and the polydispersity index , pdi . in each case , the polymerization rate was compared by determination of the pseudo - first - order rate constant , kobs , which is the gradient of the linear plots of ln([la]0/[la]t ) versus time . determined from the gradients of the plots of ln{[la]0/[la]t } versus time , where the average errors = 910% ( determined using initiator 1c ) . the potassium chloride salt was removed by centrifugation , and the stock solution was added to the polymerization mixture . we can therefore eliminate the possibility of any excess potassium tert - butoxide salt , a known active initiator for the rop reaction , initiating the polymerization . the initiator , 2b , was moderately active in the rop of rac - lactide , with the polymerization proceeding to high conversion in excess of 50 h. such rates are typical for discrete group 13 initiators , and indeed the polymerization occurs at a rate similar to that of the previously reported bis(8-quinolinolato ) aluminum ethyl initiators . the initiator gives rise to pla with a moderate degree of isotactic enchainment , with a pi value of 0.70 consistent with a bias toward a stereoblock microstructure ( supporting information figure s8 ) . there are only two other examples of iso - selective gallium initiators : compound d ( figure 2 , pi = 0.70 ) and a dimethyl gallium n - heterocyclic carbene complex , reported by horeglad and co - workers , where a pi value of up to 0.78 was claimed . the initiator bis(5,7-dichloro-2-methyl-8-quinolinolato ) gallium tert - butoxide , 2b , is analogous to the bis(5,7-dichloro-2-methyl-8-quinolinolato ) aluminum ethyl initiator , where the active alkoxide initiator is formed in situ by reaction with isopropyl alcohol during the course of the rop reaction . initiator 2b is significantly faster than its aluminum analogue , with around a 3 times higher kobs value ( 1.3 10 s for 2b vs 5.0 10 s for the al analogue ) ( figure 8) . in addition to the beneficial increase in rates , the ga complex also shows high iso - selectivity and good polymerization control ( equaling those observed with the al analogue ) . this is particularly relevant as studies comparing al and in catalysts have been hampered by a reduction in stereocontrol for the indium congeners . it was therefore somewhat surprising that complex 2d completely failed to polymerize rac - la , even in the presence of an equivalent of isopropyl alcohol . used a -n , o , n-{(c6f5n c6h4)2o}game initiator , in the presence of an equivalent of alcohol ; this system was also completely inactive toward the rop of rac - la . an nmr scale experiment between 2d and a slight excess of iso - propyl alcohol , heated at 348 k for 24 h in toluene - d8 , showed no conversion to the desired alkoxide product . in contrast , monoligated compounds , 1c and 2c , were active initiators for the rop of la , in the presence of 1 equiv of isopropyl alcohol . the polymerizations using 1c and 2c are marginally faster than those using initiator 2b , with kobs values of 1.8 and 2.6 10 s , respectively ( figure 9 ) . once again , the alcoholysis reaction was studied using compound 2c , which was heated at 348 k in toluene with 110 equiv of iso - propyl alcohol for 72 h. this experiment failed to result in any formation of a gallium alkoxide complex , leaving only compound 2c unreacted . the lack of reactivity of compound 2c toward alcoholysis indicates that the ring - opening polymerization may be likely to proceed via a different mechanism to the usual coordination insertion route . such a mechanism has been previously proposed for a series of ( phenoxy - imine)indium complexes , which were also shown to be inert to alcoholysis reactions . thus , 2c might be insufficiently lewis acidic to activate the lactide , a key step in the activated monomer polymerization pathway . all of the initiators display a high degree of polymerization control , showing a linear evolution of the mn with % conversion ( supporting information figures s9 and s10 ) and mn values that are close to those predicted on the basis of the initiator concentration ( table 2 ) . the monomodal molecular weight distribution , observed with all initiators , is especially relevant for 2b as it provides further support for the lack of any additional initiating species , such as kotbu , in the polymerization system . the end groups of the polymers formed by initiators 1c and 2c were analyzed by maldi - tof mass spectrometry , which confirmed that the chains were end - capped with iso - propyl ester groups ( supporting information figure s11 ) . however , there is also a series of low molecular weight peaks that correspond to the formation of cyclic pla ( supporting information figure s12 ) . conditions : [ la]0 1 m , 1:1:100 [ i]:[iproh]:[la ] , toluene , 348 k. a series of gallium complexes featuring one or two 8-quinolinolato ligands have been synthesized with either chloride , tert - butyl alkoxide , or methyl coligands . some of the complexes were tested as initiators ( 2b ) or as part of initiating systems , with exogenous alcohol ( 1c , 2c ) for the polymerization of rac - la . both the initiator and the initiating systems displayed moderate rates and excellent polymerization control . initiator 2b yielded stereoblock pla , with a pi value of 0.70 , from rac - la . this is an unusual example of a gallium complex capable of exerting iso - selectivity and yielding the higher value stereoblock pla . of particular note is that the complex exhibits equivalent good stereocontrol to an aluminum analogue but operates at approximately 3 times the rate . all solvents and reagents were obtained from commercial sources , and triethyl aluminum was obtained from strem . benzene - d6 was distilled from sodium , and toluene - d8 and cdcl3 were dried over cah2 ; all of the nmr solvents were degassed and stored under nitrogen . rac - lactide was obtained from purac plc . the refractive angle increment for polylactide ( dn / dc ) in thf was 0.042 ml g. 8-hydroxy-2-methylquinoline ( 0.45 g , 2.8 mmol ) in toluene ( 10 ml ) was added dropwise , with stirring , to a solution of gallium(iii)chloride , in toluene ( 0.25 g , 4.4 mmol ) at 273 k. the reaction was stirred for 12 h , after which time a pale green solid precipitated from the reaction . bis(5,7-dichloro-2-methylquinolinato)gallium chloride ( 0.10 g , 0.18 mmol ) was added portion - wise over 30 min to a suspension of potassium tert - butoxide ( 20 mg , 0.18 mmol ) in benzene - d6 ( 4 ml ) . the reaction was allowed to stir at 298 k for 24 h. the orange precipitate was removed by centrifugation , and h nmr spectroscopy of the solution revealed complete conversion to the product . trimethylgallium ( 108 mg , 0.94 mmol ) in toluene ( 3 ml ) was added dropwise to a solution of 5,7-dichloro-8-hydroxy-2-methylquinoline ( 0.15 g , 0.94 mmol ) in toluene ( 10 ml ) . the volume of the filtrate was reduced , and further product was isolated ( 0.18 g , 0.70 mmol , 74% ) . in a glovebox , a young s tap ampule was loaded with rac - lactide ( 432 mg , 3 mmol ) and ( 2-methyl-8-quinolinolato ) 1c ( 7.7 mg , 0.03 mmol ) . toluene ( 2.9 ml ) and iso - propyl alcohol ( 0.03 mmol ) were injected into the reaction , such that the overall concentration of lactide was 1 m and of initiator was 10 mm . aliquots were quenched with hexane ( 12 ml ) , and the solvent was allowed to evaporate . the conversion of la to pla was determined by integration of the methine proton peaks of the h nmr spectra , 5.005.30 . the pi value was determined by integration of the methine region of the homonuclear decoupled h nmr spectrum , 5.15.24 . the pla number - averaged molecular weight , mn , and polydispersity index ( mw / mn ; pdi ) were determined using gel permeation chromatography equipped with multiangle laser light scattering ( gpc - malls ) .

the genus stachys comprises more than 450 species mainly distributed in warm - temperate regions of the mediterranean basin and south - western asia . it represents one of the largest genera of lamiaceae family , and the majority of the species belonging to this genus have been largely used in the traditional medicine . are currently indicated as mountain tea in several regions reflecting their principal use in infusions and decoctions . the genus stachys is represented by annual or perennial herbs known for their interesting biological activities , i.e. , anti - inflammatory , antispasmodic , sedative , and diuretic [ 3 - 5 ] . several phytochemical components , which may be responsible of the biological activities , have been isolated from stachys species , i.e. , iridoids , phenylethanoid glycosides , flavonoids , acidic metabolites , polysaccharides , and terpenoids . in italy , the genus stachys encompasses about 30 species and subspecies among which five species are considered endemic . this plant is characteristic of the mountain habitat of central italy , being distributed only in a few regions ( umbria , marche , abruzzo , lazio , and molise ) , on stony mountain pastures , scrubs , and screes , and preferring a calcareous , and dry soil up to 2000 m a.s.l . the opposite leaves are petiolate and densely hairy on the edge and along the veins . the flowers are gathered in small verticillasters held in a dense spike , yellow - white in color and blooming from june to august - september . the fruits , which are brown colored , are constituted by 4 ovate nuculae . a previous study on this subspecies revealed the presence of phenypropanoid , saponin , and iridoid glycosides which are considered taxonomic markers for the genus and the family . in this paper , we reported a reassessment of the polar fraction constituents of this plant employing a patented method allowing the isolation of secondary metabolites of glycosidic nature . the fresh plant materials ( aerial parts ) were collected at the flowering stage from a spontaneous population growing at the altitude of 1520 m a.s.l . in the territory of pizzo tre vescovi , marche , italy ( the botanical recognition was performed by one of us ( f.m . ) using available literature , and a representative specimen has been deposited in the herbarium universitatis camerinensis ( came , included in the online edition of index herbariorum c / o school of biosciences and veterinary medicine , university of camerino , italy ) under the codex came 24825 . a sample of the studied species is also stored in our laboratory ( code number sad-30062012 ) for any further reference . nmr spectra were recorded on a varian ( now agilent technologies ) mercury 300 mhz instrument and/or on a bruker avance iii 400 mhz instrument using d2o or cd3od as deuterated solvents : the chemical shifts were expressed in ppm using the hdo signal to set the 1h - spectra acquired in d2o , while the internal solvent signal ( m5 ) at 3.31 ppm was set as reference for the spectra in cd3od . ms spectra were performed on a q - tof micro spectrometer ( micromass , now waters , manchester , uk ) equipped with an esi source that operated in the negative and/or positive ion mode . the flow rate of sample infusion was 10 l / min with 100 acquisitions per spectrum . solvents having rpe purity grade were all purchased from sigma - aldrich or carlo erba reagenti , silica gel 60 ( 70 - 230 mesh astm ) was purchased from fluka . bidimensional spectra were performed on a bruker avance iii 400 mhz instrument , operating at 9.4 t at 298 k . heteronuclear single quantum correlation ( hsqc ) experiments were acquired with a spectral width of 15 and 250 ppm for the proton and carbon , respectively , an average 1jc - h of 145 hz , a recycle delay of 2 s and a data matrix of 4 k 256 points . heteronuclear multiple bond correlation ( hmbc ) experiments were acquired with a spectral width of 15 and 250 ppm for the proton and carbon , respectively , a long - range coupling constant of jc - h of 8 hz , a recycle delay of 2 s and a data matrix of 4 k 256 points . a portion of 390.0 g of hair - dried plant materials was extracted with a mixture of ethanol 96% and distilled water in ratio 8:2 v / v ( 1.4 l ) . the whole was left in maceration for 5 days so that the metabolites could come into solution . the solutions , having a dark green coloration , were gathered together in a same flask and then filtered . after filtration , the organic solvent was eliminated under reduced pressure at a temperature below 45c until a water suspension was obtained . throughout this last part , ph was checked on normal litmus paper , and this was about 8 . this step is necessary to verify that ph is not too acid , meaning not under the value of 5.5 , because an extreme acidity at this step may cause secondary reactions with the formation of several artifacts coming from the hydrolysis of glycosides and esters present in the extract . the water suspension was then frozen to 20c and later lyophilized at the same temperature to preserve temperature - sensitive compounds possibly present . the final dried crude extract obtained from this methodology weighed 86.7 g and was dark green colored . it was adsorbed on a stationary phase consisting in a mixture of charcoal / celite / polyamide ( 10:1:1 ) ( 60.0 g ) until no reaction to vanillin / hcl spray reagent occured according to a method reported in the patent by ballero et al . . the resulting suspension was then stratified on a gooch funnel and eluted first with h2o to eliminate the mono- and disaccharides . these usually coelute with glycosidic constituents in silica gel chromatography and may interfere during the structure identification of the metabolites of interest . the desorption chromatography was conducted in steps using etoh / h2o mixtures gradually increasing in etoh percentages ( 30 , 60 , and 95% ) . in this manner , three fractions at different polarity were collected , and after freeze drying 23.9 , 4.70 , and 0.82 g of solids were recovered , from the 30 , 60 and 96% ethanolic elutions , respectively . after a preliminary screening on thin layer chromatographys and paper chromatography , the separation step on silica gel column was first conducted on the 60% eluate because this showed the whole iridoid components and in comparable amount among them in respect to the other fractions . the 30% fraction , containing the more polar compounds , was partitioned in a second step . the first chromatographic separation was performed on an aliquot of the 60% ethanolic elution for the weight of 4.0 g using 120.0 g of silica gel ( ratio 1:30 ) . the eluting system was a mixture of n - buoh / h2o ( 82:18 v / v ) . from this chromatographic separation ( scheme 1 ) 11 compounds were isolated and identified by comparison with data reported in literature and/or by comparison with pure compounds available in our laboratory : verbascoside ( 10 , 12.4 mg ) from the assembly of fractions 8 - 15 ; stachysoside a ( 11 , 11.2 mg ) in mixture with verbascoside ( 1:2 ) in the assembly of fractions 17 - 24 ; b - arbutin ( 9 , 3.2 mg ) from the assembly of fractions 30 - 32 ; reptoside ( 6 ) and 6-o - acetyl - ajugol ( 7 ) in mixture in ratio 1:1.5 ( 25.0 mg ) from the assembly of fractions 43 - 45 ; ajugoside ( 1 , 113.7 mg ) as pure compound from the assembly of fractions 53 - 54 ; 8-o - acetyl - harpagide ( 2 , 1.0 g ) from the assembly of fractions 70 - 88 ; chlorogenic acid ( 8) in mixture with 8-o - acetyl - harpagide ( 2 ) in ratio 1:2 ( 109.4 mg ) from the assembly of fractions 95 - 112 ; harpagide ( 3 ) and chlorogenic acid ( 8) in mixture 3:1 ( 48.3 mg ) from the assembly of fractions 118 - 122 ; allobetonicoside ( 4 ) and chlorogenic acid ( 8) in mixture 10:1 ( 29.1 mg ) from the assembly of fractions 133 - 135 ; allobetonicoside ( 4 , 100.7 mg ) as a pure compound from the assembly of fractions 144 - 163 ; 4-o - galactopyranosyl - teuhircoside ( 5 ) in mixture with allobetonicoside ( 4 ) in ratio 3:1 ( 24.7 mg ) from the assembly of fractions 166 - 169 . chromatographic steps on 60% ethonolic fraction and elution order of components a second chromatographic column was , instead , conducted on an aliquot of the 30% desorption solids ( 3.5 g ) . in particular , harpagide ( 3 ) , 8-o - acetylharpagide ( 2 ) as pure compounds , allobetonicoside ( 4 ) in mixture with 4-o - galactopyranosyl - teuhircoside ( 5 ) , and ajugoside ( 1 ) occurring in traces ( relative quantities not estimated ) . on the other hand , phenolic compounds as well as reptoside ( 6 ) and 6-o - acetyl - ajugol ( 7 ) , were not found . ajugoside ( 1 ) : nmr data see table 1 ; esi - ms : m / z 412.80 [ m+na ] . complete nmr assignment of ajugoside ( 1 ) in d2o 8-o - acetylharpagide ( 2 ) : h nmr ( 300 mhz , cd3od ) d : 6.40 ( 1h , d , j = 6.4 hz , h-3 ) , 6.08 ( 1h , br s , h-1 ) , 4.92 ( 1h , d , j = 6.5 hz , h-4 ) , 4.60 ( 1h , d , j = 7.8 hz , h-1 ) , 3.90 ( 1h , d , j = 11.8 hz , h-6a ) , 3.71 ( 2h , m , h-6b ; h-3 ) , 3.44 - 3.29 ( 2h , m , h-4 ; h-5 ) , 3.22 ( 1h , t like , j = 8.4 hz , h-2 ) , 2.86 ( 1h , br s , h-9 ) , 2.18 ( 1h , d , j = 15.1 hz , h-7a ) , 2.02 ( 3h , s , ch3co ) , 1.94 ( 1h , dd , j = 15.2 , 4.4 hz , h-7b ) , and 1.45 ( 3h , s , h-10 ) . c nmr ( apt ) ( 75 mhz , cd3od ) d : 173.3 ( ch3co ) , 143.8 ( c-3 ) , 106.8 ( c-4 ) , 99.8 ( c-1 ) , 94.5 ( c-1 ) , 88.6 ( c-8 ) , 78.0 ( c-3 ) , 77.6 ( c-5 ) , 77.4 ( c-6 ) , 74.4 ( c-2 ) , 73.3 ( c-5 ) , 71.6 ( c-4 ) , 62.8 ( c-6 ) , 55.4 ( c-9 ) , 46.0 ( c-7 ) , 22.5 ( ch3co ) , and 22.2 ( c-10 ) . esi - ms : m / z 429.04 [ m+na ] ; m / z 835.19 [ 2m+na ] . harpagide ( 3 ) : h nmr ( 300 mhz , cd3od ) d : 6.32 ( 1h , d , j = 6.3 hz , h-3 ) , 5.74 ( 1h , br s , h-1 ) , 4.95 ( 1h , d , j = 6.3 hz , h-4 ) , 4.58 ( 1h , d , j = 7.9 hz , h-1 ) , 3.90 ( 1h , d , j = 12.2 hz , h-6a ) , 3.75 - 3.60 ( 2h , m , h-6b ; h-3 ) , 3.44 - 3.33 ( 2h , m , h-4 ; h-5 ) , 3.26 - 3.16 ( 1h , dd , j = 8.8 , 8.2 hz , h-2 ) , 2.56 ( 1h , br s , h-9 ) , 1.90 ( 1h , dd , j = 13.8 , 4.6 hz , h-7a ) , 1.80 ( 1h , dd , j = 13.8 , 3.6 hz , h-7b ) , 1.25 ( 3h , s , h-10 ) . allobetonicoside ( 4 ) : h nmr ( 300 mhz , d2o ) d : 6.43 ( 1h , d , j = 6.3 hz , h-3 ) , 6.11 ( 1h , br s , h-7 ) , 5.94 ( 1h br s , h-1 ) , 5.28 ( 1h , d , j = 8.3 hz , h-1 ) , 5.01 ( 1h , d , j = 6.3 hz , h-4 ) , 4.75 ( 1h , d , j = 7.8 hz , h-1 ) , 4.19 ( 1h , t , j = 2.6 hz , h-9 ) , 3.93 ( 2h , m , h-6a ; h-6a ) , 3.83 ( 2h , m , h-6b ; h-6b ) , 3.70 - 3.33 ( 8h , m , h-3 ; h-3 ; h-4 ; h-4 ; h5 ; h-5 ; h-2 ; h-2 ) , and 2.28 ( 3h , s , h-10 ) . c nmr ( 75 mhz , d2o ) d : 204.86 ( c-6 ) , 178.50 ( c-8 ) , 144.09 ( c-3 ) , 127.77 ( c-7 ) , 102.69 ( c-4 ) , 98.89 ( c-1 ) , 96.58 ( c-1 ) , 92.25 ( c1 ) , 77.45 ( c-5 ) , 76.18 ( c-5 ) , 75.60 ( c-3 ) , 73.57 ( c-2 ) , 72.71 ( c-4 ) , 71.20 ( c-2 ) , 69.85 ( c-3 ) , 69.42 ( c-4 ) , 66.62 ( c-4 ) , 61.03 ( c-6 ) , 60.70 ( c-6 ) , 54.85 ( c-9 ) , 17.62 ( c-10 ) . esi - ms : m / z 528.73 [ m+na ] ; m / z 544.69 [ m+k ] . 4-o - galactopyranosyl - teuhircoside ( 5 ) : h nmr ( 300 mhz , d2o ) d : 6.43 ( 1h , d , j = 6.4 hz , h-3 ) , 6.13 ( 1h , br s , h-7 ) , 5.94 ( 1h , br s , h-1 ) , 4.98 ( 1h , d , j = 6.4 hz , h-4 ) , 4.80 ( 1h , partially overlapped with solvent signal , h-1 ) , 4.73 ( 1h , d , j = 8.1 hz , h-1 ) , 2.29 ( 3h , s , h-10 ) . esi - ms : m / z 529.12 [ m+na ] . reptoside ( 6 ) : h nmr ( 300 mhz , d2o ) d : 6.40 ( 1h , d , j = 6.5 hz , h-3 ) , 5.90 ( 1h , br s , h-1 ) , 5.02 ( 1h , dd , j = 6.4 , 1.3 hz , h-4 ) , 4.75 ( 1h , d , partially obscured by the solvent signal , h-1 ) , 3.92 ( 1h , dd , j = 12.4 , 1.9 hz , h-6a ) , 3.71 ( 1h , br d , j = 12.4 hz , h-6b ) , 3.26 ( 1h , dd , j = 8.3 , 8.2 hz , h-2 ) , 2.65 ( 1h , br s , h-9 ) , 2.04 ( 3h , s , ch3co ) , 1.93 ( 2h , m , h-6a ; h-7a ) , 1.71 ( 2h , m , h-6b ; h-7b ) , 1.43 ( 3h , s , h-10 ) . 6-o - acetyl - ajugol ( 7 ) : h nmr ( 300 mhz , d2o ) d : 6.34 ( 1h , dd , j = 6.4 , 1.8 hz , h-3 ) , 5.50 ( 1h , d , j = 2.8 hz , h-1 ) , 4.89 ( 1h , dd , j = 6.4 , 2.8 hz , h-4 ) , 4.81 ( 1h , obscured by the solvent signal , h-6 ) , 4.70 ( 1h , d , j = 8.0 hz , h-1 ) , 3.90 ( 1h , dd , j = 12.1 , 1.6 hz , h-6 ) , 3.68 ( 1h , br d , j = 12.2 hz , h-6b ) , 3.24 ( 1h , m , h-2 ) , 2.79 ( 1h , br t , j = 6.5 hz , h-5 ) , 2.34 ( 1h , dd , j = 7.9 , 2.7 hz , h-9 ) , 2.12 ( 1h , br dd , j = 14.0 , 6.5 hz , h-7a ) , 2.08 ( 3h , s , ch3co ) , 1.93 ( 1h , dd , j = 13.8 , 8.6 hz , h-7b ) , 1.38 ( 3h , s , h-10 ) . esi - ms : m / z 413.04 [ m+na ] . chlorogenic acid ( 8) : h nmr ( 300 mhz , cd3od ) d : 7.56 ( 1h , d , j = 15.9 hz , h - b ) , 7.05 ( 1h , d , j = 1.9 hz , h-2 ) , 6.96 ( 1h , dd , j = 8.2 , 1.9 hz , h-6 ) , 6.78 ( 1h , d , j = 8.2 hz , h-5 ) , 6.26 ( 1h , d , j = 15.9 hz , h - a ) , 5.33 ( 1h , td , j = 9.1 , 4.5 hz , h-3 ) , 4.17 ( 1h , dt , j = 6.2 , 3.2 hz , h-4 ) , 3.73 ( dd , j = 8.5 , 3.1 hz , h-5 ) , 2.27 - 2.13 ( 2h , m , h-2a ; h-6a ) , 2.13 - 1.98 ( 2h , m , h-2b;h-6b ) . b - arbutin ( 9 ) : h nmr ( 300 mhz , cd3od ) d : 7.07 ( 2h , d , j = 8.6 hz , h-3 , h-5 ) , 6.69 ( 2h , d , j = 8.6 hz , h-2 , h-6 ) , 4.95 ( 1h , partially obscured by hdo signal , h-1 ) , 3.96 - 3.79 ( m , overlapped glucose protons ) , 3.77 - 3.54 ( m , overlapped glucose protons ) , 3.18 ( 1h , br t , j = 7.9 hz , h-2 ) . verbascoside ( 10 ) : h nmr ( 300 mhz , cd3od ) d : 7.59 ( 1h , d , j = 15.9 hz , h - b ) , 7.05 ( 1h , d , j = 1.4 hz , h-2 ) , 6.96 ( 1h , dd , j = 8.2 , 1.4 hz , h-6 ) , 6.78 ( 1h , d , j = 8.2 hz , h-5 ) , 6.69 ( 1h , br s , h-2 ) , 6.68 ( 1h , d , j = 8.0 hz , h-5 ) , 6.56 ( 1h , dd , j = 8.0 , 1.6 hz , h-6 ) , 6.28 ( 1h , d , j = 15.9 hz , h - a ) , 5.19 ( 1h , d , j = 0.9 hz , h-1 ) , 4.38 ( 1h , d , j = 7.8 hz , h-1 ) , 2.79 ( 2h , br t , j = 7.3 hz , h - b ) , 1.09 ( 3h , d , j = 6.2 hz , h-6 ) . esi - ms : m / z 623.19 [ m - h ] ; m / z 647.12 [ m+na ] . stachysoside a ( 11 ) : h nmr ( 300 mhz , cd3od ) d : 7.60 ( 1h , d , j = 15.6 hz , h - b[caff . ] ) , 7.06 ( 1h , br s , h-2 ) , 6.98 ( 1h , br d , j = 8.5 hz , h-6 ) , 6.78 ( 1h , d , j = 8.5 hz , h-5 ) , 6.70 ( 1h , br s , h-2 ) , 6.68 ( 1h , d , j = 8.0 hz , h-5 ) , 6.57 ( 1h , br d , j = 8.1 hz , h-6 ) , 6.28 ( 1h , d , j = 15.6 hz , h - a ) , 5.42 ( 1h , br s , h-1 ) , 4.43 ( 1h , d , j = 7.9 hz , h-1 ) , 4.31 ( 1h , d , j = 7.2 hz , h-1 ) , 2.81 ( 2h , d , j = 7.1 hz , h - b ) , 1.16 ( 3h , d , j = 6.0 hz , h-6 ) . esi - ms : m / z 755.20 [ m - h ] ; m / z 779.16 [ m+na ] . the fresh plant materials ( aerial parts ) were collected at the flowering stage from a spontaneous population growing at the altitude of 1520 m a.s.l . in the territory of pizzo tre vescovi , marche , italy ( the botanical recognition was performed by one of us ( f.m . ) using available literature , and a representative specimen has been deposited in the herbarium universitatis camerinensis ( came , included in the online edition of index herbariorum c / o school of biosciences and veterinary medicine , university of camerino , italy ) under the codex came 24825 . a sample of the studied species is also stored in our laboratory ( code number sad-30062012 ) for any further reference . nmr spectra were recorded on a varian ( now agilent technologies ) mercury 300 mhz instrument and/or on a bruker avance iii 400 mhz instrument using d2o or cd3od as deuterated solvents : the chemical shifts were expressed in ppm using the hdo signal to set the 1h - spectra acquired in d2o , while the internal solvent signal ( m5 ) at 3.31 ppm was set as reference for the spectra in cd3od . ms spectra were performed on a q - tof micro spectrometer ( micromass , now waters , manchester , uk ) equipped with an esi source that operated in the negative and/or positive ion mode . the flow rate of sample infusion was 10 l / min with 100 acquisitions per spectrum . solvents having rpe purity grade were all purchased from sigma - aldrich or carlo erba reagenti , silica gel 60 ( 70 - 230 mesh astm ) was purchased from fluka . bidimensional spectra were performed on a bruker avance iii 400 mhz instrument , operating at 9.4 t at 298 k . heteronuclear single quantum correlation ( hsqc ) experiments were acquired with a spectral width of 15 and 250 ppm for the proton and carbon , respectively , an average 1jc - h of 145 hz , a recycle delay of 2 s and a data matrix of 4 k 256 points . heteronuclear multiple bond correlation ( hmbc ) experiments were acquired with a spectral width of 15 and 250 ppm for the proton and carbon , respectively , a long - range coupling constant of jc - h of 8 hz , a recycle delay of 2 s and a data matrix of 4 k 256 points . a portion of 390.0 g of hair - dried plant materials was extracted with a mixture of ethanol 96% and distilled water in ratio 8:2 v / v ( 1.4 l ) . the whole was left in maceration for 5 days so that the metabolites could come into solution . the solutions , having a dark green coloration , were gathered together in a same flask and then filtered . after filtration , the organic solvent was eliminated under reduced pressure at a temperature below 45c until a water suspension was obtained . throughout this last part , ph was checked on normal litmus paper , and this was about 8 . this step is necessary to verify that ph is not too acid , meaning not under the value of 5.5 , because an extreme acidity at this step may cause secondary reactions with the formation of several artifacts coming from the hydrolysis of glycosides and esters present in the extract . the water suspension was then frozen to 20c and later lyophilized at the same temperature to preserve temperature - sensitive compounds possibly present . the final dried crude extract obtained from this methodology weighed 86.7 g and was dark green colored . it was adsorbed on a stationary phase consisting in a mixture of charcoal / celite / polyamide ( 10:1:1 ) ( 60.0 g ) until no reaction to vanillin / hcl spray reagent occured according to a method reported in the patent by ballero et al . . the resulting suspension was then stratified on a gooch funnel and eluted first with h2o to eliminate the mono- and disaccharides . these usually coelute with glycosidic constituents in silica gel chromatography and may interfere during the structure identification of the metabolites of interest . the desorption chromatography was conducted in steps using etoh / h2o mixtures gradually increasing in etoh percentages ( 30 , 60 , and 95% ) . in this manner , three fractions at different polarity were collected , and after freeze drying 23.9 , 4.70 , and 0.82 g of solids were recovered , from the 30 , 60 and 96% ethanolic elutions , respectively . after a preliminary screening on thin layer chromatographys and paper chromatography , the separation step on silica gel column was first conducted on the 60% eluate because this showed the whole iridoid components and in comparable amount among them in respect to the other fractions . the 30% fraction , containing the more polar compounds , was partitioned in a second step . the first chromatographic separation was performed on an aliquot of the 60% ethanolic elution for the weight of 4.0 g using 120.0 g of silica gel ( ratio 1:30 ) . the eluting system was a mixture of n - buoh / h2o ( 82:18 v / v ) . from this chromatographic separation ( scheme 1 ) 11 compounds were isolated and identified by comparison with data reported in literature and/or by comparison with pure compounds available in our laboratory : verbascoside ( 10 , 12.4 mg ) from the assembly of fractions 8 - 15 ; stachysoside a ( 11 , 11.2 mg ) in mixture with verbascoside ( 1:2 ) in the assembly of fractions 17 - 24 ; b - arbutin ( 9 , 3.2 mg ) from the assembly of fractions 30 - 32 ; reptoside ( 6 ) and 6-o - acetyl - ajugol ( 7 ) in mixture in ratio 1:1.5 ( 25.0 mg ) from the assembly of fractions 43 - 45 ; ajugoside ( 1 , 113.7 mg ) as pure compound from the assembly of fractions 53 - 54 ; 8-o - acetyl - harpagide ( 2 , 1.0 g ) from the assembly of fractions 70 - 88 ; chlorogenic acid ( 8) in mixture with 8-o - acetyl - harpagide ( 2 ) in ratio 1:2 ( 109.4 mg ) from the assembly of fractions 95 - 112 ; harpagide ( 3 ) and chlorogenic acid ( 8) in mixture 3:1 ( 48.3 mg ) from the assembly of fractions 118 - 122 ; allobetonicoside ( 4 ) and chlorogenic acid ( 8) in mixture 10:1 ( 29.1 mg ) from the assembly of fractions 133 - 135 ; allobetonicoside ( 4 , 100.7 mg ) as a pure compound from the assembly of fractions 144 - 163 ; 4-o - galactopyranosyl - teuhircoside ( 5 ) in mixture with allobetonicoside ( 4 ) in ratio 3:1 ( 24.7 mg ) from the assembly of fractions 166 - 169 . chromatographic steps on 60% ethonolic fraction and elution order of components a second chromatographic column was , instead , conducted on an aliquot of the 30% desorption solids ( 3.5 g ) . in particular , harpagide ( 3 ) , 8-o - acetylharpagide ( 2 ) as pure compounds , allobetonicoside ( 4 ) in mixture with 4-o - galactopyranosyl - teuhircoside ( 5 ) , and ajugoside ( 1 ) occurring in traces ( relative quantities not estimated ) . on the other hand , phenolic compounds as well as reptoside ( 6 ) and 6-o - acetyl - ajugol ( 7 ) , were not found . ajugoside ( 1 ) : nmr data see table 1 ; esi - ms : m / z 412.80 [ m+na ] . complete nmr assignment of ajugoside ( 1 ) in d2o 8-o - acetylharpagide ( 2 ) : h nmr ( 300 mhz , cd3od ) d : 6.40 ( 1h , d , j = 6.4 hz , h-3 ) , 6.08 ( 1h , br s , h-1 ) , 4.92 ( 1h , d , j = 6.5 hz , h-4 ) , 4.60 ( 1h , d , j = 7.8 hz , h-1 ) , 3.90 ( 1h , d , j = 11.8 hz , h-6a ) , 3.71 ( 2h , m , h-6b ; h-3 ) , 3.44 - 3.29 ( 2h , m , h-4 ; h-5 ) , 3.22 ( 1h , t like , j = 8.4 hz , h-2 ) , 2.86 ( 1h , br s , h-9 ) , 2.18 ( 1h , d , j = 15.1 hz , h-7a ) , 2.02 ( 3h , s , ch3co ) , 1.94 ( 1h , dd , j = 15.2 , 4.4 hz , h-7b ) , and 1.45 ( 3h , s , h-10 ) . c nmr ( apt ) ( 75 mhz , cd3od ) d : 173.3 ( ch3co ) , 143.8 ( c-3 ) , 106.8 ( c-4 ) , 99.8 ( c-1 ) , 94.5 ( c-1 ) , 88.6 ( c-8 ) , 78.0 ( c-3 ) , 77.6 ( c-5 ) , 77.4 ( c-6 ) , 74.4 ( c-2 ) , 73.3 ( c-5 ) , 71.6 ( c-4 ) , 62.8 ( c-6 ) , 55.4 ( c-9 ) , 46.0 ( c-7 ) , 22.5 ( ch3co ) , and 22.2 ( c-10 ) . esi - ms : m / z 429.04 [ m+na ] ; m / z 835.19 [ 2m+na ] . harpagide ( 3 ) : h nmr ( 300 mhz , cd3od ) d : 6.32 ( 1h , d , j = 6.3 hz , h-3 ) , 5.74 ( 1h , br s , h-1 ) , 4.95 ( 1h , d , j = 6.3 hz , h-4 ) , 4.58 ( 1h , d , j = 7.9 hz , h-1 ) , 3.90 ( 1h , d , j = 12.2 hz , h-6a ) , 3.75 - 3.60 ( 2h , m , h-6b ; h-3 ) , 3.44 - 3.33 ( 2h , m , h-4 ; h-5 ) , 3.26 - 3.16 ( 1h , dd , j = 8.8 , 8.2 hz , h-2 ) , 2.56 ( 1h , br s , h-9 ) , 1.90 ( 1h , dd , j = 13.8 , 4.6 hz , h-7a ) , 1.80 ( 1h , dd , j = 13.8 , 3.6 hz , h-7b ) , 1.25 ( 3h , s , h-10 ) . allobetonicoside ( 4 ) : h nmr ( 300 mhz , d2o ) d : 6.43 ( 1h , d , j = 6.3 hz , h-3 ) , 6.11 ( 1h , br s , h-7 ) , 5.94 ( 1h br s , h-1 ) , 5.28 ( 1h , d , j = 8.3 hz , h-1 ) , 5.01 ( 1h , d , j = 6.3 hz , h-4 ) , 4.75 ( 1h , d , j = 7.8 hz , h-1 ) , 4.19 ( 1h , t , j = 2.6 hz , h-9 ) , 3.93 ( 2h , m , h-6a ; h-6a ) , 3.83 ( 2h , m , h-6b ; h-6b ) , 3.70 - 3.33 ( 8h , m , h-3 ; h-3 ; h-4 ; h-4 ; h5 ; h-5 ; h-2 ; h-2 ) , and 2.28 ( 3h , s , h-10 ) . c nmr ( 75 mhz , d2o ) d : 204.86 ( c-6 ) , 178.50 ( c-8 ) , 144.09 ( c-3 ) , 127.77 ( c-7 ) , 102.69 ( c-4 ) , 98.89 ( c-1 ) , 96.58 ( c-1 ) , 92.25 ( c1 ) , 77.45 ( c-5 ) , 76.18 ( c-5 ) , 75.60 ( c-3 ) , 73.57 ( c-2 ) , 72.71 ( c-4 ) , 71.20 ( c-2 ) , 69.85 ( c-3 ) , 69.42 ( c-4 ) , 66.62 ( c-4 ) , 61.03 ( c-6 ) , 60.70 ( c-6 ) , 54.85 ( c-9 ) , 17.62 ( c-10 ) . esi - ms : m / z 528.73 [ m+na ] ; m / z 544.69 [ m+k ] . 4-o - galactopyranosyl - teuhircoside ( 5 ) : h nmr ( 300 mhz , d2o ) d : 6.43 ( 1h , d , j = 6.4 hz , h-3 ) , 6.13 ( 1h , br s , h-7 ) , 5.94 ( 1h , br s , h-1 ) , 4.98 ( 1h , d , j = 6.4 hz , h-4 ) , 4.80 ( 1h , partially overlapped with solvent signal , h-1 ) , 4.73 ( 1h , d , j = 8.1 hz , h-1 ) , 2.29 ( 3h , s , h-10 ) . reptoside ( 6 ) : h nmr ( 300 mhz , d2o ) d : 6.40 ( 1h , d , j = 6.5 hz , h-3 ) , 5.90 ( 1h , br s , h-1 ) , 5.02 ( 1h , dd , j = 6.4 , 1.3 hz , h-4 ) , 4.75 ( 1h , d , partially obscured by the solvent signal , h-1 ) , 3.92 ( 1h , dd , j = 12.4 , 1.9 hz , h-6a ) , 3.71 ( 1h , br d , j = 12.4 hz , h-6b ) , 3.26 ( 1h , dd , j = 8.3 , 8.2 hz , h-2 ) , 2.65 ( 1h , br s , h-9 ) , 2.04 ( 3h , s , ch3co ) , 1.93 ( 2h , m , h-6a ; h-7a ) , 1.71 ( 2h , m , h-6b ; h-7b ) , 1.43 ( 3h , s , h-10 ) . 6-o - acetyl - ajugol ( 7 ) : h nmr ( 300 mhz , d2o ) d : 6.34 ( 1h , dd , j = 6.4 , 1.8 hz , h-3 ) , 5.50 ( 1h , d , j = 2.8 hz , h-1 ) , 4.89 ( 1h , dd , j = 6.4 , 2.8 hz , h-4 ) , 4.81 ( 1h , obscured by the solvent signal , h-6 ) , 4.70 ( 1h , d , j = 8.0 hz , h-1 ) , 3.90 ( 1h , dd , j = 12.1 , 1.6 hz , h-6 ) , 3.68 ( 1h , br d , j = 12.2 hz , h-6b ) , 3.24 ( 1h , m , h-2 ) , 2.79 ( 1h , br t , j = 6.5 hz , h-5 ) , 2.34 ( 1h , dd , j = 7.9 , 2.7 hz , h-9 ) , 2.12 ( 1h , br dd , j = 14.0 , 6.5 hz , h-7a ) , 2.08 ( 3h , s , ch3co ) , 1.93 ( 1h , dd , j = 13.8 , 8.6 hz , h-7b ) , 1.38 ( 3h , s , h-10 ) . chlorogenic acid ( 8) : h nmr ( 300 mhz , cd3od ) d : 7.56 ( 1h , d , j = 15.9 hz , h - b ) , 7.05 ( 1h , d , j = 1.9 hz , h-2 ) , 6.96 ( 1h , dd , j = 8.2 , 1.9 hz , h-6 ) , 6.78 ( 1h , d , j = 8.2 hz , h-5 ) , 6.26 ( 1h , d , j = 15.9 hz , h - a ) , 5.33 ( 1h , td , j = 9.1 , 4.5 hz , h-3 ) , 4.17 ( 1h , dt , j = 6.2 , 3.2 hz , h-4 ) , 3.73 ( dd , j = 8.5 , 3.1 hz , h-5 ) , 2.27 - 2.13 ( 2h , m , h-2a ; h-6a ) , 2.13 - 1.98 ( 2h , m , h-2b;h-6b ) . b - arbutin ( 9 ) : h nmr ( 300 mhz , cd3od ) d : 7.07 ( 2h , d , j = 8.6 hz , h-3 , h-5 ) , 6.69 ( 2h , d , j = 8.6 hz , h-2 , h-6 ) , 4.95 ( 1h , partially obscured by hdo signal , h-1 ) , 3.96 - 3.79 ( m , overlapped glucose protons ) , 3.77 - 3.54 ( m , overlapped glucose protons ) , 3.18 ( 1h , br t , j = 7.9 hz , h-2 ) . verbascoside ( 10 ) : h nmr ( 300 mhz , cd3od ) d : 7.59 ( 1h , d , j = 15.9 hz , h - b ) , 7.05 ( 1h , d , j = 1.4 hz , h-2 ) , 6.96 ( 1h , dd , j = 8.2 , 1.4 hz , h-6 ) , 6.78 ( 1h , d , j = 8.2 hz , h-5 ) , 6.69 ( 1h , br s , h-2 ) , 6.68 ( 1h , d , j = 8.0 hz , h-5 ) , 6.56 ( 1h , dd , j = 8.0 , 1.6 hz , h-6 ) , 6.28 ( 1h , d , j = 15.9 hz , h - a ) , 5.19 ( 1h , d , j = 0.9 hz , h-1 ) , 4.38 ( 1h , d , j = 7.8 hz , h-1 ) , 2.79 ( 2h , br t , j = 7.3 hz , h - b ) , 1.09 ( 3h , d , j = 6.2 hz , h-6 ) . esi - ms : m / z 623.19 [ m - h ] ; m / z 647.12 [ m+na ] . stachysoside a ( 11 ) : h nmr ( 300 mhz , cd3od ) d : 7.60 ( 1h , d , j = 15.6 hz , h - b[caff . ] ) , 7.06 ( 1h , br s , h-2 ) , 6.98 ( 1h , br d , j = 8.5 hz , h-6 ) , 6.78 ( 1h , d , j = 8.5 hz , h-5 ) , 6.70 ( 1h , br s , h-2 ) , 6.68 ( 1h , d , j = 8.0 hz , h-5 ) , 6.57 ( 1h , br d , j = 8.1 hz , h-6 ) , 6.28 ( 1h , d , j = 15.6 hz , h - a ) , 5.42 ( 1h , br s , h-1 ) , 4.43 ( 1h , d , j = 7.9 hz , h-1 ) , 4.31 ( 1h , d , j = 7.2 hz , h-1 ) , 2.81 ( 2h , d , j = 7.1 hz , h - b ) , 1.16 ( 3h , d , j = 6.0 hz , h-6 ) . esi - ms : m / z 755.20 [ m - h ] ; m / z 779.16 [ m+na ] . the majority of them were iridoids , followed by a phenolic glucoside , two phenylethanoid glycosides , and a caffeoylquinic acid . they were identified as ajugoside ( 1 ) , 8-o - acetylharpagide ( 2 ) , harpagide ( 3 ) , allobetonicoside ( 4 ) , 4-o - galactopyranosyl - teuhircoside ( 5 ) , reptoside ( 6 ) , 6-o - acetyl - ajugol ( 7 ) [ figure 1 ] for the iridoid glycosides constituents , and as chlorogenic acid ( 8) , b - arbutin ( 9 ) , verbascoside ( 10 ) , and stachysoside a ( 11 ) [ ( 2-(3,4-dihydroxyphenyl)ethyl - o - a - l - arabinopyranosyl-(12)-o-6-deoxy - b - l - mannopyranosyl-(13)-4-o-[(2e)-3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]-b - d - glucopyranoside ] [ figure 2 ] for the phenolic compounds . the presence of previously identified iridoid glycosides ( 2 - 5 ) has been confirmed , together with ajugoside ( 1 ) , reptoside ( 6 ) , and 6-o - acetyl - ajugol ( 7 ) which resulted to be new iridoid components for this species . among the phenolic constituents , chlorogenic acid ( 8) , b - arbutin ( 9 ) , and stachysoside a ( 11 ) the identified iridoids have all chemosystematic relevance . in particular , 2 and 3 are considered family markers , 4 is characteristic of the section betonica , and 5 has never been reported in other stachys species so that it may be regarded as a chemotaxonomic marker at a subspecific level . reptoside ( 6 ) resulted to be a rare metabolite in the stachys genus because its occurrence has been reported only in a few species , namely betonica officinalis ( syn . 6-o - acetyl - ajugol ( 7 ) , is also a rare metabolite in the lamiaceae family since it has been found only in leonurus persicus so far . it is also worth of mention the presence of ajugoside ( 1 ) and reptoside ( 6 ) which are iridoids widely distributed in the species of the ajugoideae subfamily . in fact , 1 was mainly reported in the ajuga genus ( e.g. ajuga tenorei and ajuga chamaepitys ) , although its presence was also signaled in a few lamioideae species as melittis melissophyllum subsp . the occurrence of 1 in the stachys genus is limited to a few species such as b. officinalis ( syn . of s. officinalis ( l. ) trevis . ) , s. macrantha , s. alpina , stachys germanica and stachys recta , and it has been identified also in the taxonomically related sideritis genus like for instance in sideritis romana , and sideritis perfoliata subsp . perfoliata . considering their occurrence , compounds ( 1 ) and ( 6 ) may represent chemosystematic traits of proximity between lamioideae and ajugoideae subfamilies . the identification of 1 has been carried out by extensive nmr analysis on both mono - [ figures 3 and 4 ] and bidimensional [ figures 5 and 6 ] experiments since the assignment of resonances was not immediate for several signals , in particular for protons in 5 , 6 and 9 positions on the iridoid skeleton as well as for protons in 3 and 5 positions of the glucose moiety [ figures 3 and 4 ] . h - nmr spectrum of ajugoside ( 1 ) c - nmr spectrum of ajugoside ( 1 ) heteronuclear single quantum correlation spectrum of ajugoside ( 1 ) heteronuclear multiple bond correlation spectrum of ajugoside ( 1 ) in particular , the resonance of h-6 was readily visible in the proton spectrum at 4.18 ppm , but the corresponding carbon signal was very near to a glucose carbon resonance and has been assigned by the direct hsqc correlation [ figure 5 ] with the signal resonating at 75.55 ppm . on the contrary , the assignment of protons and carbons in the position 5 and 9 was not immediate , and several long range correlations resulted to be crucial for discriminating the resonances of these nuclei . the signals relative to the positions 5 and 9 present resonances in the proton spectrum at 2.93 and 2.85 ppm , both as broad signals , and the study of the coupling constants gave no help for the structure elucidation . in the hsqc spectrum , a direct correlation between the proton signal at 2.93 ppm and the carbon resonating at 47.48 ppm was recorded as well as the correlation between the proton at 2.85 and the carbon at 39.83 ppm . this permitted to assign the respective carbons signals . in the hmbc experiments , [ figure 6 ] the only diagnostic correlation which permitted to differentiate the positions 5 and 9 was a long range correlation between h-3 ( 6.31 ppm ) and the carbon resonating at 39.83 ppm , which could be assigned to c-5 of the iridoid skeleton , since almost all the proton signals correlated with both the carbons at 39.83 and 47.48 ppm . in fact , h-3 is relatively far from c-9 , and this kind of signal could not be visualized with the coupling constant of 8 hz set in the experiment . using the hmbc correlations , it was also possible to differentiate the positions 3 and 5 on the glucopyranose unit . in particular , the resonances of the positions 1 , 2 , 4 , and 6 ( 98.25 , 72.73 , 69.65 , and 60.77 , respectively ) were readily assignable to the saccharidic moiety , while the signals relative to 3 and 5 might be differentiated on the basis of their correlation with near nuclei [ figures 5 and 6 ] . more specifically , from hmbc experiments the following long - range correlations were recognized : between the proton signal at 3.33 ppm ( h-2 ) and the carbon at 75.65 ( c-3 ) ; the proton at 3.34 ppm ( h-4 ) with carbons at 76.26 ( c-5 ) and 75.65 ( c-3 ) ; the protons at 3.98 - 3.78 ( h-6 ) with carbon resonating at 76.26 ( c-5 ) . the latter correlation is particularly crucial for the assignation of the c-5 position because h-6 can not correlate with c-3. the complete assignments of resonances are reported in table 1 . chlorogenic acid ( 8) has been isolated for the first time from s. alopecuros subsp divulsa , although it has been already found in several stachys species as s. tymphaea , s. lanata , s. officinalis , s. recta , s. byzantina , s. iberica subsp . the chlorogenic acid ( 8) resulted to be the main polyphenolic component occurring in the polar fraction just like in this case [ figure 2 ] . furthermore , b - arbutin ( 9 ) was a metabolite identified for the first time in this species . it proved to be a quite rare compound in the stachys genus since its presence has been signaled only in s. germanica subsp . the presence of verbascoside ( 10 ) , a quite common phenyletanoid glycoside ( phgs ) in lamiaceae , has been confirmed , while stachysoside a ( 11 ) , also known with the name lavandulifolioside , resulted to be as a new constituent of s. alopecuros subsp . phgs are compounds with a chemosystematic relevance in asterids since their cooccurrence with iridoid glycosides , as in the studied case , has been largely confirmed . the presence of 11 was noteworthy since it is not an ubiquitous compound , especially in the stachys genus where it has been recognized only in a few species such as s. lavandulifolia , s. sieboldii , and s. tymphaea . this compound resulted to be a constituent of other systematically related genera such as leonurus , lagochilus , and sideritis . ( pedaliaceae ) , a medicinal plant native to namibia and commonly known as devil s claw . this plant has been widely studied for its anti - inflammatory and analgesic effects which were demonstrated also in in vivo models , principally in rats [ 41 - 43 ] . the target of harpagide and its derivatives is represented by the inhibition of cox-2 , an enzyme deputed to the production of the proinflammatory mediators prostaglandins and leukotrienes . recent docking studies have demonstrated that harpagide is a selective inhibitor of cox-2 , binding its active site with a strong interaction and being stabilized by 10 hydrogen bonds . this confirms , on a molecular base , the observed anti - inflammatory and analgesic actions exerted by plants containing harpagide derivatives . beside the anti - inflammatory action , harpagide ( 3 ) results a modulator of tumor necrosis factor a ( tnf - a ) secretion and induces the expression of several proteins involved in cell migrations . the latter is a very important role because the inflammatory process is not only responsible of pain and loss of function but it is also involved in the onset and progression of degenerative diseases , including cardiovascular disorders and cancer . in a similar way , 8-o - acetyl harpagide ( 3 ) also showed a dose - related inhibition of leukocyte adhesion and transmigration in a system based on human umbilical vein endothelial cells , and human monocytic leukemia cell line thp-1 previously activated with tnf - a . it was also studied , the protective effect of harpagide ( 3 ) against bone loss in ovariectomized mouse model . it resulted that this iridoid stimulates the differentiation and maturation processes of osteoblasts and this conducted to an overall improved bone properties with the recovery of mineral density . the reduced levels of biochemical markers of bone loss in the serum of treated mice ( alkaline phosphatase , osteocalcin , and c - terminal telopeptide ) seems to be the major mode of action of harpagide ( 3 ) , thus demonstrating the potential role of this iridoid also in the prevention and treatment of age - dependent bone osteoporosis diseases . from recent studies on iridoid activities , 8-o - acetylharpagide ( 2 ) and ajugoside ( 1 ) resulted to be useful compounds in the treatment of diabetes mellitus since they were able to stimulate the cellular glucose intake and lower the glucose blood level . another target of iridoids for the antidiabetic activity is represented by the inhibitory action , verified by docking studies , on glycogen phosphorylase - a . in fact , several plant species rich in iridoids have been largely used in traditional medicine for the treatment of diabetes , e.g. , scrophularia deserti del . and leonurus sibiricus l. . the phenolic fraction of s. alopecuros subsp . divulsa was mainly composed by chlorogenic acid ( 8) , which is also endowed with interesting activities . this caffeoylquinic acid is a strong antioxidant , a good wound healer if topically applied on excisions and has also antiviral action toward hepatitis b virus . furthermore , it shows antiproliferative action , inhibits carcinogenesis , strongly reduces the risk of cardiovascular diseases and significantly improves the metabolism of glucose . the latter is the main reason why it has recently drawn the attention from nutritionists for its high antidiabetic and anti - obesity properties . the antioxidant and enzyme inhibitory activities in respect to acetylcholineserase , butrylcholinesterase , and a - amylase may have a protective role in neurologic diseases such as alzheimer s disease , and metabolic disorders as diabetes mellitus . in fact , b - arbutin ( 9 ) possesses a strong antibacterial activity and is traditionally used for urinary tract infections . it is also a tyrosinase inhibitor and is employed as a skin whitener because it inhibits the melanin synthesis . the phenylethanoid glycoside verbascoside ( 10 ) shows an antibacterial action especially toward s. aureus and anti - inflammatory activity . more recently , in an in vivo study , it resulted to be an inhibitor of xanthine oxidase which reduced uric acid concentration in rat serum . the activity of stachysoside a ( 11 ) , in literature there are mainly studies conducted on extracts obtained from species containing this compound . in this context , it is noteworthy the neuroprotective effects observed in s. sieboldii tuber extract which resulted to improve the memory dysfunction associated with vascular dementia or alzheimer s disease in induced - ischaemia in mice . the presence of antioxidant components resulted to have a primary role in neuroprotection and phgs are well - known antioxidants . species belonging to leonurus genus , all containing ( 11 ) together with iridoids , have been largely used in the traditional mongolian medicine for the treatment of diabetes mellitus and in the treatment of neurological disorders such as anxiety , depression , and nervousness . the identified iridoids have all chemosystematic relevance . in particular , 2 and 3 are considered family markers , 4 is characteristic of the section betonica , and 5 has never been reported in other stachys species so that it may be regarded as a chemotaxonomic marker at a subspecific level . reptoside ( 6 ) resulted to be a rare metabolite in the stachys genus because its occurrence has been reported only in a few species , namely betonica officinalis ( syn . 6-o - acetyl - ajugol ( 7 ) , is also a rare metabolite in the lamiaceae family since it has been found only in leonurus persicus so far . it is also worth of mention the presence of ajugoside ( 1 ) and reptoside ( 6 ) which are iridoids widely distributed in the species of the ajugoideae subfamily . in fact , 1 was mainly reported in the ajuga genus ( e.g. ajuga tenorei and ajuga chamaepitys ) , although its presence was also signaled in a few lamioideae species as melittis melissophyllum subsp . the occurrence of 1 in the stachys genus is limited to a few species such as b. officinalis ( syn . of s. officinalis ( l. ) trevis . ) , s. macrantha , s. alpina , stachys germanica and stachys recta , and it has been identified also in the taxonomically related sideritis genus like for instance in sideritis romana , and sideritis perfoliata subsp . perfoliata . considering their occurrence , compounds ( 1 ) and ( 6 ) may represent chemosystematic traits of proximity between lamioideae and ajugoideae subfamilies . the identification of 1 has been carried out by extensive nmr analysis on both mono - [ figures 3 and 4 ] and bidimensional [ figures 5 and 6 ] experiments since the assignment of resonances was not immediate for several signals , in particular for protons in 5 , 6 and 9 positions on the iridoid skeleton as well as for protons in 3 and 5 positions of the glucose moiety [ figures 3 and 4 ] . h - nmr spectrum of ajugoside ( 1 ) c - nmr spectrum of ajugoside ( 1 ) heteronuclear single quantum correlation spectrum of ajugoside ( 1 ) heteronuclear multiple bond correlation spectrum of ajugoside ( 1 ) in particular , the resonance of h-6 was readily visible in the proton spectrum at 4.18 ppm , but the corresponding carbon signal was very near to a glucose carbon resonance and has been assigned by the direct hsqc correlation [ figure 5 ] with the signal resonating at 75.55 ppm . on the contrary , the assignment of protons and carbons in the position 5 and 9 was not immediate , and several long range correlations resulted to be crucial for discriminating the resonances of these nuclei . the signals relative to the positions 5 and 9 present resonances in the proton spectrum at 2.93 and 2.85 ppm , both as broad signals , and the study of the coupling constants gave no help for the structure elucidation . in the hsqc spectrum , a direct correlation between the proton signal at 2.93 ppm and the carbon resonating at 47.48 ppm was recorded as well as the correlation between the proton at 2.85 and the carbon at 39.83 ppm . this permitted to assign the respective carbons signals . in the hmbc experiments , [ figure 6 ] the only diagnostic correlation which permitted to differentiate the positions 5 and 9 was a long range correlation between h-3 ( 6.31 ppm ) and the carbon resonating at 39.83 ppm , which could be assigned to c-5 of the iridoid skeleton , since almost all the proton signals correlated with both the carbons at 39.83 and 47.48 ppm . in fact , h-3 is relatively far from c-9 , and this kind of signal could not be visualized with the coupling constant of 8 hz set in the experiment . using the hmbc correlations , it was also possible to differentiate the positions 3 and 5 on the glucopyranose unit . in particular , the resonances of the positions 1 , 2 , 4 , and 6 ( 98.25 , 72.73 , 69.65 , and 60.77 , respectively ) were readily assignable to the saccharidic moiety , while the signals relative to 3 and 5 might be differentiated on the basis of their correlation with near nuclei [ figures 5 and 6 ] . more specifically , from hmbc experiments the following long - range correlations were recognized : between the proton signal at 3.33 ppm ( h-2 ) and the carbon at 75.65 ( c-3 ) ; the proton at 3.34 ppm ( h-4 ) with carbons at 76.26 ( c-5 ) and 75.65 ( c-3 ) ; the protons at 3.98 - 3.78 ( h-6 ) with carbon resonating at 76.26 ( c-5 ) . the latter correlation is particularly crucial for the assignation of the c-5 position because h-6 can not correlate with c-3. the complete assignments of resonances are reported in table 1 . chlorogenic acid ( 8) has been isolated for the first time from s. alopecuros subsp divulsa , although it has been already found in several stachys species as s. tymphaea , s. lanata , s. officinalis , s. recta , s. byzantina , s. iberica subsp . the chlorogenic acid ( 8) resulted to be the main polyphenolic component occurring in the polar fraction just like in this case [ figure 2 ] . furthermore , b - arbutin ( 9 ) was a metabolite identified for the first time in this species . it proved to be a quite rare compound in the stachys genus since its presence has been signaled only in s. germanica subsp . the presence of verbascoside ( 10 ) , a quite common phenyletanoid glycoside ( phgs ) in lamiaceae , has been confirmed , while stachysoside a ( 11 ) , also known with the name lavandulifolioside , resulted to be as a new constituent of s. alopecuros subsp . phgs are compounds with a chemosystematic relevance in asterids since their cooccurrence with iridoid glycosides , as in the studied case , has been largely confirmed . the presence of 11 was noteworthy since it is not an ubiquitous compound , especially in the stachys genus where it has been recognized only in a few species such as s. lavandulifolia , s. sieboldii , and s. tymphaea . this compound resulted to be a constituent of other systematically related genera such as leonurus , lagochilus , and sideritis . ( pedaliaceae ) , a medicinal plant native to namibia and commonly known as devil s claw . this plant has been widely studied for its anti - inflammatory and analgesic effects which were demonstrated also in in vivo models , principally in rats [ 41 - 43 ] . the target of harpagide and its derivatives is represented by the inhibition of cox-2 , an enzyme deputed to the production of the proinflammatory mediators prostaglandins and leukotrienes . recent docking studies have demonstrated that harpagide is a selective inhibitor of cox-2 , binding its active site with a strong interaction and being stabilized by 10 hydrogen bonds . this confirms , on a molecular base , the observed anti - inflammatory and analgesic actions exerted by plants containing harpagide derivatives . beside the anti - inflammatory action , harpagide ( 3 ) results a modulator of tumor necrosis factor a ( tnf - a ) secretion and induces the expression of several proteins involved in cell migrations . the latter is a very important role because the inflammatory process is not only responsible of pain and loss of function but it is also involved in the onset and progression of degenerative diseases , including cardiovascular disorders and cancer . in a similar way , 8-o - acetyl harpagide ( 3 ) also showed a dose - related inhibition of leukocyte adhesion and transmigration in a system based on human umbilical vein endothelial cells , and human monocytic leukemia cell line thp-1 previously activated with tnf - a . it was also studied , the protective effect of harpagide ( 3 ) against bone loss in ovariectomized mouse model . it resulted that this iridoid stimulates the differentiation and maturation processes of osteoblasts and this conducted to an overall improved bone properties with the recovery of mineral density . the reduced levels of biochemical markers of bone loss in the serum of treated mice ( alkaline phosphatase , osteocalcin , and c - terminal telopeptide ) seems to be the major mode of action of harpagide ( 3 ) , thus demonstrating the potential role of this iridoid also in the prevention and treatment of age - dependent bone osteoporosis diseases . from recent studies on iridoid activities , 8-o - acetylharpagide ( 2 ) and ajugoside ( 1 ) resulted to be useful compounds in the treatment of diabetes mellitus since they were able to stimulate the cellular glucose intake and lower the glucose blood level . another target of iridoids for the antidiabetic activity is represented by the inhibitory action , verified by docking studies , on glycogen phosphorylase - a . in fact , several plant species rich in iridoids have been largely used in traditional medicine for the treatment of diabetes , e.g. , scrophularia deserti del . and leonurus sibiricus l. . the phenolic fraction of s. alopecuros subsp . divulsa was mainly composed by chlorogenic acid ( 8) , which is also endowed with interesting activities . this caffeoylquinic acid is a strong antioxidant , a good wound healer if topically applied on excisions and has also antiviral action toward hepatitis b virus . furthermore , it shows antiproliferative action , inhibits carcinogenesis , strongly reduces the risk of cardiovascular diseases and significantly improves the metabolism of glucose . the latter is the main reason why it has recently drawn the attention from nutritionists for its high antidiabetic and anti - obesity properties . the antioxidant and enzyme inhibitory activities in respect to acetylcholineserase , butrylcholinesterase , and a - amylase may have a protective role in neurologic diseases such as alzheimer s disease , and metabolic disorders as diabetes mellitus . in fact , b - arbutin ( 9 ) possesses a strong antibacterial activity and is traditionally used for urinary tract infections . it is also a tyrosinase inhibitor and is employed as a skin whitener because it inhibits the melanin synthesis . the phenylethanoid glycoside verbascoside ( 10 ) shows an antibacterial action especially toward s. aureus and anti - inflammatory activity . more recently , in an in vivo study , it resulted to be an inhibitor of xanthine oxidase which reduced uric acid concentration in rat serum . the activity of stachysoside a ( 11 ) , in literature there are mainly studies conducted on extracts obtained from species containing this compound . in this context , it is noteworthy the neuroprotective effects observed in s. sieboldii tuber extract which resulted to improve the memory dysfunction associated with vascular dementia or alzheimer s disease in induced - ischaemia in mice . the presence of antioxidant components resulted to have a primary role in neuroprotection and phgs are well - known antioxidants . species belonging to leonurus genus , all containing ( 11 ) together with iridoids , have been largely used in the traditional mongolian medicine for the treatment of diabetes mellitus and in the treatment of neurological disorders such as anxiety , depression , and nervousness . divulsa shed light on three additional iridoid glucosides together with chlorogenic acid ( 8) , b - arbutin ( 9 ) , verbascoside ( 10 ) , and stachysoside a ( 11 ) in the phenolic fraction . the presence of verbascoside has been already reported , while the other phenolics were identified for the first time in the studied species . the monoterpene glycoside pattern owned by s. alopecuros subsp . divulsa has been disclosed with respect to literature data , showing the presence of ajugoside ( 1 ) , reptoside ( 6 ) , and 6-o - acetyl - ajugol ( 7 ) . in particular , 1 and 6 may represent a metabolic connection between lamioideae and ajugoideae subfamilies while 7 is a quite uncommon compound in the lamiaceae family since it has been found only in l. persicus so far . we also took advantage of this occasion to report a complete nmr assignment for compound 1 . from a therapeutic perspective , the isolated compounds are known for their antioxidant , antiproliferative , anti - inflammatory , analgesic , immunomodulatory , neuroprotective , and antidiabetic actions . their occurrence in the studied species provides evidence of a possible natural source of bioactive compounds for extractive purposes but also of its potential as a natural remedy of medical importance .

background : the phytochemical analysis of stachys alopecuros subsp . divulsa , an endemic italian species , has been recently reported and has showed the presence of 8-o - acetylharpagide ( 2 ) , harpagide ( 3 ) , allobetonicoside ( 4 ) , and 4-o - galactopyranosyl - teuhircoside ( 5 ) . in this paper , an in deep study of its glycosidic fraction with the aim to widen the knowledge on its secondary metabolites content is reported.materials and methods : chromatographic techniques were used for the isolation of constituents while spectroscopic and spectrometric techniques were applied for the structures elucidation.results:besides the known constituents , all of them reconfirmed , ajugoside ( 1 ) , reptoside ( 6 ) and 6-o - acetyl - ajugol ( 7 ) were also identified among the iridoids while the phenolic components resulted to be chlorogenic acid ( 8) , b - arbutin ( 9 ) , verbascoside ( 10 ) , and stachysoside a ( 11 ) , instead.conclusion:the iridoid pattern of s. alopecuros subsp . divulsa has been expanded with the identification of not previously reported compounds as well as for the phenolic fraction . except for the reconfirmed verbascoside ( 10 ) , the other phenolic compounds were recognized for the first time in the studied species . the complete nmr assignment of compound ( 1 ) by means of bidimensional techniques is reported , and both the chemotaxonomic and pharmacological relevance of the isolated compounds is largely discussed .

INTRODUCTION MATERIALS AND METHODS Plant Materials Instruments Bidimensional NMR Experiments Extraction and Isolation of Polar Compounds Adsorption Chromatography Silica Gel Column Chromatography Spectral Data of the Isolated Compounds RESULTS DISCUSSION Chemosystematic Implications of Isolated Compounds Bioactivities of Isolated Compounds CONCLUSIONS

from this chromatographic separation ( scheme 1 ) 11 compounds were isolated and identified by comparison with data reported in literature and/or by comparison with pure compounds available in our laboratory : verbascoside ( 10 , 12.4 mg ) from the assembly of fractions 8 - 15 ; stachysoside a ( 11 , 11.2 mg ) in mixture with verbascoside ( 1:2 ) in the assembly of fractions 17 - 24 ; b - arbutin ( 9 , 3.2 mg ) from the assembly of fractions 30 - 32 ; reptoside ( 6 ) and 6-o - acetyl - ajugol ( 7 ) in mixture in ratio 1:1.5 ( 25.0 mg ) from the assembly of fractions 43 - 45 ; ajugoside ( 1 , 113.7 mg ) as pure compound from the assembly of fractions 53 - 54 ; 8-o - acetyl - harpagide ( 2 , 1.0 g ) from the assembly of fractions 70 - 88 ; chlorogenic acid ( 8) in mixture with 8-o - acetyl - harpagide ( 2 ) in ratio 1:2 ( 109.4 mg ) from the assembly of fractions 95 - 112 ; harpagide ( 3 ) and chlorogenic acid ( 8) in mixture 3:1 ( 48.3 mg ) from the assembly of fractions 118 - 122 ; allobetonicoside ( 4 ) and chlorogenic acid ( 8) in mixture 10:1 ( 29.1 mg ) from the assembly of fractions 133 - 135 ; allobetonicoside ( 4 , 100.7 mg ) as a pure compound from the assembly of fractions 144 - 163 ; 4-o - galactopyranosyl - teuhircoside ( 5 ) in mixture with allobetonicoside ( 4 ) in ratio 3:1 ( 24.7 mg ) from the assembly of fractions 166 - 169 . in particular , harpagide ( 3 ) , 8-o - acetylharpagide ( 2 ) as pure compounds , allobetonicoside ( 4 ) in mixture with 4-o - galactopyranosyl - teuhircoside ( 5 ) , and ajugoside ( 1 ) occurring in traces ( relative quantities not estimated ) . on the other hand , phenolic compounds as well as reptoside ( 6 ) and 6-o - acetyl - ajugol ( 7 ) , were not found . complete nmr assignment of ajugoside ( 1 ) in d2o 8-o - acetylharpagide ( 2 ) : h nmr ( 300 mhz , cd3od ) d : 6.40 ( 1h , d , j = 6.4 hz , h-3 ) , 6.08 ( 1h , br s , h-1 ) , 4.92 ( 1h , d , j = 6.5 hz , h-4 ) , 4.60 ( 1h , d , j = 7.8 hz , h-1 ) , 3.90 ( 1h , d , j = 11.8 hz , h-6a ) , 3.71 ( 2h , m , h-6b ; h-3 ) , 3.44 - 3.29 ( 2h , m , h-4 ; h-5 ) , 3.22 ( 1h , t like , j = 8.4 hz , h-2 ) , 2.86 ( 1h , br s , h-9 ) , 2.18 ( 1h , d , j = 15.1 hz , h-7a ) , 2.02 ( 3h , s , ch3co ) , 1.94 ( 1h , dd , j = 15.2 , 4.4 hz , h-7b ) , and 1.45 ( 3h , s , h-10 ) . 4-o - galactopyranosyl - teuhircoside ( 5 ) : h nmr ( 300 mhz , d2o ) d : 6.43 ( 1h , d , j = 6.4 hz , h-3 ) , 6.13 ( 1h , br s , h-7 ) , 5.94 ( 1h , br s , h-1 ) , 4.98 ( 1h , d , j = 6.4 hz , h-4 ) , 4.80 ( 1h , partially overlapped with solvent signal , h-1 ) , 4.73 ( 1h , d , j = 8.1 hz , h-1 ) , 2.29 ( 3h , s , h-10 ) . 6-o - acetyl - ajugol ( 7 ) : h nmr ( 300 mhz , d2o ) d : 6.34 ( 1h , dd , j = 6.4 , 1.8 hz , h-3 ) , 5.50 ( 1h , d , j = 2.8 hz , h-1 ) , 4.89 ( 1h , dd , j = 6.4 , 2.8 hz , h-4 ) , 4.81 ( 1h , obscured by the solvent signal , h-6 ) , 4.70 ( 1h , d , j = 8.0 hz , h-1 ) , 3.90 ( 1h , dd , j = 12.1 , 1.6 hz , h-6 ) , 3.68 ( 1h , br d , j = 12.2 hz , h-6b ) , 3.24 ( 1h , m , h-2 ) , 2.79 ( 1h , br t , j = 6.5 hz , h-5 ) , 2.34 ( 1h , dd , j = 7.9 , 2.7 hz , h-9 ) , 2.12 ( 1h , br dd , j = 14.0 , 6.5 hz , h-7a ) , 2.08 ( 3h , s , ch3co ) , 1.93 ( 1h , dd , j = 13.8 , 8.6 hz , h-7b ) , 1.38 ( 3h , s , h-10 ) . from this chromatographic separation ( scheme 1 ) 11 compounds were isolated and identified by comparison with data reported in literature and/or by comparison with pure compounds available in our laboratory : verbascoside ( 10 , 12.4 mg ) from the assembly of fractions 8 - 15 ; stachysoside a ( 11 , 11.2 mg ) in mixture with verbascoside ( 1:2 ) in the assembly of fractions 17 - 24 ; b - arbutin ( 9 , 3.2 mg ) from the assembly of fractions 30 - 32 ; reptoside ( 6 ) and 6-o - acetyl - ajugol ( 7 ) in mixture in ratio 1:1.5 ( 25.0 mg ) from the assembly of fractions 43 - 45 ; ajugoside ( 1 , 113.7 mg ) as pure compound from the assembly of fractions 53 - 54 ; 8-o - acetyl - harpagide ( 2 , 1.0 g ) from the assembly of fractions 70 - 88 ; chlorogenic acid ( 8) in mixture with 8-o - acetyl - harpagide ( 2 ) in ratio 1:2 ( 109.4 mg ) from the assembly of fractions 95 - 112 ; harpagide ( 3 ) and chlorogenic acid ( 8) in mixture 3:1 ( 48.3 mg ) from the assembly of fractions 118 - 122 ; allobetonicoside ( 4 ) and chlorogenic acid ( 8) in mixture 10:1 ( 29.1 mg ) from the assembly of fractions 133 - 135 ; allobetonicoside ( 4 , 100.7 mg ) as a pure compound from the assembly of fractions 144 - 163 ; 4-o - galactopyranosyl - teuhircoside ( 5 ) in mixture with allobetonicoside ( 4 ) in ratio 3:1 ( 24.7 mg ) from the assembly of fractions 166 - 169 . in particular , harpagide ( 3 ) , 8-o - acetylharpagide ( 2 ) as pure compounds , allobetonicoside ( 4 ) in mixture with 4-o - galactopyranosyl - teuhircoside ( 5 ) , and ajugoside ( 1 ) occurring in traces ( relative quantities not estimated ) . on the other hand , phenolic compounds as well as reptoside ( 6 ) and 6-o - acetyl - ajugol ( 7 ) , were not found . complete nmr assignment of ajugoside ( 1 ) in d2o 8-o - acetylharpagide ( 2 ) : h nmr ( 300 mhz , cd3od ) d : 6.40 ( 1h , d , j = 6.4 hz , h-3 ) , 6.08 ( 1h , br s , h-1 ) , 4.92 ( 1h , d , j = 6.5 hz , h-4 ) , 4.60 ( 1h , d , j = 7.8 hz , h-1 ) , 3.90 ( 1h , d , j = 11.8 hz , h-6a ) , 3.71 ( 2h , m , h-6b ; h-3 ) , 3.44 - 3.29 ( 2h , m , h-4 ; h-5 ) , 3.22 ( 1h , t like , j = 8.4 hz , h-2 ) , 2.86 ( 1h , br s , h-9 ) , 2.18 ( 1h , d , j = 15.1 hz , h-7a ) , 2.02 ( 3h , s , ch3co ) , 1.94 ( 1h , dd , j = 15.2 , 4.4 hz , h-7b ) , and 1.45 ( 3h , s , h-10 ) . 4-o - galactopyranosyl - teuhircoside ( 5 ) : h nmr ( 300 mhz , d2o ) d : 6.43 ( 1h , d , j = 6.4 hz , h-3 ) , 6.13 ( 1h , br s , h-7 ) , 5.94 ( 1h , br s , h-1 ) , 4.98 ( 1h , d , j = 6.4 hz , h-4 ) , 4.80 ( 1h , partially overlapped with solvent signal , h-1 ) , 4.73 ( 1h , d , j = 8.1 hz , h-1 ) , 2.29 ( 3h , s , h-10 ) . 6-o - acetyl - ajugol ( 7 ) : h nmr ( 300 mhz , d2o ) d : 6.34 ( 1h , dd , j = 6.4 , 1.8 hz , h-3 ) , 5.50 ( 1h , d , j = 2.8 hz , h-1 ) , 4.89 ( 1h , dd , j = 6.4 , 2.8 hz , h-4 ) , 4.81 ( 1h , obscured by the solvent signal , h-6 ) , 4.70 ( 1h , d , j = 8.0 hz , h-1 ) , 3.90 ( 1h , dd , j = 12.1 , 1.6 hz , h-6 ) , 3.68 ( 1h , br d , j = 12.2 hz , h-6b ) , 3.24 ( 1h , m , h-2 ) , 2.79 ( 1h , br t , j = 6.5 hz , h-5 ) , 2.34 ( 1h , dd , j = 7.9 , 2.7 hz , h-9 ) , 2.12 ( 1h , br dd , j = 14.0 , 6.5 hz , h-7a ) , 2.08 ( 3h , s , ch3co ) , 1.93 ( 1h , dd , j = 13.8 , 8.6 hz , h-7b ) , 1.38 ( 3h , s , h-10 ) . they were identified as ajugoside ( 1 ) , 8-o - acetylharpagide ( 2 ) , harpagide ( 3 ) , allobetonicoside ( 4 ) , 4-o - galactopyranosyl - teuhircoside ( 5 ) , reptoside ( 6 ) , 6-o - acetyl - ajugol ( 7 ) [ figure 1 ] for the iridoid glycosides constituents , and as chlorogenic acid ( 8) , b - arbutin ( 9 ) , verbascoside ( 10 ) , and stachysoside a ( 11 ) [ ( 2-(3,4-dihydroxyphenyl)ethyl - o - a - l - arabinopyranosyl-(12)-o-6-deoxy - b - l - mannopyranosyl-(13)-4-o-[(2e)-3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]-b - d - glucopyranoside ] [ figure 2 ] for the phenolic compounds . the presence of previously identified iridoid glycosides ( 2 - 5 ) has been confirmed , together with ajugoside ( 1 ) , reptoside ( 6 ) , and 6-o - acetyl - ajugol ( 7 ) which resulted to be new iridoid components for this species . among the phenolic constituents , chlorogenic acid ( 8) , b - arbutin ( 9 ) , and stachysoside a ( 11 ) the identified iridoids have all chemosystematic relevance . 6-o - acetyl - ajugol ( 7 ) , is also a rare metabolite in the lamiaceae family since it has been found only in leonurus persicus so far . it is also worth of mention the presence of ajugoside ( 1 ) and reptoside ( 6 ) which are iridoids widely distributed in the species of the ajugoideae subfamily . h - nmr spectrum of ajugoside ( 1 ) c - nmr spectrum of ajugoside ( 1 ) heteronuclear single quantum correlation spectrum of ajugoside ( 1 ) heteronuclear multiple bond correlation spectrum of ajugoside ( 1 ) in particular , the resonance of h-6 was readily visible in the proton spectrum at 4.18 ppm , but the corresponding carbon signal was very near to a glucose carbon resonance and has been assigned by the direct hsqc correlation [ figure 5 ] with the signal resonating at 75.55 ppm . chlorogenic acid ( 8) has been isolated for the first time from s. alopecuros subsp divulsa , although it has been already found in several stachys species as s. tymphaea , s. lanata , s. officinalis , s. recta , s. byzantina , s. iberica subsp . furthermore , b - arbutin ( 9 ) was a metabolite identified for the first time in this species . the presence of verbascoside ( 10 ) , a quite common phenyletanoid glycoside ( phgs ) in lamiaceae , has been confirmed , while stachysoside a ( 11 ) , also known with the name lavandulifolioside , resulted to be as a new constituent of s. alopecuros subsp . from recent studies on iridoid activities , 8-o - acetylharpagide ( 2 ) and ajugoside ( 1 ) resulted to be useful compounds in the treatment of diabetes mellitus since they were able to stimulate the cellular glucose intake and lower the glucose blood level . reptoside ( 6 ) resulted to be a rare metabolite in the stachys genus because its occurrence has been reported only in a few species , namely betonica officinalis ( syn . 6-o - acetyl - ajugol ( 7 ) , is also a rare metabolite in the lamiaceae family since it has been found only in leonurus persicus so far . it is also worth of mention the presence of ajugoside ( 1 ) and reptoside ( 6 ) which are iridoids widely distributed in the species of the ajugoideae subfamily . the identification of 1 has been carried out by extensive nmr analysis on both mono - [ figures 3 and 4 ] and bidimensional [ figures 5 and 6 ] experiments since the assignment of resonances was not immediate for several signals , in particular for protons in 5 , 6 and 9 positions on the iridoid skeleton as well as for protons in 3 and 5 positions of the glucose moiety [ figures 3 and 4 ] . h - nmr spectrum of ajugoside ( 1 ) c - nmr spectrum of ajugoside ( 1 ) heteronuclear single quantum correlation spectrum of ajugoside ( 1 ) heteronuclear multiple bond correlation spectrum of ajugoside ( 1 ) in particular , the resonance of h-6 was readily visible in the proton spectrum at 4.18 ppm , but the corresponding carbon signal was very near to a glucose carbon resonance and has been assigned by the direct hsqc correlation [ figure 5 ] with the signal resonating at 75.55 ppm . chlorogenic acid ( 8) has been isolated for the first time from s. alopecuros subsp divulsa , although it has been already found in several stachys species as s. tymphaea , s. lanata , s. officinalis , s. recta , s. byzantina , s. iberica subsp . furthermore , b - arbutin ( 9 ) was a metabolite identified for the first time in this species . the presence of verbascoside ( 10 ) , a quite common phenyletanoid glycoside ( phgs ) in lamiaceae , has been confirmed , while stachysoside a ( 11 ) , also known with the name lavandulifolioside , resulted to be as a new constituent of s. alopecuros subsp . from recent studies on iridoid activities , 8-o - acetylharpagide ( 2 ) and ajugoside ( 1 ) resulted to be useful compounds in the treatment of diabetes mellitus since they were able to stimulate the cellular glucose intake and lower the glucose blood level . divulsa shed light on three additional iridoid glucosides together with chlorogenic acid ( 8) , b - arbutin ( 9 ) , verbascoside ( 10 ) , and stachysoside a ( 11 ) in the phenolic fraction . the presence of verbascoside has been already reported , while the other phenolics were identified for the first time in the studied species . divulsa has been disclosed with respect to literature data , showing the presence of ajugoside ( 1 ) , reptoside ( 6 ) , and 6-o - acetyl - ajugol ( 7 ) .

the treatment of periodontal diseases has now moved toward an antimicrobial model of disease management . with the threat of widespread antibiotic resistance rendering many antibiotics useless against important diseases , there is an increased necessity not only to minimize antibiotic use and develop novel nonantibiotic - based treatments , but also to raise the profile of disease prevention . probiotics are defined as live microorganisms that when administered in adequate amounts confer health benefits on the host . they repopulate the beneficial bacteria , which can help kill pathogenic bacteria and fight against infection . probiotics administered orally may benefit oral health by preventing the growth of harmful microbiota or by modulating mucosal immunity in the oral cavity . mechanical removal of supragingival plaque is the most effective tool to prevent gingivitis ( loe et al . 1965 ) but most individuals do not adequately control plaque accumulation and gingivitis continues to be prevalent . to overcome this hindrance , antimicrobial products in the form of dentifrices or mouthwashes have been tested for their adjunctive efficacy in reducing plaque and gingivitis . among them , chlorhexidine is regarded as a gold standard in dentistry for the prevention of dental plaque . though very effective , it has certain side effects such as brown discoloration of teeth , oral mucosal erosion , and bitter taste . as an alternative preventive tool however , only a few clinical studies have been so far conducted on the use of probiotics in the prevention of oral diseases . thus taking into consideration , all these above facts and claims , this study was carried out to test the potential antiplaque and anti - inflammatory properties of probiotic in the form of a mouthwash . a total of 45 systemically healthy subjects ( 29 males and 16 females ) visiting the department of periodontology , at the new horizon dental college and research institute bilaspur , chhattisgarh , were recruited for the study . the inclusion criteria for the study included : subjects between the age group 20 and 30 years of agesubjects with chronic gingivitissubjects are having a dentition with 20 evaluable teeth ( minimum of five teeth per quadrant ) . subjects between the age group 20 and 30 years of age subjects with chronic gingivitis subjects are having a dentition with 20 evaluable teeth ( minimum of five teeth per quadrant ) . the exclusion criteria of the study included : history of systemic diseasespregnant , lactating femaleshistory of antibiotic therapy in the past 3 monthshistory of oral prophylaxis within 6 months previous to the studysubjects with mouth breathing habitsubjects with orthodontic and prosthodontic appliancessubjects with deleterious habit such as smokinghistory of undergoing nonsurgical and surgical periodontal therapy in the last 6 months . history of systemic diseases pregnant , lactating females history of antibiotic therapy in the past 3 months history of oral prophylaxis within 6 months previous to the study subjects with mouth breathing habit subjects with orthodontic and prosthodontic appliances subjects with deleterious habit such as smoking history of undergoing nonsurgical and surgical periodontal therapy in the last 6 months . a randomized , parallel group clinical study was conducted on 45 systemically healthy patients reporting to the outpatient department of periodontics and oral implantology , at the new horizon dental college and research institute , sakri , bilaspur ( chhattisgarh ) with chronic gingivitis . the subjects were assessed for plaque and gingival inflammation by recording the plaque index ( pi ) ( loe and silness 1964 ) , gingival index ( gi ) ( loe and silness ) , and oral hygiene index - simplified ( ohi - s ) ( green and vermillion ) by a single investigator experienced with index system recording at baseline . thorough scaling and polishing were performed and the patients were randomly divided into three groups consisting of 15 patients each as under : group a - probiotic mouthrinse ( sporlac plus [ sanzyme ltd . india ] + distilled water)group b - chlorhexidine mouthwash 0.02% ( hexidine [ icpa])group c - control ( saline ) group a - probiotic mouthrinse ( sporlac plus [ sanzyme ltd . india ] + distilled water ) group b - chlorhexidine mouthwash 0.02% ( hexidine [ icpa ] ) group c - control ( saline ) an informed written consent was obtained from each patient included in the study . the patients in group a were given sporlac plus satchets ( a probiotic formulation containing lactobacillus acidophilus , lactobacillus rhamnosus , lactobacillus sporogenes , bifidobacterium longum , and saccharomyces boulardii ) and 10 ml ampules of distilled water which are commercially available at the chemist as shown in figure 1 . the patients were demonstrated and instructed to prepare the experimental probiotic mouthwash by mixing together the contents of the sachet and 10 ml of distilled water . emphasis was made to explain to the patient that the solution had to be stirred thoroughly until all the contents were completely dissolved in the distilled water . the formulation had to be prepared and rinsed immediately once prepared and could not be stored . photograph showing the sporlac plus probiotic sachet , distilled water ampules , and chlorhexidine mouthwash all the three groups were advised to rinse their mouths with the respective mouthwashes prescribed to them for 15 days without any dilution for 1 min twice daily half an hour after brushing . they were advised not to eat anything for half an hour after using the mouthwash . the clinical parameters of pi , gi , and ohi - s recorded at baseline were repeated on 14 and 28 day . descriptive analysis was done and independent sample t - test was employed between 2 groups . the study protocol was in accordance with the local ethical guidelines and approved by the local ethics committee . a randomized , parallel group clinical study was conducted on 45 systemically healthy patients reporting to the outpatient department of periodontics and oral implantology , at the new horizon dental college and research institute , sakri , bilaspur ( chhattisgarh ) with chronic gingivitis . the subjects were assessed for plaque and gingival inflammation by recording the plaque index ( pi ) ( loe and silness 1964 ) , gingival index ( gi ) ( loe and silness ) , and oral hygiene index - simplified ( ohi - s ) ( green and vermillion ) by a single investigator experienced with index system recording at baseline . thorough scaling and polishing were performed and the patients were randomly divided into three groups consisting of 15 patients each as under : group a - probiotic mouthrinse ( sporlac plus [ sanzyme ltd . india ] + distilled water)group b - chlorhexidine mouthwash 0.02% ( hexidine [ icpa])group c - control ( saline ) group a - probiotic mouthrinse ( sporlac plus [ sanzyme ltd . india ] + distilled water ) group b - chlorhexidine mouthwash 0.02% ( hexidine [ icpa ] ) group c - control ( saline ) an informed written consent was obtained from each patient included in the study . the patients in group a were given sporlac plus satchets ( a probiotic formulation containing lactobacillus acidophilus , lactobacillus rhamnosus , lactobacillus sporogenes , bifidobacterium longum , and saccharomyces boulardii ) and 10 ml ampules of distilled water which are commercially available at the chemist as shown in figure 1 . the patients were demonstrated and instructed to prepare the experimental probiotic mouthwash by mixing together the contents of the sachet and 10 ml of distilled water . emphasis was made to explain to the patient that the solution had to be stirred thoroughly until all the contents were completely dissolved in the distilled water . the formulation had to be prepared and rinsed immediately once prepared and could not be stored . photograph showing the sporlac plus probiotic sachet , distilled water ampules , and chlorhexidine mouthwash all the three groups were advised to rinse their mouths with the respective mouthwashes prescribed to them for 15 days without any dilution for 1 min twice daily half an hour after brushing . they were advised not to eat anything for half an hour after using the mouthwash . the clinical parameters of pi , gi , and ohi - s recorded at baseline were repeated on 14 and 28 day . descriptive analysis was done and independent sample t - test was employed between 2 groups . the study protocol was in accordance with the local ethical guidelines and approved by the local ethics committee . the mean pi values for all three mouthrinses were 0 at baseline as scaling and polishing were done for all tooth surfaces . a pi score of 0 represented a tooth surface that was entirely free of clinically detectable plaque . mean values of pi scores for probiotics was 0.36 0.14 , control 1.10 0.22 , and chlorhexidine 0.21 0.15 as cited in table 1 . the degree of increment of mean pi scores was more pronounced for control rinse ( saline ) as compared to probiotic and chlorhexidine rinses . the differences in the increment of the mean pi scores before and after rinse for probiotic and control were significant , whereas that between probiotic and chlorhexidine were statistically insignificant . the mean gi scores were 0.45 0.174 for probiotic , 1.03 0.142 for control , and 0.40 0.124 for chlorhexidine as cited in table 2 . in comparison to the baseline data , there was a significant decrease in mean gi scores of probiotic and chlorhexidine rinses as compared to the control rinses , whereas that between probiotic and chlorhexidine it was statistically insignificant . the degree of reduction of mean gi scores was more pronounced for the probiotic and chlorhexidine rinses as compared to control rinse . intergroup comparisons in pi intergroup comparisons in gi the mean ohi - s values were 0.35 0.106 for probiotic , 0.97 0.139 for control , and 0.48 0.341 for chlorhexidine , respectively , as cited in table 3 . in comparison to baseline data , there was a significant decrease in the ohi - s score of the probiotic and chlorhexidine group as compared to the control group . intergroup comparisons in ohi - s whereas clinical significance after intergroup comparison between control and chlorhexidine was p = 0.00 , probiotic and chlorhexidine was p = 0.16 , probiotic and control was p = 0.00 for pi . between control and chlorhexidine , it was p = 0.00 , probiotic and chlorhexidine it was p = 0.75 , probiotic and control was p = 0.00 when comparison for gi was calculated . between control and chlorhexidine , p value was at 0.00 for probiotic , and chlorhexidine p value was at 0.41 for probiotic and control p value was at 0.00 , when ohi - s was calculated . this intergroup comparison shows that there is a statistically significant difference in the control group and probiotic group in the reduction of pi , gi , and ohi - s scores . these side effects are brown discoloration of the teeth and tongue , oral mucosal erosion , and taste perturbation . several side effects associated with its use have stimulated the search for an alternative antiplaque agent . antibacterial mouthrinses act by nonspecifically reducing the levels of both friendly and harmful oral bacteria . in contrast , probiotic has been developed utilizing natural beneficial bacteria to promote a healthy balance of microorganisms in the mouth . probiotic technology represents a breakthrough approach to maintain oral health by utilizing natural beneficial bacteria commonly found in healthy mouths to provide a natural defense against those bacteria thought to be harmful to teeth and gums . probiotic species mostly belong to the genera lactobacillus and bifidobacterium . to be able to exert probiotic properties in the oral cavity , however , it is essential for the microorganism to resist the oral environmental conditions and defense mechanism , to be able to colonize and grow in the mouth , and to inhibit oral pathogens . the potential benefits of probiotics on systemic health and medical disorders , such as gastrointestinal diseases , have been elaborately described . the first species introduced into research were l. acidophilus and bifidobacterium bifidum . among a number of potential benefits that have been proposed are reduced susceptibility to infections , reductions in allergies , and lactose intolerance , as well as lowered blood pressure and serum cholesterol values . within dentistry , the previous studies with lactobacilli strains such as l. rhamnosus , lactobacillus . casei , lactobacillus reuteri , or a lactobacilli mix have revealed mixed results on oral microorganisms . a variety of mechanisms have been proposed for their actions , and some effects have been stated to be systemic rather than only local . it is likely that these mechanisms vary according to the specific strain or combinations of strains used , the presence of prebiotics and the condition that is being treated , as well as the stage of the disease process in which the probiotic is administered . there are common themes emerging in studies of the modes of action of probiotics and numerous mechanisms have been proposed including prevention of adhesion of pathogens to host tissues , stimulation , and modulation of the mucosal immune system , e.g. , by reducing production of pro - inflammatory cytokines through actions on nfkb pathways , increasing production of anti - inflammatory cytokines such as interleukin-10 ( il-10 , and host defense peptides such as beta - defensin 2 , enhancing immunoglobulin a defenses , and influencing dendritic cell maturation . killing or inhibition of growth of pathogens through production of bacteriocins or other products , such as acid or peroxide , which are antagonistic toward pathogenic bacteria has also been reported . the current concept concerning the etiology of periodontal disease considers three groups of factors which determine whether active periodontal disease will occur : a susceptible host , the presence of pathogenic species , and the absence of so - called beneficial bacteria . it is generally accepted that the oral biofilm in association with an aerobic bacteria is the main etiological factor in periodontal disease . did a study in patients with moderate to severe gingivitis who were given either freeze - dried lactobacillus salivarius wb21 ( wb21)-containing tablets or xylitol containing placebos regularly ( 3 times daily for 8 weeks ) . the intake of tablets containing l. salivarius resulted in benefits in terms of pocket probing depth and pi in individuals at high - risk of periodontal disease ( smokers ) as compared to a placebo control group . did a study in patients with moderate to severe gingivitis who were given one of the two different l. reuteri formulations ( lr-1 or lr-2 ) at a dose of 2 10 ( 8) cfu / day , or a corresponding placebo . l. reuteri was efficacious in reducing both gingivitis and plaque in patients with moderate to severe gingivitis . other studies have aimed to identify organisms which have the potential for probiotic action that may protect against periodontal diseases . some oral strains of lactobacilli , streptococci , and bifidobacteria have been reported to have in vitro inhibitory activity against periodontal pathogens while others are more active against mutans streptococci . observed that l. gasseri strains isolated from periodontally healthy subjects were more efficient at inhibiting the growth of aggregatibacter actinomycetemcomitans than strains from periodontally diseased subjects , and also inhibited the growth of porphyromonas gingivalis and porphyromonas intermedia . this correlated with an inverse relationship between carriage of homofermentative lactobacilli and subgingival colonization by a. actinomycetemcomitans , p. gingivalis , and p. intermedia . ishikawa et al . observed in vitro inhibition of p. gingivalis , p. intermedia , and prevotella nigrescens by l. salivarius . daily ingestion of l. salivarius - containing tablets resulted in reduced salivary counts of these black pigmented anaerobes . noordin and kamin conducted a trial among 90 school children and assigned them into placebo , chlorhexidine , and probiotic groups ; and plaque scores were recorded at baseline ( 0 day ) , on 15 day ( after 14 days of intervention ) , and 3 weeks ( after discontinuation of intervention ) . probiotic mouthrinse was more effective for inhibition of dental plaque accumulation after 14 days of intervention and also after 3 weeks of discontinuation of intervention . vivekananda et al . evaluated the effects of lactobacilli reuteri ( prodentis ) alone and in combination with scaling and root planing ( srp ) in patients with chronic periodontitis for a period of 42 days . their trial confirmed the plaque inhibition , anti - inflammatory , and antimicrobial effects of l. reuteri ( prodentis ) and they recommended the use of probiotic during nonsurgical and maintenance phase of periodontal treatment . harini and anegundi evaluated clinically the efficacy of a probiotic and chlorhexidine mouthrinses on plaque and gingival accumulation in children for 14 days and concluded that the probiotic mouthrinse was found effective in reducing plaque accumulation and gingival inflammation . evaluated the effects of l. reuteri - containing probiotic lozenges and placebos as an adjunct to srp in 30 patients with chronic periodontitis , monitored clinically , and microbiologically at baseline , 3 , 6 , 9 , and 12 weeks after therapy . significant improvement in all clinical parameters reduced p. gingivalis levels , more pocket depth reduction and attachment gain in moderate and deep pockets was observed in the srp + probiotic group . maekawa and hajishengallis studied whether lactobacillus brevis cd2 or placebo could inhibit periodontal inflammation and bone loss in experimentally induced periodontitis in mice . mice topically treated with l. brevis cd2 displayed significantly decreased bone loss , lower expression of tumor necrosis factor , il-1 , -6 and -17a , lower counts of anaerobic bacteria , but higher counts of aerobic bacteria as compared to placebo - treated mice . hence , l. brevis cd2 could inhibit periodontitis through modulatory effects on the host response and the periodontal microbiota . purunaik et al . aimed to investigate the efficacy of probiotic ( 1 g powder of 1.25 billion freeze dried combination , a mixture of l. acidophilus , l. rhamnosus , b. longum , and s. boulardii ) , 0.2% of chlorhexidine and placebo mouthrinses in reducing plaque and gingivitis among 90 school children aged 1516 years . it was found that both probiotic and chlorhexidine mouthrinses were able to significantly reduce plaque and gingival levels after 14 days . did a study to test and compare in vitro antimicrobial activity of l. reuteri on pathogenic bacteria involved in the formation of dental caries : streptococcus mutans , streptococcus gordonii , and periodontal disease : actinomyces naeslundii and tannerella forsythia chlorhexidine of 0.12% was used as a positive control . l. reuteri was shown to have an inhibitory effect against s. mutans , followed by t. forsythia and s. gordonii , and a less significant effect against a. naeslundii . our results indicate that probiotics could be useful in the improvement / maintenance of oral health in subjects at a high - risk of periodontal disease and add to the body of data supporting the effectiveness of both chlorhexidine and probiotic as antiplaque / antigingivitis agents . the advantages of using a probiotic mouthrinse are that as it contains friendly commensals , there is no issue of antibiotic resistance , and there are no known / proven toxicities caused due to their use . sporlac plus sachet powder for oral solution is a commercially available powder containing probiotics lactobacillus and bifidobacterium not < 1.25 billion cells ( l. acidophilus , l. rhamnosus , lactic acid bacillus , b. bifidum , b. longum , and s. boulardii ) popularly used in the treatment of diarrhea of any etiology infantile , weaning , and neonatal diarrhea along with any anti - diarrheal agent . several clinical studies have already demonstrated the effectiveness of this probiotic in the treatment of systemic diseases and infectious diseases such as acute diarrhea and crohn 's disease . very few studies till date have studied the basic / initial treatment for periodontal patients in terms of srp and use of probiotic mouthwash in the reduction of clinical parameters of gingivitis in india . an initial attempt was made in this randomized , parallel clinical trial to evaluate the benefits of scaling and sporlac plus probiotic sachet in the treatment of chronic gingivitis and to compare it with chlorhexidine , which has been regarded as the gold standard in dentistry for the prevention of plaque and gingivitis . in all the three groups namely chlorhexidine , probiotic , and saline , pi , gi , and ohi - s were significantly reduced within each treatment group over the 30 days , and thus , even the control had a significant effect . however , such placebo effects are known to occur as a simple consequence of the hawthorne effect . the results of our study are in accordance with studies done by vivekanand et al . , harini and anegundi , and noordin and kamin showing the effect of the probiotics in the management of periodontal disease . the combination of scaling and probiotics and scaling and chlorhexidine demonstrated a significant reduction of pi , gi , and ohi - s when compared with scaling and saline group . the effect of srp on plaque reduction is similar to that reported by mousques et al . and proye et al . thus , the plaque reduction brought about by scaling was enhanced by the use of probiotics and chlorhexidine , which is in accordance with the studies done by krasse et al . in the present study , using a negative ( saline ) and a positive control ( chlorhexidine ) , we were able to state that the probiotic mouth wash ( sporlac plus ) has shown a good potential as an antiplaque agent and its effectiveness in reducing the plaque accumulation and gingival inflammation is comparable to chlorhexidine . considering the local side effects of chlorhexidine including brown staining , taste disturbance , enhanced supra gingival calculus formation , and less commonly desquamation of the oral mucosa . sporlac plus probiotic mouthrinse seems a very effective and economical alternative for patients with periodontal disease . probiotic effects are strain - specific , thus , each individual bacterial strain needs to be tested separately , and the effects described for one strain can not be directly applied to others . most of the studies carried out to evaluate the effects of probiotics on periodontal disease have been associated with an individual bacterial strain . unfortunately , mislabeling of strains in probiotic products seems to be a common problem . on the other hand , multispecies or multi - strain probiotic products can be even more effective than products with only one bacterial strain . in our study , we used a probiotic mouthrinse containing the combination of lactobacillus , bifidobacterium , and sacchromyces strains . it is possible , in the complex environment of the human mouth that probiotic cocktail of multiple strains would be more effective than any single probiotic agent . this combination of probiotic strain was similar to those used by haukioja et al . and purunaik et al . the results of our study showed a significant reduction of plaque and gingival status and were in accordance with the above - mentioned studies suggesting that combinations of probiotics strains may have synergistic adhesion effect . though these strains tested maintained the oro - microbiological balance , their action in the oral cavity is dubious as oral mucosa is not their innate habitat . furthermore , there is also a need to evaluate whether these lactobacilli strains are momentary or stable oral colonizers . however , it seems plausible that prolonged administration of probiotic preparations may have a preventive role against the development of plaque and gingivitis . however , we would like to state that the major limitation of our probiotic preparation is that it needs to be used immediately once prepared and can not be stored . thus , we would recommend that a proper vehicle is needed for delivering probiotics so that patient compliance can be improved . apart from the unacceptable taste , no other adverse effects on the oral mucosa such as ulcerations were noted with probiotic mouthrinse . likewise , it would be interesting to evaluate the additional gi benefits of probiotics in studies , where the patient is instructed to ingest rather than expectorate the probiotic mouthrinse . this was not possible to evaluate in this study , since it was a comparative clinical trial assessing the efficacy of three mouthrinses . longitudinal studies involving probiotics and further microbiological evaluation are also essential when prescribing them in place of antiseptics and antimicrobials . in the recent times , when organisms are fast developing resistance to antibiotics , the emergence of probiotics appears to be a boon for the treatment of diseases . researchers have confirmed that diseases of the periodontium are not confined to the oral cavity but have strong systemic effects . hence , probiotics offer a natural and promising option to establish both a good oral and systemic health . in the present study , therefore , probiotic mouth rinse has a potential therapeutic value , and further long - term studies are recommended to determine its efficacy .

background : the aim of our clinical trial was to assess and compare the antiplaque and anti - inflammatory potential of a probiotic mouthwash with 0.2% chlorhexidine and saline.materials and methods : a randomized parallel group study was designed for a period of 4 weeks on 45 systemically healthy subjects between 20 and 30 years having chronic gingivitis . the study population was divided into three groups . group a - 15 subjects were advised experimental ( probiotic ) mouthwash . group b - 15 subjects were advised positive control ( chlorhexidine ) mouthwash and group c - 15 subjects into a negative control group ( normal saline ) . oral prophylaxis was done for all groups at baseline . after the proper oral hygiene instructions , all the three groups were instructed to rinse their mouth with 10 ml of their respective mouthrinse , undiluted for 1 min twice daily , 30 min after brushing . clinical parameters such as plaque index ( pi ) , gingival index ( gi ) , and oral hygiene index simplified ( ohi - s ) were assessed at baseline , 2 weeks and 4 weeks , respectively.results:at day 28 , the pi , gi , and ohi - s were significantly reduced by all treatment modalities ranking probiotic and chlorhexidine is greater than saline.conclusion:the probiotic mouthrinses tested was effectively used as an adjunct to mechanical plaque control in the prevention of plaque and gingivitis . thus , the probiotic mouthrinse has a great therapeutic potential .

INTRODUCTION MATERIALS AND METHODS Study design and clinical measurements Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

to overcome this hindrance , antimicrobial products in the form of dentifrices or mouthwashes have been tested for their adjunctive efficacy in reducing plaque and gingivitis . among them , chlorhexidine is regarded as a gold standard in dentistry for the prevention of dental plaque . though very effective , it has certain side effects such as brown discoloration of teeth , oral mucosal erosion , and bitter taste . as an alternative preventive tool however , only a few clinical studies have been so far conducted on the use of probiotics in the prevention of oral diseases . thus taking into consideration , all these above facts and claims , this study was carried out to test the potential antiplaque and anti - inflammatory properties of probiotic in the form of a mouthwash . a total of 45 systemically healthy subjects ( 29 males and 16 females ) visiting the department of periodontology , at the new horizon dental college and research institute bilaspur , chhattisgarh , were recruited for the study . the inclusion criteria for the study included : subjects between the age group 20 and 30 years of agesubjects with chronic gingivitissubjects are having a dentition with 20 evaluable teeth ( minimum of five teeth per quadrant ) . subjects between the age group 20 and 30 years of age subjects with chronic gingivitis subjects are having a dentition with 20 evaluable teeth ( minimum of five teeth per quadrant ) . the exclusion criteria of the study included : history of systemic diseasespregnant , lactating femaleshistory of antibiotic therapy in the past 3 monthshistory of oral prophylaxis within 6 months previous to the studysubjects with mouth breathing habitsubjects with orthodontic and prosthodontic appliancessubjects with deleterious habit such as smokinghistory of undergoing nonsurgical and surgical periodontal therapy in the last 6 months . history of systemic diseases pregnant , lactating females history of antibiotic therapy in the past 3 months history of oral prophylaxis within 6 months previous to the study subjects with mouth breathing habit subjects with orthodontic and prosthodontic appliances subjects with deleterious habit such as smoking history of undergoing nonsurgical and surgical periodontal therapy in the last 6 months . a randomized , parallel group clinical study was conducted on 45 systemically healthy patients reporting to the outpatient department of periodontics and oral implantology , at the new horizon dental college and research institute , sakri , bilaspur ( chhattisgarh ) with chronic gingivitis . the subjects were assessed for plaque and gingival inflammation by recording the plaque index ( pi ) ( loe and silness 1964 ) , gingival index ( gi ) ( loe and silness ) , and oral hygiene index - simplified ( ohi - s ) ( green and vermillion ) by a single investigator experienced with index system recording at baseline . thorough scaling and polishing were performed and the patients were randomly divided into three groups consisting of 15 patients each as under : group a - probiotic mouthrinse ( sporlac plus [ sanzyme ltd . india ] + distilled water)group b - chlorhexidine mouthwash 0.02% ( hexidine [ icpa])group c - control ( saline ) group a - probiotic mouthrinse ( sporlac plus [ sanzyme ltd . india ] + distilled water ) group b - chlorhexidine mouthwash 0.02% ( hexidine [ icpa ] ) group c - control ( saline ) an informed written consent was obtained from each patient included in the study . the patients in group a were given sporlac plus satchets ( a probiotic formulation containing lactobacillus acidophilus , lactobacillus rhamnosus , lactobacillus sporogenes , bifidobacterium longum , and saccharomyces boulardii ) and 10 ml ampules of distilled water which are commercially available at the chemist as shown in figure 1 . the patients were demonstrated and instructed to prepare the experimental probiotic mouthwash by mixing together the contents of the sachet and 10 ml of distilled water . emphasis was made to explain to the patient that the solution had to be stirred thoroughly until all the contents were completely dissolved in the distilled water . photograph showing the sporlac plus probiotic sachet , distilled water ampules , and chlorhexidine mouthwash all the three groups were advised to rinse their mouths with the respective mouthwashes prescribed to them for 15 days without any dilution for 1 min twice daily half an hour after brushing . the clinical parameters of pi , gi , and ohi - s recorded at baseline were repeated on 14 and 28 day . descriptive analysis was done and independent sample t - test was employed between 2 groups . a randomized , parallel group clinical study was conducted on 45 systemically healthy patients reporting to the outpatient department of periodontics and oral implantology , at the new horizon dental college and research institute , sakri , bilaspur ( chhattisgarh ) with chronic gingivitis . the subjects were assessed for plaque and gingival inflammation by recording the plaque index ( pi ) ( loe and silness 1964 ) , gingival index ( gi ) ( loe and silness ) , and oral hygiene index - simplified ( ohi - s ) ( green and vermillion ) by a single investigator experienced with index system recording at baseline . thorough scaling and polishing were performed and the patients were randomly divided into three groups consisting of 15 patients each as under : group a - probiotic mouthrinse ( sporlac plus [ sanzyme ltd . india ] + distilled water)group b - chlorhexidine mouthwash 0.02% ( hexidine [ icpa])group c - control ( saline ) group a - probiotic mouthrinse ( sporlac plus [ sanzyme ltd . india ] + distilled water ) group b - chlorhexidine mouthwash 0.02% ( hexidine [ icpa ] ) group c - control ( saline ) an informed written consent was obtained from each patient included in the study . the patients in group a were given sporlac plus satchets ( a probiotic formulation containing lactobacillus acidophilus , lactobacillus rhamnosus , lactobacillus sporogenes , bifidobacterium longum , and saccharomyces boulardii ) and 10 ml ampules of distilled water which are commercially available at the chemist as shown in figure 1 . the patients were demonstrated and instructed to prepare the experimental probiotic mouthwash by mixing together the contents of the sachet and 10 ml of distilled water . emphasis was made to explain to the patient that the solution had to be stirred thoroughly until all the contents were completely dissolved in the distilled water . photograph showing the sporlac plus probiotic sachet , distilled water ampules , and chlorhexidine mouthwash all the three groups were advised to rinse their mouths with the respective mouthwashes prescribed to them for 15 days without any dilution for 1 min twice daily half an hour after brushing . the clinical parameters of pi , gi , and ohi - s recorded at baseline were repeated on 14 and 28 day . descriptive analysis was done and independent sample t - test was employed between 2 groups . the mean pi values for all three mouthrinses were 0 at baseline as scaling and polishing were done for all tooth surfaces . mean values of pi scores for probiotics was 0.36 0.14 , control 1.10 0.22 , and chlorhexidine 0.21 0.15 as cited in table 1 . the degree of increment of mean pi scores was more pronounced for control rinse ( saline ) as compared to probiotic and chlorhexidine rinses . the differences in the increment of the mean pi scores before and after rinse for probiotic and control were significant , whereas that between probiotic and chlorhexidine were statistically insignificant . the mean gi scores were 0.45 0.174 for probiotic , 1.03 0.142 for control , and 0.40 0.124 for chlorhexidine as cited in table 2 . in comparison to the baseline data , there was a significant decrease in mean gi scores of probiotic and chlorhexidine rinses as compared to the control rinses , whereas that between probiotic and chlorhexidine it was statistically insignificant . the degree of reduction of mean gi scores was more pronounced for the probiotic and chlorhexidine rinses as compared to control rinse . intergroup comparisons in pi intergroup comparisons in gi the mean ohi - s values were 0.35 0.106 for probiotic , 0.97 0.139 for control , and 0.48 0.341 for chlorhexidine , respectively , as cited in table 3 . in comparison to baseline data , there was a significant decrease in the ohi - s score of the probiotic and chlorhexidine group as compared to the control group . intergroup comparisons in ohi - s whereas clinical significance after intergroup comparison between control and chlorhexidine was p = 0.00 , probiotic and chlorhexidine was p = 0.16 , probiotic and control was p = 0.00 for pi . between control and chlorhexidine , it was p = 0.00 , probiotic and chlorhexidine it was p = 0.75 , probiotic and control was p = 0.00 when comparison for gi was calculated . between control and chlorhexidine , p value was at 0.00 for probiotic , and chlorhexidine p value was at 0.41 for probiotic and control p value was at 0.00 , when ohi - s was calculated . this intergroup comparison shows that there is a statistically significant difference in the control group and probiotic group in the reduction of pi , gi , and ohi - s scores . these side effects are brown discoloration of the teeth and tongue , oral mucosal erosion , and taste perturbation . to be able to exert probiotic properties in the oral cavity , however , it is essential for the microorganism to resist the oral environmental conditions and defense mechanism , to be able to colonize and grow in the mouth , and to inhibit oral pathogens . the potential benefits of probiotics on systemic health and medical disorders , such as gastrointestinal diseases , have been elaborately described . within dentistry , the previous studies with lactobacilli strains such as l. rhamnosus , lactobacillus . a variety of mechanisms have been proposed for their actions , and some effects have been stated to be systemic rather than only local . it is likely that these mechanisms vary according to the specific strain or combinations of strains used , the presence of prebiotics and the condition that is being treated , as well as the stage of the disease process in which the probiotic is administered . there are common themes emerging in studies of the modes of action of probiotics and numerous mechanisms have been proposed including prevention of adhesion of pathogens to host tissues , stimulation , and modulation of the mucosal immune system , e.g. , by reducing production of pro - inflammatory cytokines through actions on nfkb pathways , increasing production of anti - inflammatory cytokines such as interleukin-10 ( il-10 , and host defense peptides such as beta - defensin 2 , enhancing immunoglobulin a defenses , and influencing dendritic cell maturation . the current concept concerning the etiology of periodontal disease considers three groups of factors which determine whether active periodontal disease will occur : a susceptible host , the presence of pathogenic species , and the absence of so - called beneficial bacteria . observed that l. gasseri strains isolated from periodontally healthy subjects were more efficient at inhibiting the growth of aggregatibacter actinomycetemcomitans than strains from periodontally diseased subjects , and also inhibited the growth of porphyromonas gingivalis and porphyromonas intermedia . this correlated with an inverse relationship between carriage of homofermentative lactobacilli and subgingival colonization by a. actinomycetemcomitans , p. gingivalis , and p. intermedia . observed in vitro inhibition of p. gingivalis , p. intermedia , and prevotella nigrescens by l. salivarius . noordin and kamin conducted a trial among 90 school children and assigned them into placebo , chlorhexidine , and probiotic groups ; and plaque scores were recorded at baseline ( 0 day ) , on 15 day ( after 14 days of intervention ) , and 3 weeks ( after discontinuation of intervention ) . probiotic mouthrinse was more effective for inhibition of dental plaque accumulation after 14 days of intervention and also after 3 weeks of discontinuation of intervention . evaluated the effects of lactobacilli reuteri ( prodentis ) alone and in combination with scaling and root planing ( srp ) in patients with chronic periodontitis for a period of 42 days . their trial confirmed the plaque inhibition , anti - inflammatory , and antimicrobial effects of l. reuteri ( prodentis ) and they recommended the use of probiotic during nonsurgical and maintenance phase of periodontal treatment . harini and anegundi evaluated clinically the efficacy of a probiotic and chlorhexidine mouthrinses on plaque and gingival accumulation in children for 14 days and concluded that the probiotic mouthrinse was found effective in reducing plaque accumulation and gingival inflammation . evaluated the effects of l. reuteri - containing probiotic lozenges and placebos as an adjunct to srp in 30 patients with chronic periodontitis , monitored clinically , and microbiologically at baseline , 3 , 6 , 9 , and 12 weeks after therapy . significant improvement in all clinical parameters reduced p. gingivalis levels , more pocket depth reduction and attachment gain in moderate and deep pockets was observed in the srp + probiotic group . aimed to investigate the efficacy of probiotic ( 1 g powder of 1.25 billion freeze dried combination , a mixture of l. acidophilus , l. rhamnosus , b. longum , and s. boulardii ) , 0.2% of chlorhexidine and placebo mouthrinses in reducing plaque and gingivitis among 90 school children aged 1516 years . it was found that both probiotic and chlorhexidine mouthrinses were able to significantly reduce plaque and gingival levels after 14 days . did a study to test and compare in vitro antimicrobial activity of l. reuteri on pathogenic bacteria involved in the formation of dental caries : streptococcus mutans , streptococcus gordonii , and periodontal disease : actinomyces naeslundii and tannerella forsythia chlorhexidine of 0.12% was used as a positive control . l. reuteri was shown to have an inhibitory effect against s. mutans , followed by t. forsythia and s. gordonii , and a less significant effect against a. naeslundii . our results indicate that probiotics could be useful in the improvement / maintenance of oral health in subjects at a high - risk of periodontal disease and add to the body of data supporting the effectiveness of both chlorhexidine and probiotic as antiplaque / antigingivitis agents . the advantages of using a probiotic mouthrinse are that as it contains friendly commensals , there is no issue of antibiotic resistance , and there are no known / proven toxicities caused due to their use . sporlac plus sachet powder for oral solution is a commercially available powder containing probiotics lactobacillus and bifidobacterium not < 1.25 billion cells ( l. acidophilus , l. rhamnosus , lactic acid bacillus , b. bifidum , b. longum , and s. boulardii ) popularly used in the treatment of diarrhea of any etiology infantile , weaning , and neonatal diarrhea along with any anti - diarrheal agent . several clinical studies have already demonstrated the effectiveness of this probiotic in the treatment of systemic diseases and infectious diseases such as acute diarrhea and crohn 's disease . very few studies till date have studied the basic / initial treatment for periodontal patients in terms of srp and use of probiotic mouthwash in the reduction of clinical parameters of gingivitis in india . an initial attempt was made in this randomized , parallel clinical trial to evaluate the benefits of scaling and sporlac plus probiotic sachet in the treatment of chronic gingivitis and to compare it with chlorhexidine , which has been regarded as the gold standard in dentistry for the prevention of plaque and gingivitis . in all the three groups namely chlorhexidine , probiotic , and saline , pi , gi , and ohi - s were significantly reduced within each treatment group over the 30 days , and thus , even the control had a significant effect . the results of our study are in accordance with studies done by vivekanand et al . , harini and anegundi , and noordin and kamin showing the effect of the probiotics in the management of periodontal disease . the combination of scaling and probiotics and scaling and chlorhexidine demonstrated a significant reduction of pi , gi , and ohi - s when compared with scaling and saline group . thus , the plaque reduction brought about by scaling was enhanced by the use of probiotics and chlorhexidine , which is in accordance with the studies done by krasse et al . in the present study , using a negative ( saline ) and a positive control ( chlorhexidine ) , we were able to state that the probiotic mouth wash ( sporlac plus ) has shown a good potential as an antiplaque agent and its effectiveness in reducing the plaque accumulation and gingival inflammation is comparable to chlorhexidine . considering the local side effects of chlorhexidine including brown staining , taste disturbance , enhanced supra gingival calculus formation , and less commonly desquamation of the oral mucosa . probiotic effects are strain - specific , thus , each individual bacterial strain needs to be tested separately , and the effects described for one strain can not be directly applied to others . in our study , we used a probiotic mouthrinse containing the combination of lactobacillus , bifidobacterium , and sacchromyces strains . the results of our study showed a significant reduction of plaque and gingival status and were in accordance with the above - mentioned studies suggesting that combinations of probiotics strains may have synergistic adhesion effect . however , it seems plausible that prolonged administration of probiotic preparations may have a preventive role against the development of plaque and gingivitis . thus , we would recommend that a proper vehicle is needed for delivering probiotics so that patient compliance can be improved . apart from the unacceptable taste , no other adverse effects on the oral mucosa such as ulcerations were noted with probiotic mouthrinse . likewise , it would be interesting to evaluate the additional gi benefits of probiotics in studies , where the patient is instructed to ingest rather than expectorate the probiotic mouthrinse . in the recent times , when organisms are fast developing resistance to antibiotics , the emergence of probiotics appears to be a boon for the treatment of diseases . in the present study , therefore , probiotic mouth rinse has a potential therapeutic value , and further long - term studies are recommended to determine its efficacy .

scientists world - wide are trying to unravel the mechanisms that determine vascular function and dysfunction . important objects of study in this field of research include the maintenance of vascular tone , regulation of inflammation , sprouting of new blood vessels , regulation of cell survival , and the differentiation of stem cells into vascular tissue . vascular science is a field with a strong translational focus , combining results from fundamental molecular and cell biology with in vitro models of blood vessels and in vivo tests to develop insight in vascular physiology and treatment of disease . vascular dysfunction is an important factor in major diseases like atherosclerosis , cancer , and diabetes . the basis for understanding the functioning of blood vessels lies in understanding its building blocks , the vascular cells . therefore , a lot of research is focused on how endothelial cells or smooth muscle cells react to relevant biological , chemical , or physical cues in vitro . usually , this work is carried out by using conventional methods , culturing cells of animal or human origin in wells - plates , subjecting them to the aforementioned stimuli , and analyzing the outcome by biological or biochemical techniques . however , in vivo dynamic conditions are present : vascular endothelial cells are constantly subjected to shear stress caused by the flowing blood , while smooth muscle cells are stretched because of distension of the blood vessel during the cardiac cycle . moreover , vascular cells are embedded in a three - dimensional environment consisting of an elastic extracellular matrix , other cells , and flowing blood , with its platelets , red blood cells , and leukocytes ( figure 1 ) . both the three - dimensional environment and the dynamic mechanical changes with each cardiac cycle are very important factors in vascular cell functioning . it is advantageous to design laboratory setups that allow researchers to include these factors and control the relevant parameters . the main challenge when building such setups is that they should still be easy to assemble , handle , and combine with conventional analysis techniques . in the recent years , the field of microfluidic technology has gained much scientific interest among biologists , biochemists , and biophysicists ( figure 2 ) . we feel that microfluidic technology holds great promise to overcome the challenge of performing in vitro experiments with more physiologically realistic setups that are still simple enough to be used in everyday laboratory practice . moreover , microfluidic technology allows for increasing scale and parallelization of current research , leading to more comprehensive insights into cell and tissue physiology . the advantages of microfluidic technology for cell culture in general have been reviewed elsewhere [ 16 , 17 ] . in this review , we will focus specifically on the application of microfluidic devices in vascular cell biology research . microfluidic technology deals with the design , fabrication , and application of devices for manipulation of fluids on the micrometer scale . typically , the sizes of features in these devices range from several micrometers to a few hundred micrometers . the amounts of fluid that are manipulated inside these devices are typically in the picoliter to nanoliter range . most devices that are used in combination with cell biological research are made of glass or the silicone rubber polydimethylsiloxane ( pdms ) , because these materials are cheap , biocompatible , and transparent . because all microfluidic studies that are discussed in this review use microfluidic devices of pdms ( sometimes combined with glass components ) , the process of producing these devices will be described shortly ( see also figure 3 ) . pdms devices are produced by soft lithography replica molding , which means that the devices are elastic replicas of a stiff , reusable mold . the mold is usually made of silicon with micrometer - size structures produced either by plasma - etching of the silicon plate or by building on top of the plate with the epoxy - based , photo - crosslinkable polymer su-8 . a mixture of pdms oligomers is poured on top of this mold , allowed to solidify by crosslinking , and then peeled off from the mold . in order to create sealed channels , the surface of the pdms replica is activated with oxygen plasma and bound to a pdms or glass surface . holes can be punctured to reach the closed channel structure and tubing can be connected to manipulate fluid inside the channels . the silicon master - molds need to be produced in a clean room , but replica molding can be performed under standard laboratory conditions . once the master - mold has been created , producing new microfluidic devices by this method takes only a few hours . generally speaking , pdms microfluidic devices offer a number of distinct advantages over conventional techniques for cell culturing , manipulation , and analysis . the main feature of microfluidic devices that makes them suitable for use in cell biology is that they are smaller than conventional setups ( for an impression of the size of a microfluidic channel , see figure 4(a ) ) . because of this small size first of all , if experiments are to be conducted with rare primary cell material or expensive drugs , it is quite advantageous to use only small quantities of these valuable materials . secondly , if cultures are to be maintained under conditions of constant fluid flow , small sizes are a considerable advantage . in conventional bioreactors , cell culture medium is usually collected and re - used after it has passed through the cell culture chamber . a constant flux of fresh medium can be used , because the volumes involved are orders of magnitude smaller . the third benefit is that the small , planar and transparent microfluidic setups are easily combined with bright field and fluorescence microscopy or spectroscopy , because they fit easily on stages of conventional microscopes . this facilitates monitoring cell behavior for long periods and with high magnification during the experiment . it is important to realize that cells that are cultured inside microfluidic devices need to be subjected to a constant flux of fresh medium . when the small volumes in the cell - containing devices would be left under static conditions , nutrients would be depleted quickly , whereas waste products would increase to undesirable concentrations . the fact that constant refreshment of medium is needed may seem cumbersome at first glance . however , under physiological conditions , all cell types need a flux of nutrients and waste products . the flow conditions in microfluidic devices mimic this process more closely than in vitro culturing in wells plates . because of the small dimensions of microfluidic channels , fluid flow is fully laminar , meaning that the flow patterns are completely predictable and turbulent mixing does not occur . in some applications , such as microreactors , which require mixing of different reagents , this laminar flow pattern is an obstacle that has to be overcome . however , the laminar nature of the fluid flow can also be used to perform unique experiments that are difficult or nearly impossible to perform with conventional methods . most of these experiments rely on parallel fluid flows : if two streams of fluid enter the chip in a parallel fashion , the two streams will remain separated and mixing of them will only occur by diffusion . thus , the degree of mixing can be tuned by changing the flow rate : the higher the flow rate , the shorter the residence time inside the device , the less the streams mix . therefore , as long as flow rates are sufficiently high , cells on one side of the device can be treated with one substance , whereas cells on the other side are treated with another substance . as a matter of fact another important main feature of microfluidic technology is that it is suitable for high - throughput , comprehensive studies of cell biology . this means that the effect of multiple factors and parameters on cell functioning can be screened in one assay . efforts are made to merge microfluidic technology with microarray and microtitre plate technology [ 2123 ] . also , researchers are dedicated to integrate multiple steps , such as cell culturing , lysis , and analysis in one device . numerous examples of this parallel and serial microfluidic biochemical analysis , also known as lab - on - a - chip , have already been reported and are starting to be implemented in cell - containing microfluidic devices . vascular endothelial cells are highly responsive to shear stress that is caused by the flow of fluid over their surface . this shear stress is the result of the presence of a fluid velocity gradient in the cross section of a tube . the velocity of the fluid next to the walls is zero , whereas the velocity is maximal in the center of the channel . the steeper this gradient , the higher the shear forces that act on the vessel wall . the endothelial mechanoresponse has been found to be a key process in preventing vascular disease . the mechanoresponse is usually studied in vitro by subjecting endothelial cells to shear stress in parallel plate flow chambers . because shear stress is proportional to flow rate and inversely proportional to channel dimensions , only low flow rates are needed in microfluidic channels to mimic the high shear stresses found in the human body . . took advantage of this fact by designing a microfluidic device that can subject endothelial cells to physiological levels of shear stress in multiple parallel channels . they showed that a flow rate of less than 200 l per hour is already enough to make the sheared endothelial cells elongate and orient in the direction of the flow , which is a prominent feature of the endothelial mechanoresponse that is also found in vivo . this reorientation is also reflected in the actin cytoskeleton of the cells . in our laboratory , we subjected cells to a shear stress of 1 pa for 12 hours and then stained the actin filaments with phalloidin - fitc . most filaments were aligned and oriented in the flow direction ( figure 4(b ) ) . recently , tkachenko et al . also reported the design of a microfluidic device that allows for real - time tracking of endothelial cells that are subjected to shear stress . they could generate shear stresses ranging from 0.01 to 0.9 pa in parallel channels , using flow rates in the range of several milliliters per hour . in contrast , flow rates are in the order of hundreds of milliliters per hour for the conventional , larger , parallel plate flow chambers . because of the small volumes of reagents that are needed , and the potential parallelized design of microfluidic devices , they are an ideal platform for screening of compounds that may have an impact on the mechanoresponse . we have recently developed such an assay , in which the morphological rearrangements of endothelial cells are used to quantify the mechanoresponse . using this assay , the impact of inhibitory drugs on the mechanoresponse can be detected ( paper submitted ) . another well - known effect of applying shear stress to endothelial cells is the release of the vasodilatant , nitric oxide . microfluidic assays have already been reported that can detect the production of nitric oxide in response to chemical stimuli amperometrically or by fluorescence . this provides researchers with an interesting tool to study nitric oxide release in response to both mechanical and chemical stimuli . when increasing the flow rate and the resultant shear stress , microfluidic devices can also be used to study the adhesion strength of endothelial cells to their underlying substrate . young et al . performed such an experiment with endothelial cells of different origins and two types of matrix proteins . when the cells were subjected to a shear stress that is about ten times higher than typical physiological values , a certain percentage of cells detached from the surface . in this manner , it was possible to give a semiquantitative indication of the strength of adhesion of different cells on different substrates . these types of experiments used to be performed with large , parallel plate shear devices that consumed large amounts of media , cells , and reagents . as discussed earlier , multiple parallel fluid flows can be introduced in one microfluidic channel . transport of components from one flow to the other only occurs by diffusion ( figure 5(a ) ) . if flow rates are low , there is sufficient time for the parallel streams to exchange components . if one of the streams contains a drug or active compound , stable gradients can be generated by taking advantage of this diffusion . an example of such a gradient that was produced in our laboratory is shown in figure 5(b ) . there are a number of studies that show how this phenomenon can be used when experimenting with vascular cells . most of these studies focus on migration of vascular cells in response to gradients of physical or biochemical cues . studying and understanding cell migration is important , because it is a process involved in embryogenesis , wound healing , and tumorigenesis . barkefors et al . studied migration of endothelial cells in gradients of vascular endothelial growth factor ( vegf ) . they designed a device with three inlets , generating three parallel fluid streams in the main channel . when vegf was added to the middle stream , the steepness of this gradient could be tuned by adjusting the flow rates : the slower the flow rate , the longer the residence time in the channel , the more time there is for vegf to diffuse , and the more shallow the gradient becomes . when endothelial cells were cultured in this stable gradient of vegf , they preferentially migrated towards the middle of the channel . because the researchers had control over the shape of the gradient moreover , it was found that endothelial cells migrated fastest in gradients from 0 to 50 ng / ml , whereas they were not able to sense gradients from 50 to 100 ng / ml due to saturation of the available receptors . biochemical cues , such as the growth factors used in this study , are not the only relevant stimuli for vascular cell migration . in an elegant study , zaari et al . showed that smooth muscle cells tend to migrate towards mechanically stiffer underlying substrates . to reach this conclusion , the authors designed a microfluidic device that could generate a gradient of crosslinker , mixed with a solution of acrylamide . a layer of gel with a gradient of stiffness was produced by crosslinking the mixture with uv light into a polyacrylamide network . when these gels were taken out of the microfluidic devices and smooth muscle cells were seeded on them , all cells tended to migrate towards the side of the gel with higher stiffness . apart from migration assays that rely on gradients , the parallel laminar fluid streams can also be used to bring the most conventional migration assay to a microfluidic scale . it works by growing cells in a monolayer , artificially creating a scratch and then following how this scratch is closed by directed migration of the surrounding cells . in a microfluidic device , the artificial scratch can be generated by adding the serine protease trypsin to one of the parallel fluid streams . when one side of the channel has been cleared of cells by trypsinization , the migration of the remaining cells can be followed over time to quantify directed migration . so far , this assay has only been published with data on fibroblasts , but work in our group has shown that it is also possible with endothelial cells ( figure 5(c ) ) . the advantage of carrying out this assay in a microfluidic device is that it can be more easily combined with stimuli such as shear stress or growth factor gradients . micrometer - scale patterning of cells can be achieved by stamping adhesive proteins on a substrate or by temporarily confining cells in a microfluidic device until they adhere , after which the device is removed from the surface . by using parallel streams , cells can be patterned without the need of removing the microfluidic device afterwards . when adding one cell type to one stream and another type to the parallel stream , cells can be cocultured in direct contact with each other inside a microfluidic device . for vascular research , this method could be used to pattern endothelial cells and smooth muscle cells in one device . the planar nature of microfluidic devices would provide great opportunities for studying interactions between these cell types . in literature , there are numerous reports of microfluidic setups that are used for vascular cell interaction studies . for example , song et al . studied the interaction between endothelial cells and circulating tumor cells , a process that is important for cancer metastasis . they developed a device in which a layer of endothelial cells can be stimulated with chemokines from the bottom , while being treated simultaneously with a suspension of breast cancer cells from the top . when the endothelium was stimulated with cxcl12 , a chemokine implicated in metastasis , they found that more cancer cells adhered to the layer of endothelial cells than under basal conditions . another study on metastasis used a microfluidic chip with small , gel - coated gaps , overlaid with a monolayer of endothelial cells to mimic the basement membrane and the endothelium , respectively . using this microfluidic in vitro model of a blood vessel , tumor cell migration could be quantified and studied in great detail with time - lapse microscopy . it is not just interactions between tumor cells and endothelium that are an interesting object of research in vascular science . studying the interactions between other circulating cells and endothelial cells is also important . for example , the binding of leukocytes to endothelial cells is an essential step in inflammation , while the endothelium - mediated activation of blood platelets is important in clotting and thrombosis . multiple reports have been published by groups that studied the adhesion of leukocytes or platelets [ 4345 ] to endothelial cells or endothelial cell - derived adhesion factors in microfluidic devices . these reports show that microfluidic cell interaction studies require less sample and reagents than similar , conventional studies . moreover , a number of these studies already show increased throughput by using parallel channels in one device [ 42 , 44 , 45 ] . cells are embedded in an environment that comprises other cells , extracellular matrix proteins , bodily fluids , and blood . three - dimensional cell culturing in the laboratory can be performed by incorporating cells in a hydrogel matrix ( e.g. , the commercially available , collagen - based matrigel ) , or by growing cells on top of this matrix , allowing them to migrate into the gel . still , the complex real - life , three - dimensional microenvironment is usually reduced to a two - dimensional system when experiments are carried out on cells in vitro . this is more convenient , because with such a system cells can easily be supplied with fresh growth medium , growth factors , and other soluble compounds . however , when using microfluidic devices , replenishment of medium , generation of gradients , and microscopic imaging is relatively easy to realize in a three - dimensional culturing environment . they describe a microfluidic device with two parallel channels , connected by a gel chamber . the gel chamber is filled with a collagen - based hydrogel and endothelial cells are grown in one of the channels . by generating a gradient of soluble growth factors , the endothelial cells grow into the gel and even form open capillaries that span the entire gel chamber from channel to channel . in this particular article , the gel is pipetted into the gel chamber by microinjection before assembling the device . however , using the laminar flow properties discussed earlier in this review , hydrogels can also be formed in situ and even be patterned and confined to certain regions of the microfluidic device . the great potential of these three - dimensional culturing techniques was recently underlined by barkefors et al . , who cultured ex vivo kidney tissue and followed the formation of blood vessels in response to a vegf gradient . because of the small scale of microfluidic devices , it is possible to advance this proof - of - concept study towards high - throughput assays in order to screen for compounds that affect blood vessel formation in such realistic models . a good example of this high - throughput trend is the recent study by hsiao et al . , who studied three - dimensional , spheroid cocultures of prostate cancer cells and endothelial cells in a microfluidic device with 28 side chambers that could all harbor a tumorous spheroid . in biomedical engineering , a lot of research is dedicated to developing particle systems that carry drugs , proteins , dna for gene therapy or sirna for gene silencing to their proper site of action . in this field of research , it is important to have a way to quickly screen for adhesion to endothelial cells the first barrier that particles encounter when injected intravenously . screening under static conditions in well plates ignores the mechanical forces caused by the flowing blood , which counter particle adhesion . a recent study by our group , using fluorescent sirna - containing polymer particles , shows that microfluidic technology allows for quick screening of particle adhesion to endothelial cells under dynamic conditions . they show that capillary geometry has a strong influence on local mechanical conditions and particle adhesion . the fabrication of a more complex microfluidic device that tries to mimic the tight blood - brain barrier , while still being easy - to - use in high - throughput assays , was recently reported by genes et al . . it is to be expected that high - throughput screening in these more realistic vascular models of the in vivo situation will become the norm in drug development and material science in the future . regenerative medicine is the field in which researchers try to engineer tissues in the laboratory to replace damaged or missing tissue in the human body . it is a multi disciplinary field , which combines materials science with cell biology and biomedicine . a major challenge in this field is the production of vascularized tissue for implantation . in order to achieve this , stem cells must be stimulated to differentiate into vascular cells , and these vascular cells need to arrange themselves into a vascular network . for differentiation of human stem cells to vascular tissue , many factors can be of influence . because human stem cells and the inducing factors are relatively difficult to obtain , it is advantageous to perform tests in a microfluidic setting instead of in a macroscopic assay . developed a 12-well micro - bioreactor in which human embryonic stem cells were directed towards a vascular phenotype by varying growth factors , perfusion , and cell seeding density . using such microfluidic devices instead of conventional techniques saves reagents and allows for more flexibility in terms of culture parameters . the other aspect of vascular tissue engineering in which microfluidic technology can be of help is in preparing vascular networks that can be incorporated into tissue constructs . first of all , a synthetic capillary can be engineered by using microfluidic technology . this works by designing a microfluidic channel of which the walls contain tiny gaps of only a few micrometer in diameter and tens of micrometers in length . behind these gaps , compartments are located in which tissue can be grown . when medium is pumped through the channel , the gaps act as a simple endothelium - like barrier , limiting mass transport to the tissue compartments . an example of such a microfluidic design was reported by lee et al . , who used this principle to design synthetic analogs of liver sinusoids . using this approach , mass transport over the endothelium - like barrier can be tweaked to mimic the values found in human vessels [ 56 , 57 ] . a second approach to use microfluidic technology for generation of vascular networks is based on the notion that the microfluidic device itself can be considered as a three - dimensional scaffold in which cells can be grown . when all sides of a channel are completely covered with endothelial cells , it has been shown that this approach will in principle work : pdms devices with microvascular network morphologies can be completely covered with endothelial cells to generate capillary - like structures . however , pdms is a non - biodegradable polymer . another study has shown that the same type of system can also be built with the biodegradable polymer poly ( glycerol sebacate ) . still , these microfluidic devices consist of only one flat layer of vascular structures . a major challenge will be to build biodegradable , truly three - dimensional microvascular networks that can be combined with other materials and cells in regenerative medicine . novel rapid techniques for three - dimensional device fabrication , such as stereolithography with biodegradable polymers , hold great promise to overcome this challenge . the examples given in this review clearly illustrate the fact that the use of microfluidic technology facilitates current vascular research and , more importantly , opens up novel areas of research that are not possible with more conventional setups and techniques . it is important to realize not only that microfluidic technology paves the way for more realistic in vitro models in vascular cell biology but also that the technology is still in its infancy in terms of throughput . almost all studies described in this review are proof - of - principle experiments that require a lot of personal effort and intervention by the researcher . however , automation , standardization , and increasing scale will all be natural stages in the maturation of microfluidic technology . these improvements will boost the systematic nature of vascular cell biological research in the future .

vascular cell biology is an area of research with great biomedical relevance . vascular dysfunction is involved in major diseases such as atherosclerosis , diabetes , and cancer . however , when studying vascular cell biology in the laboratory , it is difficult to mimic the dynamic , three - dimensional microenvironment that is found in vivo . microfluidic technology offers unique possibilities to overcome this difficulty . in this review , an overview of the recent applications of microfluidic technology in the field of vascular biological research will be given . examples of how microfluidics can be used to generate shear stresses , growth factor gradients , cocultures , and migration assays will be provided . the use of microfluidic devices in studying three - dimensional models of vascular tissue will be discussed . it is concluded that microfluidic technology offers great possibilities to systematically study vascular cell biology with setups that more closely mimic the in vivo situation than those that are generated with conventional methods .

1. Introduction 2. Microfluidic Technology 3. Microfluidic Technology and Vascular Cells 4. Conclusion

important objects of study in this field of research include the maintenance of vascular tone , regulation of inflammation , sprouting of new blood vessels , regulation of cell survival , and the differentiation of stem cells into vascular tissue . vascular science is a field with a strong translational focus , combining results from fundamental molecular and cell biology with in vitro models of blood vessels and in vivo tests to develop insight in vascular physiology and treatment of disease . vascular dysfunction is an important factor in major diseases like atherosclerosis , cancer , and diabetes . therefore , a lot of research is focused on how endothelial cells or smooth muscle cells react to relevant biological , chemical , or physical cues in vitro . usually , this work is carried out by using conventional methods , culturing cells of animal or human origin in wells - plates , subjecting them to the aforementioned stimuli , and analyzing the outcome by biological or biochemical techniques . however , in vivo dynamic conditions are present : vascular endothelial cells are constantly subjected to shear stress caused by the flowing blood , while smooth muscle cells are stretched because of distension of the blood vessel during the cardiac cycle . moreover , vascular cells are embedded in a three - dimensional environment consisting of an elastic extracellular matrix , other cells , and flowing blood , with its platelets , red blood cells , and leukocytes ( figure 1 ) . both the three - dimensional environment and the dynamic mechanical changes with each cardiac cycle are very important factors in vascular cell functioning . it is advantageous to design laboratory setups that allow researchers to include these factors and control the relevant parameters . the main challenge when building such setups is that they should still be easy to assemble , handle , and combine with conventional analysis techniques . in the recent years , the field of microfluidic technology has gained much scientific interest among biologists , biochemists , and biophysicists ( figure 2 ) . we feel that microfluidic technology holds great promise to overcome the challenge of performing in vitro experiments with more physiologically realistic setups that are still simple enough to be used in everyday laboratory practice . moreover , microfluidic technology allows for increasing scale and parallelization of current research , leading to more comprehensive insights into cell and tissue physiology . the advantages of microfluidic technology for cell culture in general have been reviewed elsewhere [ 16 , 17 ] . in this review , we will focus specifically on the application of microfluidic devices in vascular cell biology research . microfluidic technology deals with the design , fabrication , and application of devices for manipulation of fluids on the micrometer scale . the amounts of fluid that are manipulated inside these devices are typically in the picoliter to nanoliter range . most devices that are used in combination with cell biological research are made of glass or the silicone rubber polydimethylsiloxane ( pdms ) , because these materials are cheap , biocompatible , and transparent . because all microfluidic studies that are discussed in this review use microfluidic devices of pdms ( sometimes combined with glass components ) , the process of producing these devices will be described shortly ( see also figure 3 ) . the mold is usually made of silicon with micrometer - size structures produced either by plasma - etching of the silicon plate or by building on top of the plate with the epoxy - based , photo - crosslinkable polymer su-8 . a mixture of pdms oligomers is poured on top of this mold , allowed to solidify by crosslinking , and then peeled off from the mold . in order to create sealed channels , the surface of the pdms replica is activated with oxygen plasma and bound to a pdms or glass surface . holes can be punctured to reach the closed channel structure and tubing can be connected to manipulate fluid inside the channels . the silicon master - molds need to be produced in a clean room , but replica molding can be performed under standard laboratory conditions . generally speaking , pdms microfluidic devices offer a number of distinct advantages over conventional techniques for cell culturing , manipulation , and analysis . the main feature of microfluidic devices that makes them suitable for use in cell biology is that they are smaller than conventional setups ( for an impression of the size of a microfluidic channel , see figure 4(a ) ) . because of this small size first of all , if experiments are to be conducted with rare primary cell material or expensive drugs , it is quite advantageous to use only small quantities of these valuable materials . a constant flux of fresh medium can be used , because the volumes involved are orders of magnitude smaller . it is important to realize that cells that are cultured inside microfluidic devices need to be subjected to a constant flux of fresh medium . when the small volumes in the cell - containing devices would be left under static conditions , nutrients would be depleted quickly , whereas waste products would increase to undesirable concentrations . however , under physiological conditions , all cell types need a flux of nutrients and waste products . the flow conditions in microfluidic devices mimic this process more closely than in vitro culturing in wells plates . because of the small dimensions of microfluidic channels , fluid flow is fully laminar , meaning that the flow patterns are completely predictable and turbulent mixing does not occur . in some applications , such as microreactors , which require mixing of different reagents , this laminar flow pattern is an obstacle that has to be overcome . however , the laminar nature of the fluid flow can also be used to perform unique experiments that are difficult or nearly impossible to perform with conventional methods . thus , the degree of mixing can be tuned by changing the flow rate : the higher the flow rate , the shorter the residence time inside the device , the less the streams mix . therefore , as long as flow rates are sufficiently high , cells on one side of the device can be treated with one substance , whereas cells on the other side are treated with another substance . as a matter of fact another important main feature of microfluidic technology is that it is suitable for high - throughput , comprehensive studies of cell biology . this means that the effect of multiple factors and parameters on cell functioning can be screened in one assay . efforts are made to merge microfluidic technology with microarray and microtitre plate technology [ 2123 ] . also , researchers are dedicated to integrate multiple steps , such as cell culturing , lysis , and analysis in one device . numerous examples of this parallel and serial microfluidic biochemical analysis , also known as lab - on - a - chip , have already been reported and are starting to be implemented in cell - containing microfluidic devices . vascular endothelial cells are highly responsive to shear stress that is caused by the flow of fluid over their surface . this shear stress is the result of the presence of a fluid velocity gradient in the cross section of a tube . the velocity of the fluid next to the walls is zero , whereas the velocity is maximal in the center of the channel . because shear stress is proportional to flow rate and inversely proportional to channel dimensions , only low flow rates are needed in microfluidic channels to mimic the high shear stresses found in the human body . they showed that a flow rate of less than 200 l per hour is already enough to make the sheared endothelial cells elongate and orient in the direction of the flow , which is a prominent feature of the endothelial mechanoresponse that is also found in vivo . this reorientation is also reflected in the actin cytoskeleton of the cells . in our laboratory , we subjected cells to a shear stress of 1 pa for 12 hours and then stained the actin filaments with phalloidin - fitc . most filaments were aligned and oriented in the flow direction ( figure 4(b ) ) . also reported the design of a microfluidic device that allows for real - time tracking of endothelial cells that are subjected to shear stress . they could generate shear stresses ranging from 0.01 to 0.9 pa in parallel channels , using flow rates in the range of several milliliters per hour . in contrast , flow rates are in the order of hundreds of milliliters per hour for the conventional , larger , parallel plate flow chambers . because of the small volumes of reagents that are needed , and the potential parallelized design of microfluidic devices , they are an ideal platform for screening of compounds that may have an impact on the mechanoresponse . using this assay , the impact of inhibitory drugs on the mechanoresponse can be detected ( paper submitted ) . another well - known effect of applying shear stress to endothelial cells is the release of the vasodilatant , nitric oxide . when increasing the flow rate and the resultant shear stress , microfluidic devices can also be used to study the adhesion strength of endothelial cells to their underlying substrate . when the cells were subjected to a shear stress that is about ten times higher than typical physiological values , a certain percentage of cells detached from the surface . in this manner , it was possible to give a semiquantitative indication of the strength of adhesion of different cells on different substrates . these types of experiments used to be performed with large , parallel plate shear devices that consumed large amounts of media , cells , and reagents . as discussed earlier , multiple parallel fluid flows can be introduced in one microfluidic channel . if one of the streams contains a drug or active compound , stable gradients can be generated by taking advantage of this diffusion . there are a number of studies that show how this phenomenon can be used when experimenting with vascular cells . studying and understanding cell migration is important , because it is a process involved in embryogenesis , wound healing , and tumorigenesis . studied migration of endothelial cells in gradients of vascular endothelial growth factor ( vegf ) . they designed a device with three inlets , generating three parallel fluid streams in the main channel . when vegf was added to the middle stream , the steepness of this gradient could be tuned by adjusting the flow rates : the slower the flow rate , the longer the residence time in the channel , the more time there is for vegf to diffuse , and the more shallow the gradient becomes . when endothelial cells were cultured in this stable gradient of vegf , they preferentially migrated towards the middle of the channel . because the researchers had control over the shape of the gradient moreover , it was found that endothelial cells migrated fastest in gradients from 0 to 50 ng / ml , whereas they were not able to sense gradients from 50 to 100 ng / ml due to saturation of the available receptors . biochemical cues , such as the growth factors used in this study , are not the only relevant stimuli for vascular cell migration . when these gels were taken out of the microfluidic devices and smooth muscle cells were seeded on them , all cells tended to migrate towards the side of the gel with higher stiffness . apart from migration assays that rely on gradients , the parallel laminar fluid streams can also be used to bring the most conventional migration assay to a microfluidic scale . it works by growing cells in a monolayer , artificially creating a scratch and then following how this scratch is closed by directed migration of the surrounding cells . in a microfluidic device , the artificial scratch can be generated by adding the serine protease trypsin to one of the parallel fluid streams . when one side of the channel has been cleared of cells by trypsinization , the migration of the remaining cells can be followed over time to quantify directed migration . so far , this assay has only been published with data on fibroblasts , but work in our group has shown that it is also possible with endothelial cells ( figure 5(c ) ) . the advantage of carrying out this assay in a microfluidic device is that it can be more easily combined with stimuli such as shear stress or growth factor gradients . when adding one cell type to one stream and another type to the parallel stream , cells can be cocultured in direct contact with each other inside a microfluidic device . for vascular research , this method could be used to pattern endothelial cells and smooth muscle cells in one device . the planar nature of microfluidic devices would provide great opportunities for studying interactions between these cell types . in literature , there are numerous reports of microfluidic setups that are used for vascular cell interaction studies . studied the interaction between endothelial cells and circulating tumor cells , a process that is important for cancer metastasis . they developed a device in which a layer of endothelial cells can be stimulated with chemokines from the bottom , while being treated simultaneously with a suspension of breast cancer cells from the top . when the endothelium was stimulated with cxcl12 , a chemokine implicated in metastasis , they found that more cancer cells adhered to the layer of endothelial cells than under basal conditions . another study on metastasis used a microfluidic chip with small , gel - coated gaps , overlaid with a monolayer of endothelial cells to mimic the basement membrane and the endothelium , respectively . it is not just interactions between tumor cells and endothelium that are an interesting object of research in vascular science . for example , the binding of leukocytes to endothelial cells is an essential step in inflammation , while the endothelium - mediated activation of blood platelets is important in clotting and thrombosis . multiple reports have been published by groups that studied the adhesion of leukocytes or platelets [ 4345 ] to endothelial cells or endothelial cell - derived adhesion factors in microfluidic devices . these reports show that microfluidic cell interaction studies require less sample and reagents than similar , conventional studies . cells are embedded in an environment that comprises other cells , extracellular matrix proteins , bodily fluids , and blood . three - dimensional cell culturing in the laboratory can be performed by incorporating cells in a hydrogel matrix ( e.g. still , the complex real - life , three - dimensional microenvironment is usually reduced to a two - dimensional system when experiments are carried out on cells in vitro . this is more convenient , because with such a system cells can easily be supplied with fresh growth medium , growth factors , and other soluble compounds . however , when using microfluidic devices , replenishment of medium , generation of gradients , and microscopic imaging is relatively easy to realize in a three - dimensional culturing environment . the gel chamber is filled with a collagen - based hydrogel and endothelial cells are grown in one of the channels . in this particular article , the gel is pipetted into the gel chamber by microinjection before assembling the device . however , using the laminar flow properties discussed earlier in this review , hydrogels can also be formed in situ and even be patterned and confined to certain regions of the microfluidic device . the great potential of these three - dimensional culturing techniques was recently underlined by barkefors et al . because of the small scale of microfluidic devices , it is possible to advance this proof - of - concept study towards high - throughput assays in order to screen for compounds that affect blood vessel formation in such realistic models . a good example of this high - throughput trend is the recent study by hsiao et al . , who studied three - dimensional , spheroid cocultures of prostate cancer cells and endothelial cells in a microfluidic device with 28 side chambers that could all harbor a tumorous spheroid . in biomedical engineering , a lot of research is dedicated to developing particle systems that carry drugs , proteins , dna for gene therapy or sirna for gene silencing to their proper site of action . in this field of research , it is important to have a way to quickly screen for adhesion to endothelial cells the first barrier that particles encounter when injected intravenously . a recent study by our group , using fluorescent sirna - containing polymer particles , shows that microfluidic technology allows for quick screening of particle adhesion to endothelial cells under dynamic conditions . the fabrication of a more complex microfluidic device that tries to mimic the tight blood - brain barrier , while still being easy - to - use in high - throughput assays , was recently reported by genes et al . it is to be expected that high - throughput screening in these more realistic vascular models of the in vivo situation will become the norm in drug development and material science in the future . regenerative medicine is the field in which researchers try to engineer tissues in the laboratory to replace damaged or missing tissue in the human body . it is a multi disciplinary field , which combines materials science with cell biology and biomedicine . a major challenge in this field is the production of vascularized tissue for implantation . in order to achieve this , stem cells must be stimulated to differentiate into vascular cells , and these vascular cells need to arrange themselves into a vascular network . for differentiation of human stem cells to vascular tissue , many factors can be of influence . because human stem cells and the inducing factors are relatively difficult to obtain , it is advantageous to perform tests in a microfluidic setting instead of in a macroscopic assay . developed a 12-well micro - bioreactor in which human embryonic stem cells were directed towards a vascular phenotype by varying growth factors , perfusion , and cell seeding density . using such microfluidic devices instead of conventional techniques saves reagents and allows for more flexibility in terms of culture parameters . the other aspect of vascular tissue engineering in which microfluidic technology can be of help is in preparing vascular networks that can be incorporated into tissue constructs . first of all , a synthetic capillary can be engineered by using microfluidic technology . behind these gaps , compartments are located in which tissue can be grown . using this approach , mass transport over the endothelium - like barrier can be tweaked to mimic the values found in human vessels [ 56 , 57 ] . a second approach to use microfluidic technology for generation of vascular networks is based on the notion that the microfluidic device itself can be considered as a three - dimensional scaffold in which cells can be grown . when all sides of a channel are completely covered with endothelial cells , it has been shown that this approach will in principle work : pdms devices with microvascular network morphologies can be completely covered with endothelial cells to generate capillary - like structures . however , pdms is a non - biodegradable polymer . still , these microfluidic devices consist of only one flat layer of vascular structures . a major challenge will be to build biodegradable , truly three - dimensional microvascular networks that can be combined with other materials and cells in regenerative medicine . novel rapid techniques for three - dimensional device fabrication , such as stereolithography with biodegradable polymers , hold great promise to overcome this challenge . the examples given in this review clearly illustrate the fact that the use of microfluidic technology facilitates current vascular research and , more importantly , opens up novel areas of research that are not possible with more conventional setups and techniques . it is important to realize not only that microfluidic technology paves the way for more realistic in vitro models in vascular cell biology but also that the technology is still in its infancy in terms of throughput . almost all studies described in this review are proof - of - principle experiments that require a lot of personal effort and intervention by the researcher . however , automation , standardization , and increasing scale will all be natural stages in the maturation of microfluidic technology . these improvements will boost the systematic nature of vascular cell biological research in the future .

edentulous patients seeking dental treatment to restore function and aesthetic appearance have traditionally received removable complete or partial dentures . however , the use of removable dentures may lead to a sense of patient insecurity , reduced chewing capacity and taste and low self - esteem . thus , the way of treatment turned toward implants because of the high success rate of dental implants , which has changed the quality of life for many patients . the success rate seems to be correlated to the amount and the quality of bone . when a problem of severe atrophy of jaws was encountered , there have been many different approaches in solving this condition by prosthetic reconstruction . the more - aggressive protocols call for bone grafting , followed by placement of endosseous implants.[47 ] in the setting of reduced alveolar bone height , the short dental implants have recently become available and offer the clinicians a pragmatic option to facilitate prosthetic restoration in the face of anatomic limitation . through the years , various types of standard branemark implants ( 3.75 mm ) have been introduced for the treatment of edentulous jaws , starting with the 10-mm long implant in 1971 . owing to the need for rehabilitation of such increasing number of atrophic jaws , the 7-mm standard implant was introduced in 1979 . from the beginning , this implant was used either alone or with longer implants in edentulous jaws , but , eventually , it was used in the treatment of partial edentulism as well . when considering these implants in function , the 1- , 3- , 5- and 10-year results showed a preponderance of failures among the short implants.[1416 ] in addition , to facilitate the replacement of a failing standard implant and to improve the success rate in compromised situations , wide - diameter implants were introduced . the wide - diameter implant was first introduced to fulfill two indications : poor bone quality and/or quantity and replacement of a failing standard implant . some authors have found that wide - diameter implants were successful when the length of the implant was compromised for situations where residual alveolar height was less . reconstruction of the atrophic mandible using short implants without augmentation procedures yielded , after more than 10 years of follow - up , a cumulative implant survival rate of 92.3% . as it is clear that the shortcoming of short implant was found to be improved by some means and paucity of data regarding review of 7 mm short implant studies , they need to be investigated thoroughly for their use . thus , the aim of this review is to present a comprehensive view of 7 mm short implant studies . studies to be included in this structured review had to fulfil the following inclusion criteria : relevant data on implant lengths and diametersimplant survival rates were either clearly indicated or calculable from data reported in the paper or as percentage basiscriteria for implant failure clearly definedimplant placements in various sites of jaws with immediate or delayed loadinghuman - derived data were reportedcomplications before and after loadingtotal number of short implants with their lengths and diameterscombination with or without long implants . relevant data on implant lengths and diameters implant survival rates were either clearly indicated or calculable from data reported in the paper or as percentage basis criteria for implant failure clearly defined implant placements in various sites of jaws with immediate or delayed loading human - derived data were reported complications before and after loading total number of short implants with their lengths and diameters combination with or without long implants . no restrictions were placed concerning study design , and randomized and non - randomized clinical trials , cohort studies , case control studies and case reports were considered for inclusion in the review . the following search terms were used : dental / oral implant and length , diameter , shape and short dental implant , wide diameter implant , implant size < 10 , < 8 < 7 mm etc , reduced alveolar height implants , compromised implants . in addition , a manual search of the following journals from 1991 to 2011 was performed : clinical oral implant research , international journal of oral and maxillofacial implants , clinical implant dentistry and related research , journal of periodontology , journal of clinical periodontology , international journal of periodontics and restorative dentistry , quientessence international . a further manual search was conducted through the bibliographies of all relevant papers and review articles . the review looks on certain key aspects of short implants that will be helpful in deciding whether to use or not when they are really indicated . thus , the data obtained from each article was divided into six tables : a demographic data and the type of study [ table 1]type of implants , total number used and their dimensions [ table 2]location of implants in the jaws , type and quality of bone , type of surgery and augmentation [ table 3]number of short implants and their dimensions [ table 4]type of prosthesis , loading , follow - up and survival rate [ table 5]time of failure , complications before / after loading and crestal bone loss [ table 6 ] andoverall success rate of short implants as per each article [ table 7 ] . a demographic data and the type of study [ table 1 ] type of implants , total number used and their dimensions [ table 2 ] location of implants in the jaws , type and quality of bone , type of surgery and augmentation [ table 3 ] number of short implants and their dimensions [ table 4 ] type of prosthesis , loading , follow - up and survival rate [ table 5 ] time of failure , complications before / after loading and crestal bone loss [ table 6 ] and overall success rate of short implants as per each article [ table 7 ] . demographic data and type of study type of implants , total number used and their dimensions location of implants in the jaws , type and quality of bone , type of surgery and augmentation the number of short implants and their dimensions type of prosthesis , loading , follow - up and survival rate time of failure , complications before / after loading and crestal bone loss overall success rate of short implants as per each article this review presents a comprehensive view of short implants from 1991 to 2011 . in the present study , data on short implants from 12 prospective , non - randomized , non - controlled trials , 10 retrospective , non - randomized , non - controlled trials , one randomized controlled trial and the rest from clinical follow - up studies are presented . the data in this review has been published in peer - reviewed scientific journals and are therefore judged reliable . a metaanalysis was not performed as data from the included reports were not standardized and , therefore , a descriptive analysis is presented . in this study , short implants were reviewed from 1991 to 2011 by dividing the data into six tables , and the description of each study is as follows . in a retrospective study , implants were placed central ( closest to midline , terminal the most posteriorly inserted ) and intermedial ( all implants between central and terminal ) , and it did not include fixture failure after prostheses . the overall failure of the implants was attributed to the following reasons : placement of implant in soft bone and not achieving the primary stability at the time of placement , maxillary region , advanced resorption of maxilla and mandible and overheating of surgical site due to high bone density . the authors reported that the 7-mm implant had the highest failure rate both in the maxilla and in the mandible in completely edentulous patients , and they attributed the failure rate to the length of the implant as small bone volumes were present for fixture insertion . they also stressed the fact that majority of the 7-mm fixtures still became integrated and , subsequently , supported prosthetic restorations , whereas in partially edentulous patients , the authors reported no relationship between implant length and early failures . in another retrospective study , implants were placed anterior to the mental foramen , implant - supported prostheses given for a minimum of 1 year . the failure of the 7-mm implant was due to distal implants , and they were rigidly connected to the fixed prostheses and were not engaged to the inferior cortical border of the bone . they attributed this to excess occlusal forces as the distal most implants carry a large load and the implants were mobile because they were not engaged to the inferior cortical border . shortcomings of the study were retrospective design , inclusion of small sample size and limited long - term follow - up . they concluded that short implants can be used successfully in atrophic mandibles even without bone augmentation procedures . a prospective multicentre study was conducted in 1985 with a total of 159 edentulous patients with 558 implants . they used the applegate kennedy classification to classify jaws into class 1 , 2 and 4 . a clear correlation was noted with implant length failure , and most failures were represented by short implants of standard diameter whereas the implants of wider diameter seemed to achieve better results than the standard ones . implant loss was higher in the maxilla , particularly in type 4 bone quality , and most losses occurred in the early loading periods . this study relates implant length and maxillary bone quality for their failure . a retrospective study from 1985 to 1988 conducted on 150 patients , dividing them into four groups , i.e. bone - grafted group ( bg ) , resorption group ( sr ) , intermediate group ( i m ) and fixed prosthesis ( fp ) . most failed implants were found in the sr group ( 28.7% ) , least in the fp group ( 7.6% ) , and the i m and bg group recorded 20% . authors noted that implant failure decreased over time in all test groups except the grafted one . therefore , the bg group must be followed longer before full knowledge can be obtained regarding the potential of that procedure . authors concluded that even though failure rate was more for the sr group , it was acceptable because grafting would not have been a realistic treatment alternative ; so , to reach the survival level of about 70% after 5 years of clinical function must therefore be regarded as acceptable . a multicenter clinical trial was carried out for a 6-year period on 126 patients . the type of implant used was iti ( international team of implantologists ) and the surface characteristic was tps ( titanium , plasma - sprayed ) . there were five losses out of seven implants in the maxillary molar and in the premolar sites . they remarked that implant design and implant - bone interface play an important role , and the plasma - sprayed implant surface might have compensated for the shorter implant length . authors stressed that placing of short implants in extremely atrophic mandible and maxilla will create unfavorable crown root ratio , resulting in large prosthetic restorations that are heavy , aesthetically unsatisfying and uncomfortable for the patient . they concluded that 6 mm implants can be used successfully with patients with minimal bone height , preferably used in combination with longer implants . a prospective multicenter study was carried out in which five ( 7 mm ) implants with 3.75 mm diameter were used and were immediately placed . a total of three implants were placed in the maxilla , and all implants were lost . a total of four implants were lost , one each in the maxillary and mandibular premolar and molar areas . the losses were due to placement in type 4 bone , mechanical overload and periimplantitis . in a prospective multicenter study , 127 patients were involved according to kennedy 's class i or ii . the study presented only survival and not the success rate , as the prostheses were removed at 10 years , to assess individual implant stability . authors stated that the tendency for higher failure rate was seen for restorations supported by four or more implants than those supported by two to three units . a retrospective study conducted on 49 patients with edentulous mandible and bone resorption was shape e and quality 1 according to lekholm and zarb . none of the 5 mm 6 mm implants were lost in the present study , which may be coincidental or because of their limited number . a clinical study involved patients with the age range of 18 - 81 years receiving 660 posterior maxillary implants . the lekholm and zarb system of bone classification was modified to include additional variations of type 4a , type 4b , type 4c and type 5 , and they were described as follows : type 4a only crestal bone ( inferior cortical bone ) , type 4b both inferior and superior cortical bone , type 4c superior cortical bone , type 5 no cortical bone superiorly and inferiorly . the failure of wider implant was 5% versus 7% for the 3.75 mm implant . authors stated that the quantity and quality of bone did not have influence on the success rate , as a lower success rate was seen in type 4 bone where quality was concerned . according to bone quantity , there was a 7% failure in grade 4 and 6% failure in grades 1 , 2 and 3 . the authors stated that a sufficient number of implants have to be placed to support the occlusal load . therefore , the authors recommended the use of wide and double implants posteriorly for successful implant placement . osseotite implants were used in a prospective study for patients who were 18 years and above with a two - stage surgery and evaluated up to 5 years . one implant failed in the posterior maxilla during preloading due to mobility ( soft bone ) . authors attributed lower failure rate to the dual etched microtextures surface compared with machined implant surface . clinical investigations in a study regarding endopore implants that were placed in patients with an age range of 22 - 72 years showed that the 7-mm length implants were 32 in number with no failures . authors attributed success to the large surface area provided by the implant design , which is three- to four - times that of machined threaded implants and porous surface implant , and it allows for three - dimensional mechanical interlock with the bone for the in - growth of the bone , and this interface effectively resists interfacial shear forces and also tensile forces created by the transverse component of occlusal loading . in a study , authors used iti implants and made a special emphasis on short implants . combination of overdentures , full - arch fixed partial denture and single crowns were given for the patients in this study . authors recommend that the use of short and roughened iti implants is predictable for their success . a total of 111 patients with an age range of 22 - 80 years and their edentulism classified according to the applegate kennedy classification were enrolled in a study . 1179 3i self - tapping machined implants with external hex were incorporated on 493 patients with a 5-year follow - up . short implants failed at a higher rate ( 11% ) than long implant ( 6.9% ) , and more failures were noted in the posterior maxilla . short implant used in this study was 4.1/7 mm dimensions with 84 months follow - up . a total of 16 short implants with 15 implants in the maxillary 1 molar and one in the maxillary 2 molar sites were placed . all types of implants such as tps / sandblasted / acid etched / sls surface were used , with survival of 100% . authors stated that short implants may be successfully employed in the replacement of missing single maxillary molars . ninety - six short branemark implants ( 6 - 8.5 mm ) were installed by a one - stage procedure with delayed loading ( 3 - 6 months ) . the bone quality ranged from type 1 to type 4 . of the 96 implants , among the 7 mm implants , a total of three implants were lost , of which one was tiunite and two implants were machined . they reported inferior results for machined implants compared with oxidised implants , but the difference was statistically non - significant . authors stated that use of short implants is of greater simplicity and safety compared with bone augmentation procedures and severely resorbed maxilla . a prospective clinical trial was conducted on 74 patients with mean age around 53 years ( range 20 - 76 years ) . those implants with c / r ratios equal to or less than 1.0 had an average functional time of nearly 5 years , while those with c / r ratios > 2.0 on average had been functioning only approximately 3 years . the c / r ratios for the implants used in these two trials were assessed and categorized , and it was found that the majority of the implants had a c / r ratio of between 1.0 and 2.0 , with an average of 1.5 . authors stated that this might have been considered unfavorable for long - term implant survival , given the generally recommended c / r ratio of 0.5 . a study was conducted in a private practice on 264 patients with the age group of 13.8 - 95.7 years ( median age of 55.5 years ) using straumann implants . more than half of the 6 mm implants were placed in relatively poor quality ( type 4 ) bone . the implant types for 6 mm were solid screw standard ( 4.1 mm ) , solid - screw esthetic sla ( 4.1 mm ) , solid - screw standard sla ( 4.1 mm ) , wide - body implants ( 4.8 mm ) and wide body sla and wide neck sla . in this study , splinting was applied to implants placed in adjacent sites , irrespective of implant lengths . among 35 of the 6 mm implants , two failed , and implant losses a prospective 5-year life table analysis was carried out on wide neck ( wn ) iti implants placed in a private practice for 212 patients , with 263 implants of 4.8 mm diameter placed in the posterior regions . the quality of bone analyzed in this study was dense bone , normal bone and soft bone . the total numbers of short implants used in this study were four , with length of 6 mm and diameter of 4.8 mm . the failure of implant was due to placement in soft bone , and the reason was that he was bruxer and smoker . unfortunately , no conclusion could be drawn on the reliability of the 6 mm implant in this indication because of the limited number of implants ( only four implants ) . when short implants were placed in soft bone , i.e. with two combined risks , the survival rate was still 95.8% . a retrospective clinical trial was conducted with 272 implants that had machined surfaces , while 136 implants had oxidized surfaces ( tiunitetm , nobel biocare ab ) . of the five failed implants , two were located in the maxillary anterior and one in the maxillary posterior region , and two were placed in the mandibular posterior region . all implant losses occurred during the first 6 months of healing , before prosthetic loading , and all had machined surfaces . in most cases , the bone was rather soft , being in the maxilla . authors stated that the cumulative survival rate of 96% for 7-mm implants after 5 years indicates that short implants used in both jaws may be a viable concept with comparable survival rates to longer implant . a randomized controlled trial combined 5 mm long , 6 mm wide implant with 10 mm length implant , with a partial fixed prosthesis on them , and was carried out on non - smokers , light smokers and heavy smokers . the follow - up was 5 years with two - stage surgery in a total of 30 patients . patients were divided into two groups , namely : short implant group and augmented group . authors stated that the large implant diameter might have played an important role in the success , although this is just a hypothesis and not a fact , which therefore needs to be investigated with proper clinical trials . a retrospective study stated that the initial evidence suggesting high predictability of short implants has been reinforced by the different biomechanical studies addressing that maximum bone stress is practically independent of implant length and that implant width is more important than the additional length . a total of 1287 implants was placed in 661 consecutive patients : inclusion criteria was bone densitometry > 400 and primary stability ranged between 45 and 60 ncm . only one implant of 7 mm was lost , which was placed in the mandible in a one - stage protocol connected with long implant placed with high torque ( 60 ncm ) pushed out by the tongue . the vertical bone augmentation procedure has the potential to increase the number , length and diameter of the implants , and it is quite predictable for immediate loading , enabling a stable long - term prognosis to the implant - supported reconstruction , whereas the demerits are type of graft material to be used as autogenous being gold standard , which will not be always the choice with donor site morbidity and graft resorption , and requires higher cost to the patient . the placement of short implants is no less efficacious than placement of implants of 10 mm or longer ; however , according to a retrospective study , immediate loading of threaded implants of 10 mm or shorter with a rough surface should be performed with caution . but , investigations of immediate loading of implants of length ( 7 mm ) as per this review are not found . it seemed prudent to follow a two - stage implant placement approach when using short implants , as this approach has been linked with higher success rates . however , according to a review , there is no significant difference in failure rates between single - stage and two - stage implants . in a metaanalysis study , the single - stage approach might be preferable in partially edentulous patients , as it avoided one surgical intervention and shortened treatment times , while a two - stage submerged approach could be indicated when an implant had not obtained optimal primary stability , especially in fully edentulous patients . a surgical protocol may not play a key role in the prognosis of short implants . early loading refers to final crowns within 3 ( within 1 to a couple of weeks ) weeks from surgery in occlusion . in delayed loading , endosseous dental implants remain covered and free from loading for a period of 3 months in the mandible and 6 months in the maxilla to allow implant - bone integration . a study has demonstrated the possibility of early loading of slactive implants inserted with simultaneous osteotome sinus floor elevation without the use of grafting material . slactive implants allow early loading protocols of 3 - 4 weeks , as it maintains its chemical purity and high surface activity and increasing early cellular activity and bone apposition . but , the study used 10 mm as implant length , and there are no studies for early loading of 7 mm short implants . all the articles stated in this review are delayed loading , contributing the maximum survival rate undoubtedly . the crown / root ratio , especially when using short implants , has been considered as one of the prosthetic factors that may increase the risk of biomechanical complication because of overloading / non - axial loading , ultimately resulting in crestal bone loss . authors demonstrated that c / i ratio for short implants was not a complicating factor , such as affecting crestal bone levels . a study reports on the success of using four 5.05.0 mm bicon implants in a 56-year - old patient with partial edentulism . the success of this case study suggests a potential for decreasing the frequency of complications , such as inferior alveolar canal perforation , and of being able to provide dental implants to individuals who have inadequate alveolar bone levels for conventionally longer implants . a retrospective study evaluated various implant surface factors associated with bicon implant survival for single - tooth restoration in the healthy individual . among the many factors that may influence the failure rate of tps and ha - coated bicon implants , installation depth played an especially significant role in the success of ha - coated bicon implants , and implant length was an important prognostic factor for the tps - treated implants . a retrospective study was designed to estimate the 1- and 5-year survival of bicon dental implants and to identify risk factors associated with implant failure in an objective , statistically valid manner . the overall 1- and 5-year survival of the bicon implant system was 95.2% and 90.2% , respectively . the results of these analyses suggest that the overall survival of the bicon dental implant is comparable with other current implant systems . a total of 3046 short implants were placed , four articles documented the placement in the maxillary region , three articles documented in the mandibular region and 21 articles documented both . regarding augmentation procedures , 20 dealt with non - augmentation , three reported on augmentation procedures and the rest did not specify anything . branemark implants were used by 17 studies , two articles on straumann implants , two on iti implant systems , one on bicon implants and one on endopore implants . of the 28 articles , 17 articles dealt with 7 mm length , 11 considered on 6 mm either alone or in combination with longer implants and two articles documented 5 mm length implants . the observation period of the articles ranged from a minimum of 4 months to a maximum of 7 years . nine articles have taken the lekholm and zarb classification of bone qualities.[1516212325273652 ] the survival rates for short implant are as follows : from 1991 to 1995 , it was 82.45 - 98.48% ; 1996 to 2000 , 80 - 95.5% ; 2001 to 2011 , it was found to be 98 - 100% . the causes for the failures according to authors from 1991 to 2000 were small bone volumes , distal implants with excessive occlusal forces , maxilla , short and standard diameter implant ; from 2001 to 2011 , the reason was soft bone , smooth machined implants and osteoporotic bone . the striking aspect was implants with a sintered porous surface geometry ( endopore ) that can be suitable for restoring edentulous spaces as they yielded 100% survival . implants with rougher surface implants , i.e. sand - blasted and acid - etched implants , showed favorable results compared with machined surface , and the failure rate was higher with standard - diameter short implants compared with wide - diameter short implants . authors gave information regarding immediate placement not on immediate loading , whereas there are no articles either on immediate loading or immediate placement of short implants ( 7 mm ) . this structured review has identified 28 articles with data regarding implant length < 7 mm , of which nine articles are dedicated to short implants , 21 articles include wide - diameter implants and other are the usual ones . short implants could be a preferable choice as the treatment becomes faster and cheaper , and these are associated with less morbidity than vertical bone augmentation . even when the residual bone height over the mandibular canal is 57 mm , these short ones achieved better results .

background : short implants are manufactured for use in atrophic regions of the jaws . although many studies report on short implants as 10 mm length with considerable success , the literature regarding survival rate of 7 mm is sparse.purpose:the purpose of this study was to systematically evaluate the publications concerning short dental implants defined as an implant with a length of 7 mm placed in the maxilla or in the mandible.materials and methods : a medline and manual search was conducted to identify studies concerning short dental implants of length 7 mm published between 1991 and 2011 . the articles included in this study report data on implant length 7 mm , such as demographic variables , implant type , location in jaws , observation time , prostheses and complications.results:the 28 included studies represent one randomized controlled trial , 12 prospective studies and 10 retrospective studies . the survival rate of short implant was found to be increased from 80% to 90% gradually , with recent articles showing 100%.conclusion : when severe atrophy of jaws was encountered , short and wide implants can be placed successfully .

INTRODUCTION MATERIALS AND METHODS DISCUSSION SUMMARY AND CONCLUSION

however , the use of removable dentures may lead to a sense of patient insecurity , reduced chewing capacity and taste and low self - esteem . thus , the way of treatment turned toward implants because of the high success rate of dental implants , which has changed the quality of life for many patients . the success rate seems to be correlated to the amount and the quality of bone . when a problem of severe atrophy of jaws was encountered , there have been many different approaches in solving this condition by prosthetic reconstruction . [47 ] in the setting of reduced alveolar bone height , the short dental implants have recently become available and offer the clinicians a pragmatic option to facilitate prosthetic restoration in the face of anatomic limitation . owing to the need for rehabilitation of such increasing number of atrophic jaws , the 7-mm standard implant was introduced in 1979 . from the beginning , this implant was used either alone or with longer implants in edentulous jaws , but , eventually , it was used in the treatment of partial edentulism as well . when considering these implants in function , the 1- , 3- , 5- and 10-year results showed a preponderance of failures among the short implants. the wide - diameter implant was first introduced to fulfill two indications : poor bone quality and/or quantity and replacement of a failing standard implant . some authors have found that wide - diameter implants were successful when the length of the implant was compromised for situations where residual alveolar height was less . reconstruction of the atrophic mandible using short implants without augmentation procedures yielded , after more than 10 years of follow - up , a cumulative implant survival rate of 92.3% . as it is clear that the shortcoming of short implant was found to be improved by some means and paucity of data regarding review of 7 mm short implant studies , they need to be investigated thoroughly for their use . thus , the aim of this review is to present a comprehensive view of 7 mm short implant studies . studies to be included in this structured review had to fulfil the following inclusion criteria : relevant data on implant lengths and diametersimplant survival rates were either clearly indicated or calculable from data reported in the paper or as percentage basiscriteria for implant failure clearly definedimplant placements in various sites of jaws with immediate or delayed loadinghuman - derived data were reportedcomplications before and after loadingtotal number of short implants with their lengths and diameterscombination with or without long implants . relevant data on implant lengths and diameters implant survival rates were either clearly indicated or calculable from data reported in the paper or as percentage basis criteria for implant failure clearly defined implant placements in various sites of jaws with immediate or delayed loading human - derived data were reported complications before and after loading total number of short implants with their lengths and diameters combination with or without long implants . no restrictions were placed concerning study design , and randomized and non - randomized clinical trials , cohort studies , case control studies and case reports were considered for inclusion in the review . the following search terms were used : dental / oral implant and length , diameter , shape and short dental implant , wide diameter implant , implant size < 10 , < 8 < 7 mm etc , reduced alveolar height implants , compromised implants . in addition , a manual search of the following journals from 1991 to 2011 was performed : clinical oral implant research , international journal of oral and maxillofacial implants , clinical implant dentistry and related research , journal of periodontology , journal of clinical periodontology , international journal of periodontics and restorative dentistry , quientessence international . a further manual search was conducted through the bibliographies of all relevant papers and review articles . the review looks on certain key aspects of short implants that will be helpful in deciding whether to use or not when they are really indicated . thus , the data obtained from each article was divided into six tables : a demographic data and the type of study [ table 1]type of implants , total number used and their dimensions [ table 2]location of implants in the jaws , type and quality of bone , type of surgery and augmentation [ table 3]number of short implants and their dimensions [ table 4]type of prosthesis , loading , follow - up and survival rate [ table 5]time of failure , complications before / after loading and crestal bone loss [ table 6 ] andoverall success rate of short implants as per each article [ table 7 ] . a demographic data and the type of study [ table 1 ] type of implants , total number used and their dimensions [ table 2 ] location of implants in the jaws , type and quality of bone , type of surgery and augmentation [ table 3 ] number of short implants and their dimensions [ table 4 ] type of prosthesis , loading , follow - up and survival rate [ table 5 ] time of failure , complications before / after loading and crestal bone loss [ table 6 ] and overall success rate of short implants as per each article [ table 7 ] . demographic data and type of study type of implants , total number used and their dimensions location of implants in the jaws , type and quality of bone , type of surgery and augmentation the number of short implants and their dimensions type of prosthesis , loading , follow - up and survival rate time of failure , complications before / after loading and crestal bone loss overall success rate of short implants as per each article this review presents a comprehensive view of short implants from 1991 to 2011 . in the present study , data on short implants from 12 prospective , non - randomized , non - controlled trials , 10 retrospective , non - randomized , non - controlled trials , one randomized controlled trial and the rest from clinical follow - up studies are presented . in this study , short implants were reviewed from 1991 to 2011 by dividing the data into six tables , and the description of each study is as follows . the overall failure of the implants was attributed to the following reasons : placement of implant in soft bone and not achieving the primary stability at the time of placement , maxillary region , advanced resorption of maxilla and mandible and overheating of surgical site due to high bone density . the authors reported that the 7-mm implant had the highest failure rate both in the maxilla and in the mandible in completely edentulous patients , and they attributed the failure rate to the length of the implant as small bone volumes were present for fixture insertion . they also stressed the fact that majority of the 7-mm fixtures still became integrated and , subsequently , supported prosthetic restorations , whereas in partially edentulous patients , the authors reported no relationship between implant length and early failures . in another retrospective study , implants were placed anterior to the mental foramen , implant - supported prostheses given for a minimum of 1 year . the failure of the 7-mm implant was due to distal implants , and they were rigidly connected to the fixed prostheses and were not engaged to the inferior cortical border of the bone . shortcomings of the study were retrospective design , inclusion of small sample size and limited long - term follow - up . they concluded that short implants can be used successfully in atrophic mandibles even without bone augmentation procedures . a prospective multicentre study was conducted in 1985 with a total of 159 edentulous patients with 558 implants . a clear correlation was noted with implant length failure , and most failures were represented by short implants of standard diameter whereas the implants of wider diameter seemed to achieve better results than the standard ones . implant loss was higher in the maxilla , particularly in type 4 bone quality , and most losses occurred in the early loading periods . this study relates implant length and maxillary bone quality for their failure . most failed implants were found in the sr group ( 28.7% ) , least in the fp group ( 7.6% ) , and the i m and bg group recorded 20% . therefore , the bg group must be followed longer before full knowledge can be obtained regarding the potential of that procedure . authors concluded that even though failure rate was more for the sr group , it was acceptable because grafting would not have been a realistic treatment alternative ; so , to reach the survival level of about 70% after 5 years of clinical function must therefore be regarded as acceptable . there were five losses out of seven implants in the maxillary molar and in the premolar sites . authors stressed that placing of short implants in extremely atrophic mandible and maxilla will create unfavorable crown root ratio , resulting in large prosthetic restorations that are heavy , aesthetically unsatisfying and uncomfortable for the patient . they concluded that 6 mm implants can be used successfully with patients with minimal bone height , preferably used in combination with longer implants . a prospective multicenter study was carried out in which five ( 7 mm ) implants with 3.75 mm diameter were used and were immediately placed . a total of three implants were placed in the maxilla , and all implants were lost . a total of four implants were lost , one each in the maxillary and mandibular premolar and molar areas . none of the 5 mm 6 mm implants were lost in the present study , which may be coincidental or because of their limited number . the failure of wider implant was 5% versus 7% for the 3.75 mm implant . the authors stated that a sufficient number of implants have to be placed to support the occlusal load . therefore , the authors recommended the use of wide and double implants posteriorly for successful implant placement . osseotite implants were used in a prospective study for patients who were 18 years and above with a two - stage surgery and evaluated up to 5 years . one implant failed in the posterior maxilla during preloading due to mobility ( soft bone ) . clinical investigations in a study regarding endopore implants that were placed in patients with an age range of 22 - 72 years showed that the 7-mm length implants were 32 in number with no failures . authors attributed success to the large surface area provided by the implant design , which is three- to four - times that of machined threaded implants and porous surface implant , and it allows for three - dimensional mechanical interlock with the bone for the in - growth of the bone , and this interface effectively resists interfacial shear forces and also tensile forces created by the transverse component of occlusal loading . in a study , authors used iti implants and made a special emphasis on short implants . combination of overdentures , full - arch fixed partial denture and single crowns were given for the patients in this study . authors recommend that the use of short and roughened iti implants is predictable for their success . 1179 3i self - tapping machined implants with external hex were incorporated on 493 patients with a 5-year follow - up . short implants failed at a higher rate ( 11% ) than long implant ( 6.9% ) , and more failures were noted in the posterior maxilla . short implant used in this study was 4.1/7 mm dimensions with 84 months follow - up . a total of 16 short implants with 15 implants in the maxillary 1 molar and one in the maxillary 2 molar sites were placed . all types of implants such as tps / sandblasted / acid etched / sls surface were used , with survival of 100% . authors stated that short implants may be successfully employed in the replacement of missing single maxillary molars . of the 96 implants , among the 7 mm implants , a total of three implants were lost , of which one was tiunite and two implants were machined . authors stated that use of short implants is of greater simplicity and safety compared with bone augmentation procedures and severely resorbed maxilla . a prospective clinical trial was conducted on 74 patients with mean age around 53 years ( range 20 - 76 years ) . the c / r ratios for the implants used in these two trials were assessed and categorized , and it was found that the majority of the implants had a c / r ratio of between 1.0 and 2.0 , with an average of 1.5 . a study was conducted in a private practice on 264 patients with the age group of 13.8 - 95.7 years ( median age of 55.5 years ) using straumann implants . more than half of the 6 mm implants were placed in relatively poor quality ( type 4 ) bone . the implant types for 6 mm were solid screw standard ( 4.1 mm ) , solid - screw esthetic sla ( 4.1 mm ) , solid - screw standard sla ( 4.1 mm ) , wide - body implants ( 4.8 mm ) and wide body sla and wide neck sla . in this study , splinting was applied to implants placed in adjacent sites , irrespective of implant lengths . among 35 of the 6 mm implants , two failed , and implant losses a prospective 5-year life table analysis was carried out on wide neck ( wn ) iti implants placed in a private practice for 212 patients , with 263 implants of 4.8 mm diameter placed in the posterior regions . the quality of bone analyzed in this study was dense bone , normal bone and soft bone . the total numbers of short implants used in this study were four , with length of 6 mm and diameter of 4.8 mm . unfortunately , no conclusion could be drawn on the reliability of the 6 mm implant in this indication because of the limited number of implants ( only four implants ) . when short implants were placed in soft bone , i.e. with two combined risks , the survival rate was still 95.8% . a retrospective clinical trial was conducted with 272 implants that had machined surfaces , while 136 implants had oxidized surfaces ( tiunitetm , nobel biocare ab ) . of the five failed implants , two were located in the maxillary anterior and one in the maxillary posterior region , and two were placed in the mandibular posterior region . in most cases , the bone was rather soft , being in the maxilla . authors stated that the cumulative survival rate of 96% for 7-mm implants after 5 years indicates that short implants used in both jaws may be a viable concept with comparable survival rates to longer implant . a randomized controlled trial combined 5 mm long , 6 mm wide implant with 10 mm length implant , with a partial fixed prosthesis on them , and was carried out on non - smokers , light smokers and heavy smokers . patients were divided into two groups , namely : short implant group and augmented group . authors stated that the large implant diameter might have played an important role in the success , although this is just a hypothesis and not a fact , which therefore needs to be investigated with proper clinical trials . a retrospective study stated that the initial evidence suggesting high predictability of short implants has been reinforced by the different biomechanical studies addressing that maximum bone stress is practically independent of implant length and that implant width is more important than the additional length . a total of 1287 implants was placed in 661 consecutive patients : inclusion criteria was bone densitometry > 400 and primary stability ranged between 45 and 60 ncm . only one implant of 7 mm was lost , which was placed in the mandible in a one - stage protocol connected with long implant placed with high torque ( 60 ncm ) pushed out by the tongue . the vertical bone augmentation procedure has the potential to increase the number , length and diameter of the implants , and it is quite predictable for immediate loading , enabling a stable long - term prognosis to the implant - supported reconstruction , whereas the demerits are type of graft material to be used as autogenous being gold standard , which will not be always the choice with donor site morbidity and graft resorption , and requires higher cost to the patient . the placement of short implants is no less efficacious than placement of implants of 10 mm or longer ; however , according to a retrospective study , immediate loading of threaded implants of 10 mm or shorter with a rough surface should be performed with caution . but , investigations of immediate loading of implants of length ( 7 mm ) as per this review are not found . in a metaanalysis study , the single - stage approach might be preferable in partially edentulous patients , as it avoided one surgical intervention and shortened treatment times , while a two - stage submerged approach could be indicated when an implant had not obtained optimal primary stability , especially in fully edentulous patients . a surgical protocol may not play a key role in the prognosis of short implants . in delayed loading , endosseous dental implants remain covered and free from loading for a period of 3 months in the mandible and 6 months in the maxilla to allow implant - bone integration . but , the study used 10 mm as implant length , and there are no studies for early loading of 7 mm short implants . all the articles stated in this review are delayed loading , contributing the maximum survival rate undoubtedly . the crown / root ratio , especially when using short implants , has been considered as one of the prosthetic factors that may increase the risk of biomechanical complication because of overloading / non - axial loading , ultimately resulting in crestal bone loss . authors demonstrated that c / i ratio for short implants was not a complicating factor , such as affecting crestal bone levels . the success of this case study suggests a potential for decreasing the frequency of complications , such as inferior alveolar canal perforation , and of being able to provide dental implants to individuals who have inadequate alveolar bone levels for conventionally longer implants . among the many factors that may influence the failure rate of tps and ha - coated bicon implants , installation depth played an especially significant role in the success of ha - coated bicon implants , and implant length was an important prognostic factor for the tps - treated implants . a retrospective study was designed to estimate the 1- and 5-year survival of bicon dental implants and to identify risk factors associated with implant failure in an objective , statistically valid manner . the overall 1- and 5-year survival of the bicon implant system was 95.2% and 90.2% , respectively . the results of these analyses suggest that the overall survival of the bicon dental implant is comparable with other current implant systems . a total of 3046 short implants were placed , four articles documented the placement in the maxillary region , three articles documented in the mandibular region and 21 articles documented both . of the 28 articles , 17 articles dealt with 7 mm length , 11 considered on 6 mm either alone or in combination with longer implants and two articles documented 5 mm length implants . the observation period of the articles ranged from a minimum of 4 months to a maximum of 7 years . [1516212325273652 ] the survival rates for short implant are as follows : from 1991 to 1995 , it was 82.45 - 98.48% ; 1996 to 2000 , 80 - 95.5% ; 2001 to 2011 , it was found to be 98 - 100% . the causes for the failures according to authors from 1991 to 2000 were small bone volumes , distal implants with excessive occlusal forces , maxilla , short and standard diameter implant ; from 2001 to 2011 , the reason was soft bone , smooth machined implants and osteoporotic bone . the striking aspect was implants with a sintered porous surface geometry ( endopore ) that can be suitable for restoring edentulous spaces as they yielded 100% survival . authors gave information regarding immediate placement not on immediate loading , whereas there are no articles either on immediate loading or immediate placement of short implants ( 7 mm ) . this structured review has identified 28 articles with data regarding implant length < 7 mm , of which nine articles are dedicated to short implants , 21 articles include wide - diameter implants and other are the usual ones . short implants could be a preferable choice as the treatment becomes faster and cheaper , and these are associated with less morbidity than vertical bone augmentation . even when the residual bone height over the mandibular canal is 57 mm , these short ones achieved better results .

the dramatic increase in the global incidence of obesity has been accompanied by an increase in incident type 2 diabetes ( t2d ) ( 1 ) . anthropometric measures capturing abdominal adiposity , such as waist circumference or waist - to - hip ratio ( whr ) , have been related to diabetes risk independent of bmi across various ethnic groups ( 25 ) . compelling evidence shows that accumulation of visceral fat plays a pivotal role in the etiology of diabetes by overexposing the liver to free fatty acids , which subsequently result in insulin resistance and hyperinsulinemia ( 6 ) . however , whether central obesity plays a causal role in the development of t2d remains unclear . a meta - analysis of the whr genome - wide association studies ( gwas ) comprising > 100,000 individuals of european ancestry established a comprehensive genetic profile for modulating body fat distribution independent of overall adiposity ( 7 ) . information on the association of genetic predisposition to central obesity with risk of t2d is limited . in the current study , we calculated the genetic score based on 14 well - established single nucleotide polymorphisms ( snps ) for whr ( 7 ) as a proxy of genetic predisposition to central obesity . we examined the association of this genetic score with t2d risk in women and men of european ancestry from two prospective cohorts : the nurses health study ( nhs ) and the health professionals follow - up study ( hpfs ) . the nhs is a prospective cohort study of 121,700 female registered nurses aged 3055 years at study inception in 1976 when all participants completed a mailed questionnaire about medical history and lifestyle ( 8) . between 1989 and 1990 , 32,826 women provided blood samples . male health professionals who were aged 4075 years at study inception in 1986 ( 9 ) . between 1993 and 1999 , medical and lifestyle information has been collected biennially by self - administered questionnaires since inception . both studies were approved by the human research committee at the brigham and women s hospital ( boston , ma ) , and all participants provided written informed consent . participants for the current study were selected among those with a blood sample , using a nested case - control study design ( 10,11 ) . diabetes cases were defined as self - reported diabetes confirmed by a validated supplementary questionnaire ( 12,13 ) . for cases before 1998 , we used the national diabetes data group criteria to define diabetes ( 14 ) , which included one of the following : one or more classic symptoms ( excessive thirst , polyuria , weight loss , hunger , pruritus , or coma ) plus a fasting plasma glucose level of 7.8 mmol / l ( 140 mg / dl ) , a random plasma glucose level of 11.1 mmol / l ( 200 mg / dl ) , or a plasma glucose level 2 h after an oral glucose tolerance test of 11.1 mmol / l ( 200 mg / dl ) ; at least two elevated plasma glucose levels on different occasions in the absence of symptoms ; or treatment with hypoglycemia medication ( insulin or oral hypoglycemic agent ) . we used the american diabetes association diagnostic criteria for diabetes diagnosis since 1998 ( 15 ) . these criteria were the same as those proposed by the national diabetes data group except for the elevated fasting plasma glucose criterion for which the cut point was changed from 7.8 mmol / l ( 140 mg / dl ) to 7.0 mmol / l ( 126 mg / dl ) . the validity of diabetes self - report was verified in a subsample from the hpfs and the nhs cohorts . a physician blinded to the information reported on the supplementary questionnaire reviewed the medical records according to the diagnostic criteria . ninety - seven percent of the diabetes cases were confirmed in the hpfs cohort ( 13 ) . similarly , 98% of the diabetes cases reported by the supplementary questionnaire were confirmed by medical record review in a subsample of nhs participants ( 12 ) . control participants were defined as those free of diabetes at the time of the diagnosis of cases and remained unaffected through follow - up until 2006 . we matched the case participants to control participants by age , month and year of blood draw , and fasting status within their respective cohorts . after applying a quality control filter in the nhs and hpfs t2d gwas ( 11 ) , duplicate samples , samples with misidentified sex , related samples ( siblings or possible first cousins ) , samples with evidence of contamination , samples with highly variable intensity data , and samples with missing call rates 2% were excluded . the population structure was investigated by principal component analysis ( 16 ) . in this analysis , participants clustered with the hapmap ceu ( utah residents with northern and western european ancestry ) samples were genetically inferred to have european ancestry . a total of 3,221 women ( 1,467 case and 1,754 control participants ) and 2,422 men ( 1,124 case and 1,298 control participants ) of genetically inferred european ancestry were included in the current analysis . waist and hip circumference were reported to the nearest one - quarter inch in 1986 ( nhs ) , and participants were instructed to measure their waists while standing relaxed at the navel and their hips at the largest circumference , including the buttocks in 1987 ( hpfs ) ( 17 ) . self - reported anthropometric measures were validated against technician measurements among a subset of participants residing in the boston area ( 18 ) . crude pearson correlations between self - reported waist circumferences and the average of two technician - measured waist circumferences were 0.95 for men and 0.89 for women . similarly , correlations for hip measurements were 0.88 for men and 0.84 for women and for whrs , 0.69 for men and 0.70 for women . correlations became stronger after correcting for random within - person variability from daily or seasonal fluctuations ( 18 ) . bmi was calculated as weight in kilograms divided by the square of height in meters . information about diet and lifestyle factors ( e.g. , smoking status , alcohol intake ) , menopausal status and postmenopausal hormone therapy ( women only ) , and medications was derived from the baseline questionnaires ( 8,9 ) . for men , physical activity was expressed as metabolic equivalents per week by using the reported time spent doing various activities , weighting each activity by its intensity level . for women , physical activity was expressed as hours per week because met hours were not measured at baseline in the nhs . the healthy eating index , which measures adherence to the u.s . department of agriculture dietary guidelines for americans and mypyramid , was calculated among men and women ( 19 ) . the validity of the self - reported body weight and physical activity data has been described previously ( 18,20,21 ) . self - reported and measured weights were highly correlated at 0.97 for men and 0.97 for women ( 18 ) . snp genotyping and imputation have been described in detail elsewhere ( the nhs and hpfs t2d gwa scans ) ( 11 ) . in brief , samples were genotyped and analyzed using the affymetrix genome - wide human array 6.0 ( santa clara , ca ) and the birdseed calling algorithm . we used mach ( http://www.sph.umich.edu/csg/abecasis/mach ) to impute snps on chromosomes 122 , with national center for biotechnology information build 36 of phase ii hapmap ceu data ( release 22 ) as the reference panel . to estimate the genetic predisposition to central obesity , a genetic score was calculated on the basis of the well - established snps in 14 loci ( supplementary table 1 ) for the whr reported by a meta - analysis of gwas ( 7 ) . we assumed that each snp in the panel acts independently in an additive manner , and the genetic score was calculated by using a weighted method . each snp was weighted by its relative effect size ( -coefficient ) obtained from the reported meta - analysis data ( 7 ) . using the same method for the previously reported bmi genetic score ( 2224 ) , we first created a weighted score using the following equation : weighted score = 1 snp1 + 2 snp2 + + n snpn , where is the -coefficient for each individual snp , and n is number of snps . to reflect the number of whr - increasing alleles , we rescaled the weighted score using the following equation : weighted genetic risk score ( grs ) = weighted score ( total number of snps / sum of the -coefficients ) . in addition , a bmi genetic score based on 32 snps was calculated using a similar method to that described previously ( 2224 ) . whr genetic score distribution and its association with whr in the nhs and hpfs cohorts . the histograms represent the percentage of participants , and the mean ( se ) whrs are plotted with the trend lines across the grs . the slope of the trend line represents the correction between ghr and whr in control participants . comparisons of proportions and means between case and control participants were calculated by and t tests . we used logistic regression to estimate odds ratios ( ors ) for t2d risk , adjusting for age and bmi . to examine the accumulative effect of the genetic score , we compared the t2d risk across the quartiles of the genetic score . in multivariate analysis , we further adjusted for smoking ( never , past , or current ) , alcohol intake ( 0 , 0.14.9 , 5.09.9 , 10.014.9 , or 15.0 g / day ) , menopausal hormone therapy use ( never , past , or current [ women only ] ) , healthy eating index , and physical activity ( quintiles ) . bmi genetic score and whr were further adjusted for association with whr genetic score and risk of t2d . results in women and men were pooled by using inverse variance weights under a fixed model because there was no heterogeneity . a restricted cubic spline regression model was used to test linear relation between the genetic score ( as a continuous variable ) and risk of t2d ( 25 ) . we further examined the genetic association with risk of t2d according to joint classification of bmi - grs and whr - grs in which both variables were classified into three categories ( tertiles ) . all reported p values are nominal and two sided , and p 0.05 was considered statistically significant . the study had 80% power to detect an association with an or of 1.03 for risk of t2d at a significance level of 0.05 . statistical analyses were performed in sas version 9.3 software ( sas institute inc . , cary , nc ) . participants for the current study were selected among those with a blood sample , using a nested case - control study design ( 10,11 ) . diabetes cases were defined as self - reported diabetes confirmed by a validated supplementary questionnaire ( 12,13 ) . for cases before 1998 , we used the national diabetes data group criteria to define diabetes ( 14 ) , which included one of the following : one or more classic symptoms ( excessive thirst , polyuria , weight loss , hunger , pruritus , or coma ) plus a fasting plasma glucose level of 7.8 mmol / l ( 140 mg / dl ) , a random plasma glucose level of 11.1 mmol / l ( 200 mg / dl ) , or a plasma glucose level 2 h after an oral glucose tolerance test of 11.1 mmol / l ( 200 mg / dl ) ; at least two elevated plasma glucose levels on different occasions in the absence of symptoms ; or treatment with hypoglycemia medication ( insulin or oral hypoglycemic agent ) . we used the american diabetes association diagnostic criteria for diabetes diagnosis since 1998 ( 15 ) . these criteria were the same as those proposed by the national diabetes data group except for the elevated fasting plasma glucose criterion for which the cut point was changed from 7.8 mmol / l ( 140 mg / dl ) to 7.0 mmol / l ( 126 mg / dl ) . the validity of diabetes self - report was verified in a subsample from the hpfs and the nhs cohorts . a physician blinded to the information reported on the supplementary questionnaire reviewed the medical records according to the diagnostic criteria . ninety - seven percent of the diabetes cases were confirmed in the hpfs cohort ( 13 ) . similarly , 98% of the diabetes cases reported by the supplementary questionnaire were confirmed by medical record review in a subsample of nhs participants ( 12 ) . control participants were defined as those free of diabetes at the time of the diagnosis of cases and remained unaffected through follow - up until 2006 . we matched the case participants to control participants by age , month and year of blood draw , and fasting status within their respective cohorts . after applying a quality control filter in the nhs and hpfs t2d gwas ( 11 ) , duplicate samples , samples with misidentified sex , related samples ( siblings or possible first cousins ) , samples with evidence of contamination , samples with highly variable intensity data , and samples with missing call rates 2% were excluded . the population structure was investigated by principal component analysis ( 16 ) . in this analysis , participants clustered with the hapmap ceu ( utah residents with northern and western european ancestry ) samples were genetically inferred to have european ancestry . a total of 3,221 women ( 1,467 case and 1,754 control participants ) and 2,422 men ( 1,124 case and 1,298 control participants ) of genetically inferred european ancestry were included in the current analysis . waist and hip circumference were reported to the nearest one - quarter inch in 1986 ( nhs ) , and participants were instructed to measure their waists while standing relaxed at the navel and their hips at the largest circumference , including the buttocks in 1987 ( hpfs ) ( 17 ) . each supplemental questionnaire included a tape measure and detailed instructions , including a diagram . self - reported anthropometric measures were validated against technician measurements among a subset of participants residing in the boston area ( 18 ) . crude pearson correlations between self - reported waist circumferences and the average of two technician - measured waist circumferences were 0.95 for men and 0.89 for women . similarly , correlations for hip measurements were 0.88 for men and 0.84 for women and for whrs , 0.69 for men and 0.70 for women . correlations became stronger after correcting for random within - person variability from daily or seasonal fluctuations ( 18 ) . bmi was calculated as weight in kilograms divided by the square of height in meters . information about diet and lifestyle factors ( e.g. , smoking status , alcohol intake ) , menopausal status and postmenopausal hormone therapy ( women only ) , and medications was derived from the baseline questionnaires ( 8,9 ) . for men , physical activity was expressed as metabolic equivalents per week by using the reported time spent doing various activities , weighting each activity by its intensity level . for women , physical activity was expressed as hours per week because met hours were not measured at baseline in the nhs . the healthy eating index , which measures adherence to the u.s . department of agriculture dietary guidelines for americans and mypyramid , was calculated among men and women ( 19 ) . the validity of the self - reported body weight and physical activity data has been described previously ( 18,20,21 ) . self - reported and measured weights were highly correlated at 0.97 for men and 0.97 for women ( 18 ) . snp genotyping and imputation have been described in detail elsewhere ( the nhs and hpfs t2d gwa scans ) ( 11 ) . in brief , samples were genotyped and analyzed using the affymetrix genome - wide human array 6.0 ( santa clara , ca ) and the birdseed calling algorithm . we used mach ( http://www.sph.umich.edu/csg/abecasis/mach ) to impute snps on chromosomes 122 , with national center for biotechnology information build 36 of phase ii hapmap ceu data ( release 22 ) as the reference panel . to estimate the genetic predisposition to central obesity , a genetic score was calculated on the basis of the well - established snps in 14 loci ( supplementary table 1 ) for the whr reported by a meta - analysis of gwas ( 7 ) . we assumed that each snp in the panel acts independently in an additive manner , and the genetic score was calculated by using a weighted method . each snp was weighted by its relative effect size ( -coefficient ) obtained from the reported meta - analysis data ( 7 ) . using the same method for the previously reported bmi genetic score ( 2224 ) , we first created a weighted score using the following equation : weighted score = 1 snp1 + 2 snp2 + + n snpn , where is the -coefficient for each individual snp , and n is number of snps . to reflect the number of whr - increasing alleles , we rescaled the weighted score using the following equation : weighted genetic risk score ( grs ) = weighted score ( total number of snps / sum of the -coefficients ) . in addition , a bmi genetic score based on 32 snps was calculated using a similar method to that described previously ( 2224 ) . whr genetic score distribution and its association with whr in the nhs and hpfs cohorts . the histograms represent the percentage of participants , and the mean ( se ) whrs are plotted with the trend lines across the grs . the slope of the trend line represents the correction between ghr and whr in control participants . comparisons of proportions and means between case and control participants were calculated by and t tests . we used logistic regression to estimate odds ratios ( ors ) for t2d risk , adjusting for age and bmi . to examine the accumulative effect of the genetic score , we compared the t2d risk across the quartiles of the genetic score . in multivariate analysis , we further adjusted for smoking ( never , past , or current ) , alcohol intake ( 0 , 0.14.9 , 5.09.9 , 10.014.9 , or 15.0 g / day ) , menopausal hormone therapy use ( never , past , or current [ women only ] ) , healthy eating index , and physical activity ( quintiles ) . bmi genetic score and whr were further adjusted for association with whr genetic score and risk of t2d . results in women and men were pooled by using inverse variance weights under a fixed model because there was no heterogeneity . a restricted cubic spline regression model was used to test linear relation between the genetic score ( as a continuous variable ) and risk of t2d ( 25 ) . we further examined the genetic association with risk of t2d according to joint classification of bmi - grs and whr - grs in which both variables were classified into three categories ( tertiles ) . all reported p values are nominal and two sided , and p 0.05 was considered statistically significant . the study had 80% power to detect an association with an or of 1.03 for risk of t2d at a significance level of 0.05 . statistical analyses were performed in sas version 9.3 software ( sas institute inc . , cary , nc ) . table 1 shows the baseline characteristics of participants of two nested case - control studies from the nhs ( women ) and hpfs ( men ) . participants with t2d had a significantly higher bmi and lower physical activity level and were more likely to smoke and have a family history of diabetes than participants without t2d . female participants with t2d consumed less alcohol and were more likely to be postmenopausal than those without diabetes . in addition , the genetic score was not associated with age , bmi , or lifestyle factors , including smoking , alcohol intake , and physical activity ( all p > 0.05 ) . the mean genetic scores among men and women were 14.39 2.4 , and 14.53 2.3 , respectively . the range of genetic scores among men and women was 6.2623.01 and 5.3322.32 , respectively . the genetic score was significantly associated with the whr among men ( 0.005 ) and marginally related to the whr among women ( p = 0.06 ) ( fig . baseline characteristics among 2,422 men in hpfs and 3,221 women in nhs data are mean sd , % , or n ( % ) . as shown in table 2 , the central obesity genetic score was significantly associated with an increased t2d risk in women ( or 1.03 [ 95% ci 1.001.06 ] per 1-point genetic score increase ) and men ( 1.03 [ 1.001.07 ] ) . multivariate adjustment for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index showed a significant association among men ( p = 0.02 ) but a borderline of significance among women ( p = 0.08 ) . the pooled or for t2d was 1.03 ( 1.011.05 ) per 1-point genetic score increase , adjusting for age and bmi . the ors for t2d increased across the quartiles of the genetic score ( p for trend = 0.014 ) . compared with those in the lowest quartile of the genetic score , participants in the highest quartile had an or of 1.22 ( 1.021.42 ) . multivariate adjustment for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index did not change the association . further adjustment of the bmi genetic score did not significantly change the results ( p = 0.01 ) , whereas the association was abolished after further adjustment for the whr ( p = 0.12 ) . association between the genetic predisposition score of the whr and t2d risk * adjusted for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index . results of men and women were combined using inverse variance weights under a fixed model because no heterogeneity existed between women and men ( all p for heterogeneity > 0.17 ) . we further examined whether the association between the genetic score and t2d risk varied across subgroups stratified by bmi and lifestyle risk factors for t2d ( supplementary table 2 ) . although the associations appeared to be more pronounced in participants with a higher bmi and lower physical activity and who consumed modest levels of alcohol and currently smoked , no significant interaction between the genetic score and these risk factors in the combined samples of men and women was found ( all p for interaction > 0.16 ) . results were similar in both sexes when analyses were performed in men and women separately ( data not shown ) . the central obesity genetic predisposition score showed a linear relationship with increasing t2d risk ( p for linearity = 0.006 in the combined samples ) ( supplementary fig . we found that the grs for bmi was significantly associated with risk of t2d in both the nhs ( pcontinuous = 0.01 ) and the hpfs ( pcontinuous = 0.02 ) cohorts ( fig . we further examined the joint effects of bmi - grs and whr - grs on risk of t2d ( fig . , the risk of t2d was increased by 23% , 47% , and 46% within subgroups defined by increasing tertiles of whr - grs . among individuals with the lowest tertile bmi - grs , the or for t2d was 0% , 0% , and 39% within subgroups defined by increasing tertiles of whr - grs . data are pooled from women and men and adjusted for age , genotype data source , family history of diabetes , smoking , alcohol intake , menopausal hormone therapy use ( women only ) , healthy eating index , and total energy intake . data are pooled from women and men and adjusted for age , genotype data source , family history of diabetes , smoking , alcohol intake , menopausal hormone therapy use ( women only ) , healthy eating index , and total energy intake . table 1 shows the baseline characteristics of participants of two nested case - control studies from the nhs ( women ) and hpfs ( men ) . participants with t2d had a significantly higher bmi and lower physical activity level and were more likely to smoke and have a family history of diabetes than participants without t2d . female participants with t2d consumed less alcohol and were more likely to be postmenopausal than those without diabetes . in addition , the genetic score was not associated with age , bmi , or lifestyle factors , including smoking , alcohol intake , and physical activity ( all p > 0.05 ) . the mean genetic scores among men and women were 14.39 2.4 , and 14.53 2.3 , respectively . the range of genetic scores among men and women was 6.2623.01 and 5.3322.32 , respectively . the genetic score was significantly associated with the whr among men ( 0.005 ) and marginally related to the whr among women ( p = 0.06 ) ( fig . baseline characteristics among 2,422 men in hpfs and 3,221 women in nhs data are mean sd , % , or n ( % ) . as shown in table 2 , the central obesity genetic score was significantly associated with an increased t2d risk in women ( or 1.03 [ 95% ci 1.001.06 ] per 1-point genetic score increase ) and men ( 1.03 [ 1.001.07 ] ) . multivariate adjustment for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index showed a significant association among men ( p = 0.02 ) but a borderline of significance among women ( p = 0.08 ) . the pooled or for t2d was 1.03 ( 1.011.05 ) per 1-point genetic score increase , adjusting for age and bmi . the ors for t2d increased across the quartiles of the genetic score ( p for trend = 0.014 ) . compared with those in the lowest quartile of the genetic score , participants in the highest quartile had an or of 1.22 ( 1.021.42 ) . multivariate adjustment for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index did not change the association . further adjustment of the bmi genetic score did not significantly change the results ( p = 0.01 ) , whereas the association was abolished after further adjustment for the whr ( p = 0.12 ) . association between the genetic predisposition score of the whr and t2d risk * adjusted for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index . results of men and women were combined using inverse variance weights under a fixed model because no heterogeneity existed between women and men ( all p for heterogeneity > 0.17 ) . we further examined whether the association between the genetic score and t2d risk varied across subgroups stratified by bmi and lifestyle risk factors for t2d ( supplementary table 2 ) . although the associations appeared to be more pronounced in participants with a higher bmi and lower physical activity and who consumed modest levels of alcohol and currently smoked , no significant interaction between the genetic score and these risk factors in the combined samples of men and women was found ( all p for interaction > 0.16 ) . results were similar in both sexes when analyses were performed in men and women separately ( data not shown ) . the central obesity genetic predisposition score showed a linear relationship with increasing t2d risk ( p for linearity = 0.006 in the combined samples ) ( supplementary fig . we found that the grs for bmi was significantly associated with risk of t2d in both the nhs ( pcontinuous = 0.01 ) and the hpfs ( pcontinuous = 0.02 ) cohorts ( fig . we further examined the joint effects of bmi - grs and whr - grs on risk of t2d ( fig . , the risk of t2d was increased by 23% , 47% , and 46% within subgroups defined by increasing tertiles of whr - grs . among individuals with the lowest tertile bmi - grs , the or for t2d was 0% , 0% , and 39% within subgroups defined by increasing tertiles of whr - grs . data are pooled from women and men and adjusted for age , genotype data source , family history of diabetes , smoking , alcohol intake , menopausal hormone therapy use ( women only ) , healthy eating index , and total energy intake . data are pooled from women and men and adjusted for age , genotype data source , family history of diabetes , smoking , alcohol intake , menopausal hormone therapy use ( women only ) , healthy eating index , and total energy intake . in two well - established , prospective , nested case - control studies of u.s . women and men , we examined the association between a genetic score comprising 14 independent central obesity associated variants and risk of t2d . the results indicate that the genetic predisposition to central obesity was significantly associated with an increased t2d risk independent of bmi , dietary , and lifestyle risk factors . consistent with our previous analyses ( 22,26 ) , we estimated a genetic score to evaluate the overall susceptibility to central obesity based on 14 well - established whr - predisposing variants identified from gwas . the current study shows robust associations between the central obesity genetic score and risk of t2d in pooled results . although the genetic association with t2d was weaker in women than in men , there was no significant sex difference . of note , the findings may partly support the potential causal relationship between central obesity and t2d risk . because genetic variants are randomly assigned and generally uncorrelated with environmental factors , the observed association between the genetic score and t2d is free of risk for reverse causation and less likely to be affected by confounding ( 2729 ) . the current findings provide consistent evidence from two cohorts to show associations between the whr genetic score and risk of t2d . the visceral depots of fat most likely contribute to the creation of insulin resistance , with additional effects of elevated fatty acids from central fat depots ( 3 ) . the higher visceral adipose tissue - to - subcutaneous adipose tissue ratio , a measure of relative body fat distribution , is associated with higher dyslipidemia , insulin resistance , and prevalence of diabetes independent of overall obesity and absolute visceral fat mass ( 4 ) . therefore , the waist circumference and whr have been described as superior measures in predicting diabetes risk in the diabetes prevention program ( 30 ) . the potential causal relation between central obesity and diabetes is also supported by evidence from randomized clinical trials . for example , an intervention study showed that twice - weekly progressive resistance training significantly decreases abdominal fat and improves insulin sensitivity and glycemia in older men with t2d ( 31 ) . a shift of fat distribution from visceral to adipose depots after pioglitazone treatment has been associated with improvements in hepatic and peripheral tissue sensitivity to insulin ( 32 ) . in the stratified analysis by lifestyle risk factors for t2d , the associations of whr genetic score and risk of t2d appeared to be more pronounced in participants who had low physical activity and modest alcohol intake and who currently smoked , although there was no significant interaction between the genetic score and these risk factors . these results suggest that lifestyle factors might modify the genetic association with risk of t2d . the current findings are in line with a previous study showing that genetic risk for t2d modifies the overall protective effect of physical activity on t2d ( 33 ) . furthermore , the joint effects of bmi and whr genetic scores on t2d suggest that a high bmi genetic score might accentuate the whr genetic effect on risk of t2d . of note , the current results highlight the importance in considering the gene - environment interaction when studying the risk factors for diabetes . the major strengths of this study are the prospective design , high - quality genetic data , and minimal population stratification ( 11 ) . although the central obesity genetic score captured the combined information from most of the established genetic variants for the whr , these variants only explained 4% variation of the whr ( 7 ) . first , although this nested case - control study was conducted in well - established prospective cohorts , some degree of measurement error in self - reported waist and hip circumferences and covariates is inevitable . however , the self - reported waist and hip circumferences were validated with high correlation , and we confirmed the association between genetic score and whr in this study . second , residual and unmeasured confounding from other lifestyle behaviors or factors is still possible . third , although the meta - analysis of gwas for whr showed that most individual snps had a much stronger genetic association with whr in women than in men , the study found a weak association of whr genetic score with whr and t2d among women . the reason for this discrepancy might be partly due to population variation , which was also observed in the original gwas meta - analysis and warrants further investigation . therefore , further investigations in other ethnic groups are needed to validate the findings . in conclusion , the findings indicate that the genetic predisposition to central adiposity , as estimated by the whr genetic score , is associated with a higher risk of t2d among women and men from two prospective cohorts .

objectiveabdominal obesity is a major risk factor for type 2 diabetes ( t2d ) . we aimed to examine the association between the genetic predisposition to central obesity , assessed by the waist - to - hip ratio ( whr ) genetic score , and t2d risk.research design and methodsthe current study included 2,591 participants with t2d and 3,052 participants without t2d of european ancestry from the nurses health study ( nhs ) and the health professionals follow - up study ( hpfs ) . genetic predisposition to central obesity was estimated using a genetic score based on 14 established loci for the whr.resultswe found that the central obesity genetic score was linearly related to higher t2d risk . results were similar in the nhs ( women ) and hpfs ( men ) . in combined results , each point of the central obesity genetic score was associated with an odds ratio ( or ) of 1.04 ( 95% ci 1.011.07 ) for developing t2d , and the or was 1.24 ( 1.031.45 ) when comparing extreme quartiles of the genetic score after multivariate adjustment.conclusionsthe data indicate that genetic predisposition to central obesity is associated with higher t2d risk . this association is mediated by central obesity .

Introduction Research Design and Methods Ascertainment of T2D Assessment of WHR and Covariates Genotyping Genetic Score Calculation Statistical Analysis Results Characteristics of the Participants at Baseline Central Obesity Genetic Score and T2D Stratified Analyses by Lifestyle Risk Factors Linear Relationship Between Genetic Predisposition Score and Risk of T2D The Joint Effects of BMI-GRS and WHR-GRS on Risk of T2D Conclusions

the dramatic increase in the global incidence of obesity has been accompanied by an increase in incident type 2 diabetes ( t2d ) ( 1 ) . anthropometric measures capturing abdominal adiposity , such as waist circumference or waist - to - hip ratio ( whr ) , have been related to diabetes risk independent of bmi across various ethnic groups ( 25 ) . a meta - analysis of the whr genome - wide association studies ( gwas ) comprising > 100,000 individuals of european ancestry established a comprehensive genetic profile for modulating body fat distribution independent of overall adiposity ( 7 ) . information on the association of genetic predisposition to central obesity with risk of t2d is limited . in the current study , we calculated the genetic score based on 14 well - established single nucleotide polymorphisms ( snps ) for whr ( 7 ) as a proxy of genetic predisposition to central obesity . we examined the association of this genetic score with t2d risk in women and men of european ancestry from two prospective cohorts : the nurses health study ( nhs ) and the health professionals follow - up study ( hpfs ) . both studies were approved by the human research committee at the brigham and women s hospital ( boston , ma ) , and all participants provided written informed consent . participants for the current study were selected among those with a blood sample , using a nested case - control study design ( 10,11 ) . for cases before 1998 , we used the national diabetes data group criteria to define diabetes ( 14 ) , which included one of the following : one or more classic symptoms ( excessive thirst , polyuria , weight loss , hunger , pruritus , or coma ) plus a fasting plasma glucose level of 7.8 mmol / l ( 140 mg / dl ) , a random plasma glucose level of 11.1 mmol / l ( 200 mg / dl ) , or a plasma glucose level 2 h after an oral glucose tolerance test of 11.1 mmol / l ( 200 mg / dl ) ; at least two elevated plasma glucose levels on different occasions in the absence of symptoms ; or treatment with hypoglycemia medication ( insulin or oral hypoglycemic agent ) . the validity of diabetes self - report was verified in a subsample from the hpfs and the nhs cohorts . ninety - seven percent of the diabetes cases were confirmed in the hpfs cohort ( 13 ) . similarly , 98% of the diabetes cases reported by the supplementary questionnaire were confirmed by medical record review in a subsample of nhs participants ( 12 ) . control participants were defined as those free of diabetes at the time of the diagnosis of cases and remained unaffected through follow - up until 2006 . after applying a quality control filter in the nhs and hpfs t2d gwas ( 11 ) , duplicate samples , samples with misidentified sex , related samples ( siblings or possible first cousins ) , samples with evidence of contamination , samples with highly variable intensity data , and samples with missing call rates 2% were excluded . a total of 3,221 women ( 1,467 case and 1,754 control participants ) and 2,422 men ( 1,124 case and 1,298 control participants ) of genetically inferred european ancestry were included in the current analysis . waist and hip circumference were reported to the nearest one - quarter inch in 1986 ( nhs ) , and participants were instructed to measure their waists while standing relaxed at the navel and their hips at the largest circumference , including the buttocks in 1987 ( hpfs ) ( 17 ) . , smoking status , alcohol intake ) , menopausal status and postmenopausal hormone therapy ( women only ) , and medications was derived from the baseline questionnaires ( 8,9 ) . for women , physical activity was expressed as hours per week because met hours were not measured at baseline in the nhs . in brief , samples were genotyped and analyzed using the affymetrix genome - wide human array 6.0 ( santa clara , ca ) and the birdseed calling algorithm . to estimate the genetic predisposition to central obesity , a genetic score was calculated on the basis of the well - established snps in 14 loci ( supplementary table 1 ) for the whr reported by a meta - analysis of gwas ( 7 ) . we assumed that each snp in the panel acts independently in an additive manner , and the genetic score was calculated by using a weighted method . using the same method for the previously reported bmi genetic score ( 2224 ) , we first created a weighted score using the following equation : weighted score = 1 snp1 + 2 snp2 + + n snpn , where is the -coefficient for each individual snp , and n is number of snps . in addition , a bmi genetic score based on 32 snps was calculated using a similar method to that described previously ( 2224 ) . whr genetic score distribution and its association with whr in the nhs and hpfs cohorts . we used logistic regression to estimate odds ratios ( ors ) for t2d risk , adjusting for age and bmi . to examine the accumulative effect of the genetic score , we compared the t2d risk across the quartiles of the genetic score . a restricted cubic spline regression model was used to test linear relation between the genetic score ( as a continuous variable ) and risk of t2d ( 25 ) . the study had 80% power to detect an association with an or of 1.03 for risk of t2d at a significance level of 0.05 . participants for the current study were selected among those with a blood sample , using a nested case - control study design ( 10,11 ) . for cases before 1998 , we used the national diabetes data group criteria to define diabetes ( 14 ) , which included one of the following : one or more classic symptoms ( excessive thirst , polyuria , weight loss , hunger , pruritus , or coma ) plus a fasting plasma glucose level of 7.8 mmol / l ( 140 mg / dl ) , a random plasma glucose level of 11.1 mmol / l ( 200 mg / dl ) , or a plasma glucose level 2 h after an oral glucose tolerance test of 11.1 mmol / l ( 200 mg / dl ) ; at least two elevated plasma glucose levels on different occasions in the absence of symptoms ; or treatment with hypoglycemia medication ( insulin or oral hypoglycemic agent ) . these criteria were the same as those proposed by the national diabetes data group except for the elevated fasting plasma glucose criterion for which the cut point was changed from 7.8 mmol / l ( 140 mg / dl ) to 7.0 mmol / l ( 126 mg / dl ) . the validity of diabetes self - report was verified in a subsample from the hpfs and the nhs cohorts . similarly , 98% of the diabetes cases reported by the supplementary questionnaire were confirmed by medical record review in a subsample of nhs participants ( 12 ) . control participants were defined as those free of diabetes at the time of the diagnosis of cases and remained unaffected through follow - up until 2006 . after applying a quality control filter in the nhs and hpfs t2d gwas ( 11 ) , duplicate samples , samples with misidentified sex , related samples ( siblings or possible first cousins ) , samples with evidence of contamination , samples with highly variable intensity data , and samples with missing call rates 2% were excluded . a total of 3,221 women ( 1,467 case and 1,754 control participants ) and 2,422 men ( 1,124 case and 1,298 control participants ) of genetically inferred european ancestry were included in the current analysis . waist and hip circumference were reported to the nearest one - quarter inch in 1986 ( nhs ) , and participants were instructed to measure their waists while standing relaxed at the navel and their hips at the largest circumference , including the buttocks in 1987 ( hpfs ) ( 17 ) . , smoking status , alcohol intake ) , menopausal status and postmenopausal hormone therapy ( women only ) , and medications was derived from the baseline questionnaires ( 8,9 ) . for women , physical activity was expressed as hours per week because met hours were not measured at baseline in the nhs . snp genotyping and imputation have been described in detail elsewhere ( the nhs and hpfs t2d gwa scans ) ( 11 ) . in brief , samples were genotyped and analyzed using the affymetrix genome - wide human array 6.0 ( santa clara , ca ) and the birdseed calling algorithm . to estimate the genetic predisposition to central obesity , a genetic score was calculated on the basis of the well - established snps in 14 loci ( supplementary table 1 ) for the whr reported by a meta - analysis of gwas ( 7 ) . we assumed that each snp in the panel acts independently in an additive manner , and the genetic score was calculated by using a weighted method . using the same method for the previously reported bmi genetic score ( 2224 ) , we first created a weighted score using the following equation : weighted score = 1 snp1 + 2 snp2 + + n snpn , where is the -coefficient for each individual snp , and n is number of snps . in addition , a bmi genetic score based on 32 snps was calculated using a similar method to that described previously ( 2224 ) . whr genetic score distribution and its association with whr in the nhs and hpfs cohorts . the histograms represent the percentage of participants , and the mean ( se ) whrs are plotted with the trend lines across the grs . to examine the accumulative effect of the genetic score , we compared the t2d risk across the quartiles of the genetic score . a restricted cubic spline regression model was used to test linear relation between the genetic score ( as a continuous variable ) and risk of t2d ( 25 ) . table 1 shows the baseline characteristics of participants of two nested case - control studies from the nhs ( women ) and hpfs ( men ) . participants with t2d had a significantly higher bmi and lower physical activity level and were more likely to smoke and have a family history of diabetes than participants without t2d . female participants with t2d consumed less alcohol and were more likely to be postmenopausal than those without diabetes . in addition , the genetic score was not associated with age , bmi , or lifestyle factors , including smoking , alcohol intake , and physical activity ( all p > 0.05 ) . the genetic score was significantly associated with the whr among men ( 0.005 ) and marginally related to the whr among women ( p = 0.06 ) ( fig . as shown in table 2 , the central obesity genetic score was significantly associated with an increased t2d risk in women ( or 1.03 [ 95% ci 1.001.06 ] per 1-point genetic score increase ) and men ( 1.03 [ 1.001.07 ] ) . multivariate adjustment for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index showed a significant association among men ( p = 0.02 ) but a borderline of significance among women ( p = 0.08 ) . the pooled or for t2d was 1.03 ( 1.011.05 ) per 1-point genetic score increase , adjusting for age and bmi . the ors for t2d increased across the quartiles of the genetic score ( p for trend = 0.014 ) . compared with those in the lowest quartile of the genetic score , participants in the highest quartile had an or of 1.22 ( 1.021.42 ) . multivariate adjustment for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index did not change the association . further adjustment of the bmi genetic score did not significantly change the results ( p = 0.01 ) , whereas the association was abolished after further adjustment for the whr ( p = 0.12 ) . association between the genetic predisposition score of the whr and t2d risk * adjusted for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index . we further examined whether the association between the genetic score and t2d risk varied across subgroups stratified by bmi and lifestyle risk factors for t2d ( supplementary table 2 ) . although the associations appeared to be more pronounced in participants with a higher bmi and lower physical activity and who consumed modest levels of alcohol and currently smoked , no significant interaction between the genetic score and these risk factors in the combined samples of men and women was found ( all p for interaction > 0.16 ) . results were similar in both sexes when analyses were performed in men and women separately ( data not shown ) . the central obesity genetic predisposition score showed a linear relationship with increasing t2d risk ( p for linearity = 0.006 in the combined samples ) ( supplementary fig . we found that the grs for bmi was significantly associated with risk of t2d in both the nhs ( pcontinuous = 0.01 ) and the hpfs ( pcontinuous = 0.02 ) cohorts ( fig . data are pooled from women and men and adjusted for age , genotype data source , family history of diabetes , smoking , alcohol intake , menopausal hormone therapy use ( women only ) , healthy eating index , and total energy intake . data are pooled from women and men and adjusted for age , genotype data source , family history of diabetes , smoking , alcohol intake , menopausal hormone therapy use ( women only ) , healthy eating index , and total energy intake . table 1 shows the baseline characteristics of participants of two nested case - control studies from the nhs ( women ) and hpfs ( men ) . participants with t2d had a significantly higher bmi and lower physical activity level and were more likely to smoke and have a family history of diabetes than participants without t2d . in addition , the genetic score was not associated with age , bmi , or lifestyle factors , including smoking , alcohol intake , and physical activity ( all p > 0.05 ) . the genetic score was significantly associated with the whr among men ( 0.005 ) and marginally related to the whr among women ( p = 0.06 ) ( fig . as shown in table 2 , the central obesity genetic score was significantly associated with an increased t2d risk in women ( or 1.03 [ 95% ci 1.001.06 ] per 1-point genetic score increase ) and men ( 1.03 [ 1.001.07 ] ) . multivariate adjustment for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index showed a significant association among men ( p = 0.02 ) but a borderline of significance among women ( p = 0.08 ) . the pooled or for t2d was 1.03 ( 1.011.05 ) per 1-point genetic score increase , adjusting for age and bmi . the ors for t2d increased across the quartiles of the genetic score ( p for trend = 0.014 ) . compared with those in the lowest quartile of the genetic score , participants in the highest quartile had an or of 1.22 ( 1.021.42 ) . multivariate adjustment for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index did not change the association . further adjustment of the bmi genetic score did not significantly change the results ( p = 0.01 ) , whereas the association was abolished after further adjustment for the whr ( p = 0.12 ) . association between the genetic predisposition score of the whr and t2d risk * adjusted for age , family history of diabetes , smoking , menopausal hormone therapy use ( women only ) , physical activity , alcohol intake , and healthy eating index . we further examined whether the association between the genetic score and t2d risk varied across subgroups stratified by bmi and lifestyle risk factors for t2d ( supplementary table 2 ) . although the associations appeared to be more pronounced in participants with a higher bmi and lower physical activity and who consumed modest levels of alcohol and currently smoked , no significant interaction between the genetic score and these risk factors in the combined samples of men and women was found ( all p for interaction > 0.16 ) . results were similar in both sexes when analyses were performed in men and women separately ( data not shown ) . the central obesity genetic predisposition score showed a linear relationship with increasing t2d risk ( p for linearity = 0.006 in the combined samples ) ( supplementary fig . we found that the grs for bmi was significantly associated with risk of t2d in both the nhs ( pcontinuous = 0.01 ) and the hpfs ( pcontinuous = 0.02 ) cohorts ( fig . data are pooled from women and men and adjusted for age , genotype data source , family history of diabetes , smoking , alcohol intake , menopausal hormone therapy use ( women only ) , healthy eating index , and total energy intake . data are pooled from women and men and adjusted for age , genotype data source , family history of diabetes , smoking , alcohol intake , menopausal hormone therapy use ( women only ) , healthy eating index , and total energy intake . women and men , we examined the association between a genetic score comprising 14 independent central obesity associated variants and risk of t2d . the results indicate that the genetic predisposition to central obesity was significantly associated with an increased t2d risk independent of bmi , dietary , and lifestyle risk factors . consistent with our previous analyses ( 22,26 ) , we estimated a genetic score to evaluate the overall susceptibility to central obesity based on 14 well - established whr - predisposing variants identified from gwas . the current study shows robust associations between the central obesity genetic score and risk of t2d in pooled results . of note , the findings may partly support the potential causal relationship between central obesity and t2d risk . because genetic variants are randomly assigned and generally uncorrelated with environmental factors , the observed association between the genetic score and t2d is free of risk for reverse causation and less likely to be affected by confounding ( 2729 ) . the current findings provide consistent evidence from two cohorts to show associations between the whr genetic score and risk of t2d . the higher visceral adipose tissue - to - subcutaneous adipose tissue ratio , a measure of relative body fat distribution , is associated with higher dyslipidemia , insulin resistance , and prevalence of diabetes independent of overall obesity and absolute visceral fat mass ( 4 ) . in the stratified analysis by lifestyle risk factors for t2d , the associations of whr genetic score and risk of t2d appeared to be more pronounced in participants who had low physical activity and modest alcohol intake and who currently smoked , although there was no significant interaction between the genetic score and these risk factors . although the central obesity genetic score captured the combined information from most of the established genetic variants for the whr , these variants only explained 4% variation of the whr ( 7 ) . however , the self - reported waist and hip circumferences were validated with high correlation , and we confirmed the association between genetic score and whr in this study . in conclusion , the findings indicate that the genetic predisposition to central adiposity , as estimated by the whr genetic score , is associated with a higher risk of t2d among women and men from two prospective cohorts .

chikungunya virus ( chikv ) is a positive - strand rna virus belonging to the genus alphavirus ( 1 , 2 ) . transmitted by the aedes mosquito , while chikungunya fever ( chikf ) outbreaks were previously restricted to tropical regions , the range of chikv has since expanded to temperate countries such as italy ( 6 ) and france ( 7 ) . more recently , outbreaks of chikf have been reported in the caribbean islands ( 8 , 9 ) . since the first autochthonous case reported in december 2013 , a total of 1,155,354 cases have been recorded as of 30 january 2015 ( 810 ) , with more than 300 imported cases in the united states ( 11 ) . the hallmark feature in chikf patients is incapacitating arthralgia affecting multiple joints during the acute phase of disease ( 12 ) . however , it is not uncommon for chikf patients to experience persistent joint pain that can last up to several months or years after the initial infection ( 13 , 14 ) . previous studies have suggested that the pathogenesis of chikf is an immediate result of excessive immune attacks ( 13 , 15 ) . although reports have shown the involvement of t cells in chikv immunopathology ( 1620 ) , the fine mechanism remains undefined . regulatory t cells ( tregs ) are a distinct subset of cd4 t cells that prevent exacerbated proinflammatory responses ( 21 , 22 ) by maintaining tolerance and restoring immune homeostasis during an inflammatory response ( 23 ) . in various infection models , tregs have been reported to dampen excessive immune responses triggered by pathogens and minimize damage to the host ( 2426 ) . therefore , elucidating the intriguing role of tregs would no doubt allow a further understanding of chikv immunopathogenesis . studies in the chikv adult mouse model ( 27 ) have shown that cd4 mice experienced a reduced chikv - induced joint pathology , despite no differences in viremia throughout the course of disease ( 16 ) , demonstrating a role for cd4 effector t ( teff ) cells . since these cells are a prime target for tregs , it is crucial to further define how these cells could influence the course of disease by inhibiting pathology ( 16 , 23 , 28 ) . in this study , we took advantage of the observation that mouse tregs can be selectively expanded in vivo through the use of interleukin 2 ( il-2 ) in complex with the anti - il-2 antibody jes6 - 1 ( il-2 ab cx ) ( 29 ) to demonstrate the importance of tregs in regulating chikv pathogenesis . all animals were handled in strict accordance with good animal practice as defined by the national advisory committee for laboratory animal research ( naclar ) guidelines under in facilities licensed by the agri - food and veterinary authority of singapore ( ava ) . three - week - old c57/bl6j mice were housed in the absl3 facility at the biological resource center ( brc ) at biopolis , singapore . animals are fed daily and monitored closely by technical officers in charge of the animal facilities . all studies were reviewed and approved by the institutional animal care and use committee ( iacuc approval no . chikv isolate ( chikv - sgp011 ) used for in vitro and in vivo infections in mouse studies was isolated from an outbreak in singapore in 2008 at the national university hospital and propagated in c6/36 ( 30 , 31 ) . the titer of sgp011 was determined using standard plaque assays with vero - e6 cells ( 30 , 31 ) . three - week - old wild - type ( wt ) c57/bl6 female mice were inoculated subcutaneously ( s.c . ) in the ventral side of the right hind footpad with 10 pfu chikv in 30 l phosphate - buffered saline ( pbs ) . footpad joint swelling was determined daily by the measurement of the height and the breadth of the footpad using a caliper and quantified as height times breadth . the degree of inflammation was expressed as the increase relative to the preinfection measurement ( day 0 ) , obtained with the following formula : [ ( x day 0)/day 0 ] , where x is the footpad size measurement for a given day postinfection ( dpi ) ( 16 , 3133 ) . viral rna was extracted using a qiaamp viral rna minikit ( qiagen ) and quantified using quantitect probe rt - pcr ( qiagen ) with conditions as previously described ( 16 , 3134 ) . all reactions were performed using a 7900ht fast real - time pcr system machine ( applied biosciences ) with thermal cycling conditions as described previously ( 16 , 3133 ) . for total rna extraction from tissues , mice were anesthetized with ketamine ( 150 mg / kg)-xylazine ( 10 mg / kg ) and perfused with pbs ( 33 ) . joint footpads ( the ankle joint and footpad ) were obtained and stored in trizol ( invitrogen ) at 80c . tissues were homogenized using a rotor - stator homogenizer ( xiril dispomix ) at 4,000 rpm for 15 s. homogenized tissues were mixed with 230 l of chloroform and centrifuged at 12,000 rpm for 10 min at 4c . the aqueous phase was collected and isolated for total rna using an rneasy minikit ( qiagen ) according to the manufacturer 's instructions . quantification of extracted total mrna was measured by nanodrop 1000 spectrophotometer ( thermo scientific ) . qualitative real - time pcr ( qrt - pcr ) was performed using a kapa sybr fast one - step qrt - pcr kit ( kapa biosystems ) according to the manufacturer 's recommendations in a 10-l reaction volume . all reactions were performed using a 7900ht fast real - time pcr system machine ( applied biosciences ) . thermal cycling conditions were as follows : 95c for 5 min followed by 40 cycles of 95c for 5 s , 60c for 30 s , and 95c for 15 min . the fold change for each gene was calculated relative to the value for noninfected ( ni ) mice and presented as 2 . pbs , 1.5 g murine il-2 ( peprotech ) , 50 g anti - mouse il-2 ( jes6 - 1 ) ( ebioscience ) , or 1.5 g murine il-2 complexed with 50 g anti - mouse il-2 were injected intrperitoneally ( i.p . ) fifteen microliters of blood were collected from the tail vein to check for treg expansion . briefly , blood was lysed and fixed using bd fluorescence - activated cell sorting ( facs ) lysing buffer ( bd biosciences ) and washed twice with pbs . cells were stained with anti - cd4 ( bd biosciences ) and anti - cd25 ( ebioscience ) . intracellular staining of foxp3 was done using a foxp3 staining buffer set ( ebioscience ) and anti - foxp3 following the manufacturer 's instructions . dead cells and duplets were excluded in all analyses using forward and side scatter gating . results were analyzed with flowjo version x software ( tree star , inc . ) . mice were anesthetized with ketamine ( 150 mg / kg)-xylazine ( 10 mg / kg ) and perfused by intracardiac injection with pbs followed by 4% paraformaldehyde . tissues were stored in 4% paraformaldehyde , decalcified , and embedded in paraffin wax before 5-m - thick sections were cut . hematoxylin and eosin ( h&e ) staining was done using established protocols as previously described ( 16 , 33 ) . imagej software ( national institutes of health ) was used for binary image conversion of histological images and for particle analysis ( average number of particles per 30,000 square pixels of area selected ) to determine the number of cellular infiltrates ( 35 ) . depletion of regulatory t cell ( dereg ) ( 36 ) mice were treated with il-2 ab cx for 3 days , following which 0.5 g of diphtheria toxin ( dt ) was administered daily to each mouse for 2 days . treg depletion was checked by taking 10 l of blood from the tail vein , staining it with antibodies against cd4 , cd25 , and foxp3 , and analyzing the result with flow cytometry . mice were then infected with chikv - sgp011 s.c . , and depletion of tregs was maintained by dt administration every other day . spleens , popliteal lymph nodes ( pln ) , and joint footpads of chikv - infected mice were harvested at 6 dpi . isolation of splenocytes and joint footpad cells was carried out as previously described ( 16 ) . pln cells were isolated by first incubating pln in digestion medium containing 2 u / ml dispase ( invitrogen ) , 20 mg / ml collagenase iv ( sigma - aldrich ) and 50 mg / ml dnase i ( roche applied science ) mix in complete rpmi for 30 min . cells were then released from the digested pln into the medium by gentle pipetting motions . cell suspensions were then passed through a 70-m nylon cloth ( sefar ) , washed , and resuspended in complete rpmi . isolated cells from spleens , joint footpads , and pln were first stained with a live / dead fixable aqua dead cell staining kit ( 1:400 ) ( life technologies ) for 20 min . cell were then washed and resuspended in 50 l blocking buffer ( 1% rat and hamster serum in pbs ) . staining was performed using anti - cd45 ( bd pharmingen ) , anti - cd4 ( bd pharmingen ) , anti - cd8 ( ebioscience ) , anti - cd25 ( ebioscience ) , anti - cd11b ( ebioscience ) , anti - ly6c ( ebioscience ) , and anti - ly6 g ( biolegend ) antibodies for 20 min . stained cells were then washed with pbs and fixed using ic fixation buffer ( ebioscience ) . intracellular staining of anti - foxp3 ( ebioscience ) was done according to the manufacturer 's instructions . polyvinylidene difluoride ( pvdf ) membrane plates ( millipore ) were humidified with 35% ethanol and washed with water . wells were coated with anti - gamma interferon ( ifn- ) capture antibody ( clone an18 ; mabtech ) overnight at 4c . mice were sacrificed at 6 dpi , and joint footpad and pln cells were isolated as described above . splenocytes , joint footpad cells , and pln cells were added at 2 10 , 2.5 10 , and 5 10 cells per well , respectively . ex vivo cd4 enrichment was performed using a mouse cd4 t cell isolation kit ii ( miltenyi biotec ) according to the manufacturer 's instructions . stimulation of chikv - specific t cells was done in complete rpmi containing 30 u / ml il-2 and 1.5 10 veroe6-derived sgp011 virions per well . for all wells containing joint footpad cells , 1.5 10 splenocytes from ni mice were added to serve as antigen - presenting cells ( apcs ) . the plates were incubated at 37c and 5% co2 for 18 h. after incubation , cells were removed and wells were washed six times using pbs . spot detection was done using a mouse ifn- enzyme - linked immunospot ( elispot ) kit with alkaline phosphatase ( alp ) ( mabtech ) following the manufacturer 's instructions . ex vivo cd4 enrichment was performed using an easysep mouse cd4 cell enrichment kit ( stemcell ) according to the manufacturer 's instructions . enriched cd4 t cells were then labeled with 2.5 m carboxyfluorescein succinimidyl ester ( cfse for 10 min at 37c in the dark . labeled cells were plated at 1 10 cells / well and stimulated with either anti - cd3/cd28 dynabeads ( life technologies ) or cd4-depleted splenocytes from naive mice pulsed with 1.5 10 sgp011 virions per well . all analyses between the pbs - chikv , il-2-chikv , jes6 - 1-chikv , and il-2 ab cx - chikv groups were done using one - way analysis of variance ( anova ) followed by dunnett 's multiple - comparison test comparing all other groups to the pbs - chikv group . comparison between il-2 ab cx with treg depletion and il-2 ab cx without depletion were done using an unpaired t test . comparison between particle analysis of the pbs - chikv and il-2 ab cx - chikv groups was done using a mann - whitney u test . in order to assess if tregs have a role in chikv - induced pathology , we made use of an in vivo method that allows the selective expansion of tregs by administering il-2 to form a complex with the anti - il-2 antibody jes6 - 1 , termed il-2 ab cx ( 29 ) . pbs , il-2 only , or jes6 - 1 only was administered to mice prior to chikv infection as an experimental control ( referred to as infected control groups ) . a significant increase in the proportion of circulating tregs was observed only with il-2 ab cx treatment ( fig . this treatment was highly effective , as it increased the average frequency of tregs in the blood from less than 10% to nearly 70% ( fig . furthermore , the expanded tregs were also activated , as indicated by high cd25 expression ( fig . the impact of treg expansion on chikv infection was next explored in mice pretreated with pbs ( control ) , il-2 , jes6 - 1 , or il-2 ab cx for 3 days followed by virus inoculation into the footpad of the hind limb ( 27 ) . compared to noninfected ( ni ) mice , chikv infection resulted in substantial swelling of the joint that peaked at 6 days postinfection ( dpi ) ( fig . differences were not observed within the control groups of chikv - infected mice ( fig . however , a marked reduction in swelling of the joint footpad was observed in mice pretreated with il-2 ab cx , especially at 6 dpi ( fig . notably , these mice also recovered faster , with undetectable levels of swelling by 8 dpi , than the other control groups , where swelling subsided only after 14 dpi ( fig . pretreatment with il-2 ab cx reduces joint swelling in chikv - infected mice . wt mice ( n = 5 per group ) were i.p . injected with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx daily for 3 days . ( a ) representative scatter plots ( left ) and bar chart of the foxp3 cd25 peripheral treg population in mice . numbers indicate the frequency of foxp3 cd25 tregs in total peripheral cd4 cells . following pbs , il-2 only , jes6 - 1 only , or il-2 ab cx treatment , the mice were infected s.c . with 10 pfu chikv - sgp011 . ( b ) representative images showing joint footpad swelling at 6 dpi in chikv - infected mice . ( d ) viremia was determined from blood collected from tail vein from 1 to 14 dpi . ( e ) viral load of the infected joint footpad was measured at 6 dpi . all data are means and standard deviations ( sd ) and are representative of three independent experiments . statistical analysis was done across all chikv - infected groups using one - way anova , followed by dunnett 's posttest comparing each group to the pbs - chikv group . ( f ) histological analysis of chikv - infected footpad samples from 6 dpi pretreated with either pbs or il-2 ab cx and stained with h&e . * , edema ; arrows , infiltrates ; b , bone ; m , muscle ; t , tendon . statistical analysis was carried out using the mann - whitney two - tailed test . * * , p = 0.0017 ( 2 dpi joint swelling ) or 0.0059 ( 4 dpi joint swelling ) ; * * * , p < 0.0001 ( 3 , 5 , 7 , 8 , 9 , 10 , 11 , 12 , 14 , and 15 dpi joint swelling and il-2 ab cx treg expansion ) or 0.0004 ( 6 dpi joint swelling ) . the action of tregs on reducing joint pathology did not have any effect on virus clearance , as differences in viral load were not observed between il-2 ab cx - treated mice and chikv - infected control groups ( fig . 1d and e ) . tissue sections of infected joint footpad were obtained for histological assessments , where pbs - treated chikv - infected mice exhibited typical necrotizing myositis with massive immune infiltrates as well as extensive edema ( 18 , 37 ) in the loose connective tissue layer of the dermis layer ( fig . however , treatment with il-2 ab cx markedly reduced edema within the dermis layer ( fig . to ascertain that the reduced pathology observed in the il-2 ab cx - treated mice was caused by the suppressive activity of tregs and not due to a direct impact of the il-2 ab cx on the effector cells , dereg mice were explored ( 36 ) . these mice express diphtheria toxin receptor ( dtr ) under the control of foxp3 promoter , which allows the specific ablation of tregs with dt administration ( fig . treatment with il-2 ab cx protected the chikv - infected dereg mice from pronounced joint swelling ( fig . however , this protection was completely lost upon depletion of tregs with dt ( fig . joint swelling was comparable in dt - treated wt chikv - infected mice and in dt - treated dereg mice with il-2 ab cx treatment ( fig . 2b ) , verifying that the protection against chikv - induced swelling is mediated primarily by tregs ( fig . treg depletion in il-2 ab cx - treated mice results in a loss of protection against chikv - induced pathology . wt or dereg mice ( n = 4 per group ) were treated with il-2 ab cx for 3 days ( 5 , 4 , and 3 dpi ) followed by dt administration for 2 days to deplete tregs . following depletion , mice were infected s.c . with 10 pfu chikv - sgp011 . ( a ) representative scatter plot showing peripheral treg population from dereg animals with or without il-2 ab cx treatment , followed by dt or no dt treatment . numbers in scatter plots indicate the treg population percentage within total cd4 t cells . data are means and sd . statistical analysis was done using one - tailed unpaired t test . * * , p = 0.001 ( wt+dt versus dereg+il-2 ab cx+dt ) ; * * * , p < 0.0001 ( dereg+il-2 ab cx+dt versus dereg+il-2 ab cx ) . ( b ) joint swelling of mice was measured from 0 to 12 dpi following chikv infection after il-2 ab cx and dt treatment . statistical analysis was done using a two - tailed unpaired t test comparing dereg+il-2 ab cx+dt and dereg+il-2 ab cx . * * * , p = 0.0007 ( 6 dpi ) , < 0.0001 ( 7 dpi ) , < 0.0001 ( 8 dpi ) , 0.0004 ( 9 dpi ) , 0.0003 ( 10 dpi ) , < 0.0001 ( 11 dpi ) , or 0.0001 ( 12 dpi ) . in order to decipher how il-2 ab cx ameliorates chikv - induced joint swelling , immune infiltrates at the site of infection were analyzed from harvested joint footpad samples isolated during the peak of inflammation at 6 dpi . a significant increase in the cd45 leukocyte population was observed across all groups of chikv - infected mice ( fig . flow cytometry analysis revealed that the infiltrates consisted mainly of cd11b myeloid cells that include macrophages and neutrophils ( fig . 3b ) . while chikv infection had no effect on the number of neutrophils ( cd11b ly6 g ) ( fig . 3c ) increased dramatically . while the expansion of tregs did not impact the infiltration of macrophages induced by chikv infection , a striking selective effect was observed for cd4 t cells infiltration on 6 dpi . while il-2 and jes6 - 1 did not affect the influx of cd4 t cells into the infected footpad , their infiltration was significantly reduced with il-2 ab cx treatment ( fig . wt mice ( n = 5 per group ) were infected s.c . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . joint footpad cells from these treated animals were isolated at 6 dpi , enriched by percoll , and analyzed by flow cytometry . cells were stained with live / dead aqua and with antibodies to cd3 , cd4 , cd11b , ly6c , and ly6 g . ( a ) representative scatter plots showing cd45 and cd4 expression , gated on live cells . bar charts show average numbers of total leukocytes ( left ) and cd4 t cell infiltrates ( right ) . ( b ) representative scatter plots showing cd11b and ly6 g expression gated on live cd45 cells . numbers in scatter plots indicate the percentages of cells in respective quadrants of total live cd45 cells . bar charts show average numbers of total myeloid cells ( left ) and neutrophil infiltrates ( right ) . ( c ) representative scatter plots showing cd11b and ly6c expression gated on live cd45 cells . numbers in scatter plots indicate the percentages of cells in respective quadrants of total live cd45 cells . bar charts show average number of ly6c macrophage ( left ) and ly6c macrophage ( right ) infiltrates . all data are means and sd from 3 independent experiments . statistical analysis was performed using one - way anova across all chikv - infected groups , followed by dunnett 's posttest comparing to the pbs - chikv group . * * * , p < 0.0001 ( il-2 ab cx cd4 t cells ) . ifn- elispot analysis on cells isolated from chikv - infected joint footpad samples further revealed that the reduced infiltrating cd4 t cells correlated with a reduction of the antigen - specific cd4 t cell response ( fig . transcript analysis performed on total mrna isolated from joint footpad cells indicated that although chikv infection induced high expression of proinflammatory genes , such as those encoding il-6 , il-10 , entpd1 , ifn- , stat1 , and cxcl10 , addition of il-2 ab cx reversed this effect ( fig . 4c ) . a plausible explanation for this protective effect could be the interference by expanded tregs on the priming of chikv - specific cd4 teff cells . to verify this , ifn- elispot and flow cytometry analyses were carried out on cells isolated from the draining pln . flow cytometry detected only minor differences in the absolute number of pln cells ( fig . in contrast , ifn- elispot showed that chikv - specific stimulation yielded a high frequency of antigen - specific responses from total pln and isolated cd4 t cells in all chikv - infected groups except for the il-2 ab cx - treated group ( fig . this suggests that generation of chikv - specific cd4 teff cells is likely to be perturbed in the presence of expanded tregs . pretreatment with il-2 ab cx reduces production of proinflammatory cytokines in joint footpads of chikv - infected mice . wt mice ( n = 5 per group ) were infected s.c . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . ( a and b ) representative images of elispot wells depicting the number of ifn--producing cells in total cells ( a ) and enriched cd4 t cells ( b ) from joint footpads . bar charts show average numbers of ifn--producing cells per infected joint footpad after subtraction of the background of the il-2-only stimulation control . ( c ) mrna was extracted from joint footpads , and qrt - pcr was performed to detect the expression of il-6 , il-10 , entpd1 , ifn- , stat1 , and cxcl10 . statistical analysis was done using one - way anova across all chikv - infected groups , followed by dunnett 's posttest comparing to the pbs - chikv group . data are means and sd from three independent experiments . * , p = 0.0127 ( il-2 ab cx il-6 ) or 0.0113 ( il-2 ab cx il-10 ) ; * * , p = 0.0037 ( il-2 ab cx entpd1 ) , 0.0036 ( il-2 ab cx ifn- ) , 0.0032 ( il-2 ab cx stat1 ) , or 0.0067 ( il-2 ab cx cxcl10 ) ; * * * , p = 0.0008 ( il-2 ab cx ifn--producing cells per joint footpad ) or 0.0126 ( il-2 ab cx ifn--producing cd4 t cells per joint footpad ) . pretreatment with il-2 wt mice ( n = 5 per group ) were infected s.c . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . ( a through d ) the bar charts show cd45 t cells ( a ) , cd4 t cells ( b ) , cd4 teff cells ( c ) , and cd4 tregs ( d ) per pln . ( e and f ) representative images of elispot wells depicting the number of ifn--producing cells in total cells ( e ) and enriched cd4 t cells ( f ) from pln . the bar charts show the average numbers of ifn--producing cells per pln after subtraction of the background of the il-2-only stimulation control . statistical analysis was done using one - way anova across all chikv - infected groups , followed by dunnett 's posttest comparing to the pbs - chikv group . data are means and sd from three independent experiments . * * * , p < 0.0001 ( il-2 ab cx tregs per pln ) , 0.0008 ( il-2 ab cx ifn--producing cells per pln ) , or 0.0007 ( il-2 ab cx ifn--producing cd4 t cells per pln ) . to further address how tregs suppress the initial priming of cd4 teff cells in the pln , bromodeoxyuridine ( brdu ) was administered before the peak of joint swelling at 4 dpi and 5 dpi to detect in vivo proliferation of cd4 teff cells . about 10% of cd4 teff cells from chikv - infected pbs - treated control mice were brdu ( fig . 6a ) , suggesting that these cells were actively proliferating in response to virus infection . on the other hand , teff cells from il-2 ab cx - treated mice had significantly lower detectable levels of brdu cells ( fig . if the lack of brdu uptake was due to a disruption in the proliferation of cd4 teff cells , ki-67 staining was performed to assess cell cycle advancement ( 38 ) . draining pln from chikv - infected control mice revealed that approximately 30% of cd4 teff cells expressed ki-67 , indicating active proliferation of these lymphocytes ( fig . however , only 20% of cd4 teff cells from il-2 ab cx - treated mice expressed ki-67 ( fig . more importantly , teff cells from the il-2 ab cx - treated group showed significantly reduced ki-67 detection compared to the chikv - infected control groups ( fig . 6b ) . taken together , these findings suggest that cd4 teff cells with il-2 ab cx treatment did proliferate but did not enter s phase ( fig . wt mice ( n = 5 per group ) were infected s.c . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . ( a ) representative scatterplot of cd25 expression and brdu staining of teff cells . brdu was injected i.p . into mice daily for 2 days before euthanasia to detect proliferating cells . cells isolated from draining pln at 6 dpi were isolated for flow cytometry and gated on cd4 teff cells for analysis . ( b ) histogram showing ki-67 expression on cd4 teff cells ( left ) and bar charts showing mean fluorescence intensity ( mfi ) of ki-67 on teff cells ( middle ) and average percentage of ki-67 teff cells ( right ) . statistical analysis was done using one - way anova across all chikv - infected groups , followed by dunnett 's posttest comparing to the pbs - chikv group . p = 0.0324 ( il-2 ab cx percentage brdu teff cells ) ; * * * , p < 0.0001 ( il-2 ab cx ki-67 mfi and il-2 ab cx percentage ki-67 teff cells ) . finally , to address proliferation arrest ( 3941 ) , cd4 teff cells were isolated from pln of chikv - infected mice on 6 dpi and challenged with either chikv - pulsed apcs or anti - cd3/cd28 dynabeads . tregs were depleted using anti - cd25 positive selection , as they are known to exhibit suppressive effects during mixed lymphocyte reactions ( 42 ) . to monitor proliferation , isolated cd4 teff cells were labeled with carboxyfluorescein succinimidyl ester ( cfse ) , a tracking dye for proliferation ( 43 ) . cd4 teff cells from all groups responded to nonspecific anti - cd3/cd28 stimulation ( fig . 7 ) . while cd4 teff cells from il-2 ab cx showed a weaker proliferation that was comparable to that in the ni group ( fig . proliferation was observed only in the chikv - infected control groups , not in the il-2 ab cx - treated mice ( fig . this demonstrates that anergy induction is specific to chikv - specific cd4 teff cells and not due to a global shutdown of t cell responses . pretreatment with il-2 ab cx prevents proliferation of activated chikv - specific cd4 t cells in draining lymph nodes . wt mice ( n = 5 per group ) were infected s.c . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . splenocytes from ni mice were depleted of cd4 t cells and used as antigen - presenting cells ( apcs ) . enriched cd4 t cells were depleted of tregs , labeled with cfse , and stimulated for 4 days with medium only , anti - cd3/cd28 dynabeads , apcs only , or chikv - pulsed apcs . ( b ) representative scatter plot ( right ) showing major histocompatibility complex class ii ( mhcii ) and cd11c expression gated on total live cd45 leukocytes from pln . the boxed area corresponds to inflammatory dcs , which were further analyzed for cd80 expression , as shown in the histogram plot ( middle ) . statistical analysis was done using one - way anova across all chikv - infected groups , followed by dunnett 's posttest comparing to the pbs - chikv group . data are means and sd from two independent experiments . * * * , p = 0.0007 ( il-2 ab cx cd80 ) . flow cytometry was further performed to assess if the induction of anergy was due to a lack of costimulation on the apcs during t cell activation ( 3941 , 4447 ) . although migratory dendritic cells ( dcs ) ( cd11b cd11c mhcii ) ( 48 ) isolated from the draining pln of chikv - infected control groups revealed a typical increased in cd80 ( fig . 7b ) , treatment with il-2 ab cx resulted in a significant reduction of cd80 ( fig . these observations further substantiate the idea that tregs disrupted the response of chikv - specific cd4 teff cells by altering the priming capacity of apcs ( 4952 ) . arboviral infections have illustrated the importance of t cells in regulating disease pathology ( 5355 ) . cd8 t cells were demonstrated to mediate encephalitis during murray valley encephalitis virus ( mve ) infection ( 54 ) . cross - reactive cd4 t cells were found to exacerbate dengue hemorrhagic fever ( dhf ) during secondary infections ( 55 ) , while cd8 t cells have an ambivalent role , as they exhibit both protective and pathogenic affects in wnv - infected mice ( 56 ) . more recently , cd4 t cells were showed to be responsible in mediating chikv - induced pathology ( 16 ) . while t cell - mediated immunopathology in arbovirus infections has been extensively reported ( 54 , 55 , 57 , 58 ) , information on the role of tregs remains limited . here , aberrant immune responses responsible for the development of chikv - induced immunopathology were controlled by selectively expanding tregs in vivo through administering il-2 ab cx prior to virus infection ( 29 ) . the expansion and activation of tregs led to the effective amelioration of the characteristic chikv - induced joint swelling . the reduced inflamed joint pathology was similar to that observed in chikv - infected cd4 mice , where a decline in viremia was not affected by the absence of cd4 t cells ( 16 ) . further characterization of the action of these expanded tregs during chikv infection established that tregs selectively inhibit cd4 teff cells . ifn- elispot performed on splenocytes and cells from draining pln isolated from il-2 ab cx - treated chikv - infected mice resulted in a sharp reduction in the frequency of chikv - specific cd4 t cells . consequently , only a low number of chikv - specific cd4 t cells could migrate into infected tissues to maintain inflammation and inflict further damage . notably , the influx of other immune subsets , such as macrophages and cd8 t cells , was not affected , indicating no impairment in the overall immune response . brdu and ki-67 analysis suggested cell cycle arrest in cd4 teff cells , a hallmark feature of anergic t cells ( 5961 ) . in line with this , a reduction in the level of cd80 in migratory dcs further suggested the indirect immunosuppressive effect of tregs on teff cells via the effect of tregs on apcs ( 4952 ) . during virus infection , viral antigens are picked up by tissue - resident dcs that mature into migratory dcs and are transported to the draining ln to prime virus - specific t cells ( 62 ) . these activated t cells then migrate back to the site of infection and exacerbate the proinflammatory microenvironment . findings in this study suggest that tregs expansion perturbs this inflammatory cycle by interacting with the apcs to inhibit the upregulation of costimulatory molecules that cause induction of anergy due to incomplete t cell activation in the primed cells ( fig . proposed mechanism for reduction of chikv - induced pathology by il-2 ab cx . upon chikv infection in the joint footpad , resident sentinels such as macrophages and dcs sense the virus . virus stimulation induces activation of macrophages , which secrete proinflammatory cytokines and chemokines to attract other immune cell populations . meanwhile , dcs phagocytose viral particles and differentiate into mature migratory dcs to upregulate mhcii and costimulatory molecules . the matured dcs migrate to the draining pln and present viral antigen to naive t cells . upon interaction with their cognate antigen , naive t cells are activated into teff cells and enter the cell cycle to expand clonally . these expanded chikv - specific teff cells migrate toward the site of infection in response to secreted t cell chemokines . the presence of chikv - specific teff cells exacerbates the proinflammatory milieu and gives rise to the inflamed joint pathology observed . in the event of il-2 ab cx pretreatment , expanded tregs will be present in large number in all the secondary lymphoid organs . as the mature dcs carrying chikv antigen arrive at the draining ln , tregs compete with naive t cells to interact with these dcs . treg - dc interaction then causes mature dcs to downregulate their costimulatory molecules and results in the dcs presenting chikv antigens to naive t cells in the absence of costimulatory signals . activation of t cells without costimulation is likely to drive the t cells into g1 arrest , resulting in anergy . anergic t cells are not able to migrate to the site of infection in response to chemoattractants . abrogation of chikv - specific teff cells infiltration in the site of infection prevents exacerbation of proinflammatory environment , thus preventing the inflamed - joint pathology . the protective role of tregs in reducing immunopathology seen in this study could be further extended to other clinically important arboviruses such as o'nyong - nyong virus ( onnv ) , ross river virus ( rrv ) , and dengue virus ( denv ) ( 6365 ) as a means to prevent or reduce pathology ( 53 , 66 ) . nonetheless , observations in this study imply that pharmaceutical immunosuppressants blocking t cell activation and/or proliferation have a wealth of therapeutic potential ( 67 ) and could represent an alternative way to control virus - induced pathology during the acute phase of infection .

abstractchikungunya virus ( chikv ) infection is a reemerging pandemic human arboviral disease . cd4 + t cells were previously shown to contribute to joint inflammation in the course of chikv infection in mice . the jes6 - 1 anti - il-2 antibody selectively expands mouse regulatory t cells ( tregs ) by forming a complex with il-2 . in this study , we show that the il-2 jes6 - 1-mediated expansion of tregs ameliorates chikv - induced joint pathology . it does so by inhibiting the infiltration of cd4 + t cells due to the induction of anergy in chikv - specific cd4 + effector t cells . these findings suggest that activation of tregs could also become an alternative approach to control chikv - mediated disease . importance chikungunya virus ( chikv ) has reemerged as a pathogen of global significance . patients infected with chikv suffer from incapacitating joint pain that severely affects their daily functioning . despite the best efforts , treatment is still inadequate . while t cell - mediated immunopathology in chikv infections has been reported , the role of regulatory t cells ( tregs ) has not been explored . the jes6 - 1 anti - interleukin 2 ( il-2 ) antibody has been demonstrated to selectively expand mouse tregs by forming a complex with il-2 . we reveal here that il-2 jes6 - 1-mediated expansion of tregs ameliorates chikv - induced joint pathology in mice by neutralizing virus - specific cd4 + effector t ( teff ) cells . we show that this treatment abrogates the infiltration of pathogenic cd4 + t cells through induction of anergy in chikv - specific cd4 + teff cells . this is the first evidence where the role of tregs is demonstrated in chikv pathogenesis , and its expansion could control virus - mediated immunopathology .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

chikungunya virus ( chikv ) is a positive - strand rna virus belonging to the genus alphavirus ( 1 , 2 ) . transmitted by the aedes mosquito , while chikungunya fever ( chikf ) outbreaks were previously restricted to tropical regions , the range of chikv has since expanded to temperate countries such as italy ( 6 ) and france ( 7 ) . since the first autochthonous case reported in december 2013 , a total of 1,155,354 cases have been recorded as of 30 january 2015 ( 810 ) , with more than 300 imported cases in the united states ( 11 ) . although reports have shown the involvement of t cells in chikv immunopathology ( 1620 ) , the fine mechanism remains undefined . regulatory t cells ( tregs ) are a distinct subset of cd4 t cells that prevent exacerbated proinflammatory responses ( 21 , 22 ) by maintaining tolerance and restoring immune homeostasis during an inflammatory response ( 23 ) . therefore , elucidating the intriguing role of tregs would no doubt allow a further understanding of chikv immunopathogenesis . studies in the chikv adult mouse model ( 27 ) have shown that cd4 mice experienced a reduced chikv - induced joint pathology , despite no differences in viremia throughout the course of disease ( 16 ) , demonstrating a role for cd4 effector t ( teff ) cells . since these cells are a prime target for tregs , it is crucial to further define how these cells could influence the course of disease by inhibiting pathology ( 16 , 23 , 28 ) . in this study , we took advantage of the observation that mouse tregs can be selectively expanded in vivo through the use of interleukin 2 ( il-2 ) in complex with the anti - il-2 antibody jes6 - 1 ( il-2 ab cx ) ( 29 ) to demonstrate the importance of tregs in regulating chikv pathogenesis . chikv isolate ( chikv - sgp011 ) used for in vitro and in vivo infections in mouse studies was isolated from an outbreak in singapore in 2008 at the national university hospital and propagated in c6/36 ( 30 , 31 ) . pbs , 1.5 g murine il-2 ( peprotech ) , 50 g anti - mouse il-2 ( jes6 - 1 ) ( ebioscience ) , or 1.5 g murine il-2 complexed with 50 g anti - mouse il-2 were injected intrperitoneally ( i.p . ) depletion of regulatory t cell ( dereg ) ( 36 ) mice were treated with il-2 ab cx for 3 days , following which 0.5 g of diphtheria toxin ( dt ) was administered daily to each mouse for 2 days . mice were then infected with chikv - sgp011 s.c . spleens , popliteal lymph nodes ( pln ) , and joint footpads of chikv - infected mice were harvested at 6 dpi . staining was performed using anti - cd45 ( bd pharmingen ) , anti - cd4 ( bd pharmingen ) , anti - cd8 ( ebioscience ) , anti - cd25 ( ebioscience ) , anti - cd11b ( ebioscience ) , anti - ly6c ( ebioscience ) , and anti - ly6 g ( biolegend ) antibodies for 20 min . splenocytes , joint footpad cells , and pln cells were added at 2 10 , 2.5 10 , and 5 10 cells per well , respectively . ex vivo cd4 enrichment was performed using a mouse cd4 t cell isolation kit ii ( miltenyi biotec ) according to the manufacturer 's instructions . stimulation of chikv - specific t cells was done in complete rpmi containing 30 u / ml il-2 and 1.5 10 veroe6-derived sgp011 virions per well . enriched cd4 t cells were then labeled with 2.5 m carboxyfluorescein succinimidyl ester ( cfse for 10 min at 37c in the dark . all analyses between the pbs - chikv , il-2-chikv , jes6 - 1-chikv , and il-2 ab cx - chikv groups were done using one - way analysis of variance ( anova ) followed by dunnett 's multiple - comparison test comparing all other groups to the pbs - chikv group . in order to assess if tregs have a role in chikv - induced pathology , we made use of an in vivo method that allows the selective expansion of tregs by administering il-2 to form a complex with the anti - il-2 antibody jes6 - 1 , termed il-2 ab cx ( 29 ) . pbs , il-2 only , or jes6 - 1 only was administered to mice prior to chikv infection as an experimental control ( referred to as infected control groups ) . a significant increase in the proportion of circulating tregs was observed only with il-2 ab cx treatment ( fig . this treatment was highly effective , as it increased the average frequency of tregs in the blood from less than 10% to nearly 70% ( fig . the impact of treg expansion on chikv infection was next explored in mice pretreated with pbs ( control ) , il-2 , jes6 - 1 , or il-2 ab cx for 3 days followed by virus inoculation into the footpad of the hind limb ( 27 ) . differences were not observed within the control groups of chikv - infected mice ( fig . pretreatment with il-2 ab cx reduces joint swelling in chikv - infected mice . following pbs , il-2 only , jes6 - 1 only , or il-2 ab cx treatment , the mice were infected s.c . ( f ) histological analysis of chikv - infected footpad samples from 6 dpi pretreated with either pbs or il-2 ab cx and stained with h&e . the action of tregs on reducing joint pathology did not have any effect on virus clearance , as differences in viral load were not observed between il-2 ab cx - treated mice and chikv - infected control groups ( fig . tissue sections of infected joint footpad were obtained for histological assessments , where pbs - treated chikv - infected mice exhibited typical necrotizing myositis with massive immune infiltrates as well as extensive edema ( 18 , 37 ) in the loose connective tissue layer of the dermis layer ( fig . to ascertain that the reduced pathology observed in the il-2 ab cx - treated mice was caused by the suppressive activity of tregs and not due to a direct impact of the il-2 ab cx on the effector cells , dereg mice were explored ( 36 ) . 2b ) , verifying that the protection against chikv - induced swelling is mediated primarily by tregs ( fig . in order to decipher how il-2 ab cx ameliorates chikv - induced joint swelling , immune infiltrates at the site of infection were analyzed from harvested joint footpad samples isolated during the peak of inflammation at 6 dpi . a significant increase in the cd45 leukocyte population was observed across all groups of chikv - infected mice ( fig . while the expansion of tregs did not impact the infiltration of macrophages induced by chikv infection , a striking selective effect was observed for cd4 t cells infiltration on 6 dpi . while il-2 and jes6 - 1 did not affect the influx of cd4 t cells into the infected footpad , their infiltration was significantly reduced with il-2 ab cx treatment ( fig . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . cells were stained with live / dead aqua and with antibodies to cd3 , cd4 , cd11b , ly6c , and ly6 g . * * * , p < 0.0001 ( il-2 ab cx cd4 t cells ) . ifn- elispot analysis on cells isolated from chikv - infected joint footpad samples further revealed that the reduced infiltrating cd4 t cells correlated with a reduction of the antigen - specific cd4 t cell response ( fig . transcript analysis performed on total mrna isolated from joint footpad cells indicated that although chikv infection induced high expression of proinflammatory genes , such as those encoding il-6 , il-10 , entpd1 , ifn- , stat1 , and cxcl10 , addition of il-2 ab cx reversed this effect ( fig . a plausible explanation for this protective effect could be the interference by expanded tregs on the priming of chikv - specific cd4 teff cells . in contrast , ifn- elispot showed that chikv - specific stimulation yielded a high frequency of antigen - specific responses from total pln and isolated cd4 t cells in all chikv - infected groups except for the il-2 ab cx - treated group ( fig . this suggests that generation of chikv - specific cd4 teff cells is likely to be perturbed in the presence of expanded tregs . pretreatment with il-2 ab cx reduces production of proinflammatory cytokines in joint footpads of chikv - infected mice . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . statistical analysis was done using one - way anova across all chikv - infected groups , followed by dunnett 's posttest comparing to the pbs - chikv group . * , p = 0.0127 ( il-2 ab cx il-6 ) or 0.0113 ( il-2 ab cx il-10 ) ; * * , p = 0.0037 ( il-2 ab cx entpd1 ) , 0.0036 ( il-2 ab cx ifn- ) , 0.0032 ( il-2 ab cx stat1 ) , or 0.0067 ( il-2 ab cx cxcl10 ) ; * * * , p = 0.0008 ( il-2 ab cx ifn--producing cells per joint footpad ) or 0.0126 ( il-2 ab cx ifn--producing cd4 t cells per joint footpad ) . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . ( a through d ) the bar charts show cd45 t cells ( a ) , cd4 t cells ( b ) , cd4 teff cells ( c ) , and cd4 tregs ( d ) per pln . * * * , p < 0.0001 ( il-2 ab cx tregs per pln ) , 0.0008 ( il-2 ab cx ifn--producing cells per pln ) , or 0.0007 ( il-2 ab cx ifn--producing cd4 t cells per pln ) . to further address how tregs suppress the initial priming of cd4 teff cells in the pln , bromodeoxyuridine ( brdu ) was administered before the peak of joint swelling at 4 dpi and 5 dpi to detect in vivo proliferation of cd4 teff cells . about 10% of cd4 teff cells from chikv - infected pbs - treated control mice were brdu ( fig . on the other hand , teff cells from il-2 ab cx - treated mice had significantly lower detectable levels of brdu cells ( fig . if the lack of brdu uptake was due to a disruption in the proliferation of cd4 teff cells , ki-67 staining was performed to assess cell cycle advancement ( 38 ) . draining pln from chikv - infected control mice revealed that approximately 30% of cd4 teff cells expressed ki-67 , indicating active proliferation of these lymphocytes ( fig . more importantly , teff cells from the il-2 ab cx - treated group showed significantly reduced ki-67 detection compared to the chikv - infected control groups ( fig . taken together , these findings suggest that cd4 teff cells with il-2 ab cx treatment did proliferate but did not enter s phase ( fig . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . p = 0.0324 ( il-2 ab cx percentage brdu teff cells ) ; * * * , p < 0.0001 ( il-2 ab cx ki-67 mfi and il-2 ab cx percentage ki-67 teff cells ) . finally , to address proliferation arrest ( 3941 ) , cd4 teff cells were isolated from pln of chikv - infected mice on 6 dpi and challenged with either chikv - pulsed apcs or anti - cd3/cd28 dynabeads . to monitor proliferation , isolated cd4 teff cells were labeled with carboxyfluorescein succinimidyl ester ( cfse ) , a tracking dye for proliferation ( 43 ) . cd4 teff cells from all groups responded to nonspecific anti - cd3/cd28 stimulation ( fig . proliferation was observed only in the chikv - infected control groups , not in the il-2 ab cx - treated mice ( fig . this demonstrates that anergy induction is specific to chikv - specific cd4 teff cells and not due to a global shutdown of t cell responses . pretreatment with il-2 ab cx prevents proliferation of activated chikv - specific cd4 t cells in draining lymph nodes . with 10 pfu chikv - sgp011 after treatment with pbs , il-2 only , jes6 - 1 only , or il-2 ab cx . splenocytes from ni mice were depleted of cd4 t cells and used as antigen - presenting cells ( apcs ) . enriched cd4 t cells were depleted of tregs , labeled with cfse , and stimulated for 4 days with medium only , anti - cd3/cd28 dynabeads , apcs only , or chikv - pulsed apcs . statistical analysis was done using one - way anova across all chikv - infected groups , followed by dunnett 's posttest comparing to the pbs - chikv group . flow cytometry was further performed to assess if the induction of anergy was due to a lack of costimulation on the apcs during t cell activation ( 3941 , 4447 ) . although migratory dendritic cells ( dcs ) ( cd11b cd11c mhcii ) ( 48 ) isolated from the draining pln of chikv - infected control groups revealed a typical increased in cd80 ( fig . these observations further substantiate the idea that tregs disrupted the response of chikv - specific cd4 teff cells by altering the priming capacity of apcs ( 4952 ) . cd8 t cells were demonstrated to mediate encephalitis during murray valley encephalitis virus ( mve ) infection ( 54 ) . cross - reactive cd4 t cells were found to exacerbate dengue hemorrhagic fever ( dhf ) during secondary infections ( 55 ) , while cd8 t cells have an ambivalent role , as they exhibit both protective and pathogenic affects in wnv - infected mice ( 56 ) . more recently , cd4 t cells were showed to be responsible in mediating chikv - induced pathology ( 16 ) . while t cell - mediated immunopathology in arbovirus infections has been extensively reported ( 54 , 55 , 57 , 58 ) , information on the role of tregs remains limited . here , aberrant immune responses responsible for the development of chikv - induced immunopathology were controlled by selectively expanding tregs in vivo through administering il-2 ab cx prior to virus infection ( 29 ) . the expansion and activation of tregs led to the effective amelioration of the characteristic chikv - induced joint swelling . the reduced inflamed joint pathology was similar to that observed in chikv - infected cd4 mice , where a decline in viremia was not affected by the absence of cd4 t cells ( 16 ) . further characterization of the action of these expanded tregs during chikv infection established that tregs selectively inhibit cd4 teff cells . ifn- elispot performed on splenocytes and cells from draining pln isolated from il-2 ab cx - treated chikv - infected mice resulted in a sharp reduction in the frequency of chikv - specific cd4 t cells . consequently , only a low number of chikv - specific cd4 t cells could migrate into infected tissues to maintain inflammation and inflict further damage . notably , the influx of other immune subsets , such as macrophages and cd8 t cells , was not affected , indicating no impairment in the overall immune response . brdu and ki-67 analysis suggested cell cycle arrest in cd4 teff cells , a hallmark feature of anergic t cells ( 5961 ) . in line with this , a reduction in the level of cd80 in migratory dcs further suggested the indirect immunosuppressive effect of tregs on teff cells via the effect of tregs on apcs ( 4952 ) . during virus infection , viral antigens are picked up by tissue - resident dcs that mature into migratory dcs and are transported to the draining ln to prime virus - specific t cells ( 62 ) . these activated t cells then migrate back to the site of infection and exacerbate the proinflammatory microenvironment . findings in this study suggest that tregs expansion perturbs this inflammatory cycle by interacting with the apcs to inhibit the upregulation of costimulatory molecules that cause induction of anergy due to incomplete t cell activation in the primed cells ( fig . proposed mechanism for reduction of chikv - induced pathology by il-2 ab cx . upon chikv infection in the joint footpad , resident sentinels such as macrophages and dcs sense the virus . the matured dcs migrate to the draining pln and present viral antigen to naive t cells . these expanded chikv - specific teff cells migrate toward the site of infection in response to secreted t cell chemokines . the presence of chikv - specific teff cells exacerbates the proinflammatory milieu and gives rise to the inflamed joint pathology observed . activation of t cells without costimulation is likely to drive the t cells into g1 arrest , resulting in anergy . anergic t cells are not able to migrate to the site of infection in response to chemoattractants . abrogation of chikv - specific teff cells infiltration in the site of infection prevents exacerbation of proinflammatory environment , thus preventing the inflamed - joint pathology . the protective role of tregs in reducing immunopathology seen in this study could be further extended to other clinically important arboviruses such as o'nyong - nyong virus ( onnv ) , ross river virus ( rrv ) , and dengue virus ( denv ) ( 6365 ) as a means to prevent or reduce pathology ( 53 , 66 ) . nonetheless , observations in this study imply that pharmaceutical immunosuppressants blocking t cell activation and/or proliferation have a wealth of therapeutic potential ( 67 ) and could represent an alternative way to control virus - induced pathology during the acute phase of infection .

chromatin is the complex of dna , histories , and associated nonhistone proteins in the cell nucleus . dna wraps around histone octamers made up of two copies of histone h2a , h2b , h3 , and h4 , which then supercoil to form a highly condensed structure ( figure 1 ) . initially , it was thought that this elaborate chromatin structure only functioned to condense meters of dna into the microscopic cell nucleus , but it is now known to participate directly in gene regulation . because dna is tightly associated with histones and often embedded deep within chromatin supercoils , , cellular mechanisms exist to modify and remodel chromatin structure to allow for the coordinated expression of specific transcriptional programs and the silencing of others . such modifications typically occur on n - terminal histone tails and include acetylation , phosphorylation , methylation , or several other covalent modifications of histones , methylation of dna , and many others , with each modification either directly altering histone - dna interactions or serving as a mark that recruits specific proteins to positively or negatively regulate the underlying gene 's activity . ultimately , dozens of potential modifications that occur at many distinct histone residues summate to determine the final transcriptional output of a given gene . as mentioned earlier , genetic mutations in many of these chromatin remodeling enzymes an especially important aspect of certain chromatin modifications is their apparent stability , as is seen with genetic imprinting or x - inactivation , where dna methylation contributes to lifelong gene silencing . however , despite the apparent stability of some epigenetic mechanisms in vivo , all types of chromatin modifications identified to date are potentially reversible and have specific enzymes or processes which mediate the addition or removal of each mark . the in vivo mechanisms which maintain easily reversible histone modifications ( eg , acetylation ) on some genes or delete highly stable marks ( eg , dna methylation ) on other genes are currently not clear . acetylation of histone lysine residues reduces the electrostatic interaction between histone proteins and dna , which relaxes chromatin structure and improves access of transcriptional regulators to dna ( figure 1 ) . genome - wide studies indicate that high levels of histone acetylation in gene promoter regions are generally associated with higher gene activity , while low levels of acetylation correlating with reduced gene activity . most genome - wide studies of histone acetylation have focused on acetylation of the n - terminal lysine residues in histones h3 and h4 , but histone acetylation can occur on other histone proteins as well as in their globular domains . histone acetylation is a dynamic process , controlled by specific enzymes which either add or remove the acetyl mark . there are over a dozen known histone acetyltransferases ( hats ) which catalyze the addition of acetyl groups onto lysine residues of histones with varying degrees of specificity . many hats can also acetylate nonhistone proteins such as transcription factors ( eg , p53 ) , and some transcription factors ( eg , atf2 [ activating transcription factor 2 ] , clock ) even possess intrinsic hat activity that contributes to gene activation . , histone deacetylases ( hdacs ) , which remove acetyl groups from histones , are divided into four classes . class i hdacs ( eg , hdac1 , 2 , 3 , and 8) are ubiquitously expressed and likely mediate the majority of deacetylase activity within cells . class ii hdacs ( eg , hdac4 , 5 , 7 , 9 , 10 ) are only expressed in specific tissues such as heart and brain and are much larger enzymes that also contain an n - terminal regulatory domain that enables them to be shuttled in and out of the nucleus in a neural activitydependent manner . while class ii hdacs can deacetylate histones , they are much less efficient enzymes than class i hdacs , and may also deacetylate other cellular substrates . , there is currently one class iv hdac , hdac11 , and it has characteristics of both class i and class ii enzymes . class iii iidacs ( also referred to as sirtuins ) are mechanistically distinct from the other hdacs , and have been implicated in the regulation of lifespan and metabolism . histone phosphorylation is generally associated with transcriptional activation ; it can be observed on the promoters of immediate early genes such as c - fos when they are induced after cyclic adenosine monophosphate ( camp ) stimulation or glutamate treatment in cultured striatal neurons . , one of the best - characterized histone phosphorylation sites is serine 10 on histone h3 ( h3s10).this modification stabilizes the hat , gcn5 , on associated gene promoters while antagonizing the repressive modification - methylation of lysine 9 on histone h3 ( h3k9 ) and its subsequent recruitment of hp1 ( heterochromatin protein 1 , see below ) . since phosphorylation at h3s10 recruits a hat , the neighboring lysine residue at h3k9 is often acetylated in concert with phosphorylation , a process called phosphoacetylation that further potentiates gene activation . the mitogen - activated protein kinase , msk1 , and the dopamine and cyclic - amp regulated protein phosphatase inhibitor , darrp-32 , are elegant examples shown to regulate h3s10 phosphorylation in the adult brain in response to cocaine exposure . furthermore , genetic disruption of the histone - modifying ability of msk1 or darrp-32 in vivo has dramatic effects on behavioral responses to cocaine . thus , histone phosphorylation likely plays an important role in the regulation of brain function . histone methylation occurs on lysine residues in mono- , di- , or trimethylated states , enabling each state to recruit unique coregulators and exert distinct effects on transcriptional activity . additionally , methylation of different histone lysine residues can exert opposite effects on transcription . in gene promoter regions for example , trimethylation of it3k4 is highly associated with gene activation , whereas trimethylation of h3k9 or h3k27 is repressive . the repression caused by trimethylation of ii3k9 is mediated in part via the recruitment of corepressors , such as hp1 , as stated earlier . however , even this is an oversimplification , as methylated h3k9 is often found in the coding region downstream of a gene promoter and may be involved in transcriptional elongation . thus , histone methyiation provides each cell with exquisite control over an individual gene 's activity through numerous combinatorial possibilities . histone methyltransferases ( hmts ) add methyl groups to specific lysine residues of histones , and histone demelhylases ( hdms ) remove them ( figure 1 ) . like hats and hdacs , hmts and hdms not only discriminate between various histone lysine residues , but each enzyme is also unique in its ability to catalyze mono- , di- , or trimethylation or demethylation at that site . for example , the hmt , kmt1c ( g9a ) , is specific for histone h3k9 but only adds 1 or 2 methyl groups , with the distinct hmt , kmt1a ( suv39h1 ) , catalyzing trimethylation of this site . similarly , the hdm , kdm3a ( jhdm2a ) , can demethylate 1 or 2 methyl groups on h3k9 , requiring a distinct demethylase , eg , kdm4d ( jmjd2d ) to fully demethylate the trimethylated state . thus , large complexes of enzymes are required to move between the unmethylated and fully trimethylated states . proper balance of histone methylation has already been strongly implicated in normal brain function , as the hdm , kmt5c ( smcx ) , controls dendritic spine density and is mutated in patients with mental retardation . , dna methylation refers to the enzymatic methylation of cytosine bases , a fundamental cellular process required for development , tissue - specific gene expression , x - inactivation , and genetic imprinting , to name a few examples . dna methylation is thought to repress gene expression by interfering with the binding of transcription factors to their target sequences or by initiating the recruitment of corepressors . for example , the camp - response element ( cre ) contains a cytosine - guanine dinucleotide in the middle of its consensus sequence , which , when methylated , prevents the transcription factor cre - binding protein ( creb ) from binding . thus , for genes at which creb is necessary to initiate transcription , methylation at this site is repressive . methylated dna can also recruit methyl - binding domain - containing proteins , such as mecp2 , which can then recruit and stabilize transcriptional corepressors such as hdacs on specific gene promoters . mutations in mecp2 cause the autistic spectrum disorder , rett syndrome , illustrating the importance of dna methylation in normal brain development . while there is a strong correlation between methylated dna and repressed gene activity , recent studies of mecp2 indicate it may also serve to activate gene activity under some circumstances , suggesting that the context in which dna methylation occurs is an important factor in its ultimate effect on transcription . there are three known enzymes which catalyze dna cytosine methylation : dnmt1 , dnmt3a , and dnmt3b . together , these enzymes establish and maintain the unique methylation patterns that exist within each cell type . while the regulation of these enzymes in brain remains unclear , pharmacological inhibition of dna methylation in the brain in vivo results in rapid demethylation of specific gene targets and severe deficits in learning and memory . the mechanism by which this occurs , however , remains unclear because , unlike other chromatin modifications , the existence of dna demethylases remains controversial . nevertheless , regulation of dna methylation by environmental stimuli remains an attractive mediator of long - lasting changes in transcription in adult neurons . acetylation of histone lysine residues reduces the electrostatic interaction between histone proteins and dna , which relaxes chromatin structure and improves access of transcriptional regulators to dna ( figure 1 ) . genome - wide studies indicate that high levels of histone acetylation in gene promoter regions are generally associated with higher gene activity , while low levels of acetylation correlating with reduced gene activity . most genome - wide studies of histone acetylation have focused on acetylation of the n - terminal lysine residues in histones h3 and h4 , but histone acetylation can occur on other histone proteins as well as in their globular domains . histone acetylation is a dynamic process , controlled by specific enzymes which either add or remove the acetyl mark . there are over a dozen known histone acetyltransferases ( hats ) which catalyze the addition of acetyl groups onto lysine residues of histones with varying degrees of specificity . many hats can also acetylate nonhistone proteins such as transcription factors ( eg , p53 ) , and some transcription factors ( eg , atf2 [ activating transcription factor 2 ] , clock ) even possess intrinsic hat activity that contributes to gene activation . , histone deacetylases ( hdacs ) , which remove acetyl groups from histones , are divided into four classes . class i hdacs ( eg , hdac1 , 2 , 3 , and 8) are ubiquitously expressed and likely mediate the majority of deacetylase activity within cells . class ii hdacs ( eg , hdac4 , 5 , 7 , 9 , 10 ) are only expressed in specific tissues such as heart and brain and are much larger enzymes that also contain an n - terminal regulatory domain that enables them to be shuttled in and out of the nucleus in a neural activitydependent manner . while class ii hdacs can deacetylate histones , they are much less efficient enzymes than class i hdacs , and may also deacetylate other cellular substrates . , there is currently one class iv hdac , hdac11 , and it has characteristics of both class i and class ii enzymes . class iii iidacs ( also referred to as sirtuins ) are mechanistically distinct from the other hdacs , and have been implicated in the regulation of lifespan and metabolism . histone phosphorylation is generally associated with transcriptional activation ; it can be observed on the promoters of immediate early genes such as c - fos when they are induced after cyclic adenosine monophosphate ( camp ) stimulation or glutamate treatment in cultured striatal neurons . , one of the best - characterized histone phosphorylation sites is serine 10 on histone h3 ( h3s10).this modification stabilizes the hat , gcn5 , on associated gene promoters while antagonizing the repressive modification - methylation of lysine 9 on histone h3 ( h3k9 ) and its subsequent recruitment of hp1 ( heterochromatin protein 1 , see below ) . since phosphorylation at h3s10 recruits a hat , the neighboring lysine residue at h3k9 is often acetylated in concert with phosphorylation , a process called phosphoacetylation that further potentiates gene activation . the mitogen - activated protein kinase , msk1 , and the dopamine and cyclic - amp regulated protein phosphatase inhibitor , darrp-32 , are elegant examples shown to regulate h3s10 phosphorylation in the adult brain in response to cocaine exposure . furthermore , genetic disruption of the histone - modifying ability of msk1 or darrp-32 in vivo has dramatic effects on behavioral responses to cocaine . thus , histone phosphorylation likely plays an important role in the regulation of brain function . histone methylation occurs on lysine residues in mono- , di- , or trimethylated states , enabling each state to recruit unique coregulators and exert distinct effects on transcriptional activity . additionally , methylation of different histone lysine residues can exert opposite effects on transcription . in gene promoter regions for example , trimethylation of it3k4 is highly associated with gene activation , whereas trimethylation of h3k9 or h3k27 is repressive . the repression caused by trimethylation of ii3k9 is mediated in part via the recruitment of corepressors , such as hp1 , as stated earlier . however , even this is an oversimplification , as methylated h3k9 is often found in the coding region downstream of a gene promoter and may be involved in transcriptional elongation . thus , histone methyiation provides each cell with exquisite control over an individual gene 's activity through numerous combinatorial possibilities . histone methyltransferases ( hmts ) add methyl groups to specific lysine residues of histones , and histone demelhylases ( hdms ) remove them ( figure 1 ) . like hats and hdacs , hmts and hdms not only discriminate between various histone lysine residues , but each enzyme is also unique in its ability to catalyze mono- , di- , or trimethylation or demethylation at that site . for example , the hmt , kmt1c ( g9a ) , is specific for histone h3k9 but only adds 1 or 2 methyl groups , with the distinct hmt , kmt1a ( suv39h1 ) , catalyzing trimethylation of this site . similarly , the hdm , kdm3a ( jhdm2a ) , can demethylate 1 or 2 methyl groups on h3k9 , requiring a distinct demethylase , eg , kdm4d ( jmjd2d ) to fully demethylate the trimethylated state . thus , large complexes of enzymes are required to move between the unmethylated and fully trimethylated states . proper balance of histone methylation has already been strongly implicated in normal brain function , as the hdm , kmt5c ( smcx ) , controls dendritic spine density and is mutated in patients with mental retardation . , dna methylation refers to the enzymatic methylation of cytosine bases , a fundamental cellular process required for development , tissue - specific gene expression , x - inactivation , and genetic imprinting , to name a few examples . dna methylation is thought to repress gene expression by interfering with the binding of transcription factors to their target sequences or by initiating the recruitment of corepressors . for example , the camp - response element ( cre ) contains a cytosine - guanine dinucleotide in the middle of its consensus sequence , which , when methylated , prevents the transcription factor cre - binding protein ( creb ) from binding . thus , for genes at which creb is necessary to initiate transcription , methylation at this site is repressive . methylated dna can also recruit methyl - binding domain - containing proteins , such as mecp2 , which can then recruit and stabilize transcriptional corepressors such as hdacs on specific gene promoters . mutations in mecp2 cause the autistic spectrum disorder , rett syndrome , illustrating the importance of dna methylation in normal brain development . while there is a strong correlation between methylated dna and repressed gene activity , recent studies of mecp2 indicate it may also serve to activate gene activity under some circumstances , suggesting that the context in which dna methylation occurs is an important factor in its ultimate effect on transcription . there are three known enzymes which catalyze dna cytosine methylation : dnmt1 , dnmt3a , and dnmt3b . together , these enzymes establish and maintain the unique methylation patterns that exist within each cell type . while the regulation of these enzymes in brain remains unclear , pharmacological inhibition of dna methylation in the brain in vivo results in rapid demethylation of specific gene targets and severe deficits in learning and memory . the mechanism by which this occurs , however , remains unclear because , unlike other chromatin modifications , the existence of dna demethylases remains controversial . nevertheless , regulation of dna methylation by environmental stimuli remains an attractive mediator of long - lasting changes in transcription in adult neurons . drug addiction is a chronic relapsing disorder where motivation to seek and take drugs of abuse becomes compulsive and pathological . the process by which repeated drug experimentation transitions into a chronically addicted state is the focus of intense research , as clues into these mechanisms may help better manage or perhaps fully treat addicted patients . another avenue of intense research focuses on the mechanisms driving drug relapse , which occurs even after long periods of drug abstinence and is a major clinical challenge for successful treatment . the exciting new possibility that druginduced alterations in chromatin structure may contribute to long - lasting behavioral changes provides a new avenue for novel therapeutics that improve drug rehabilitation . the first studies to implicate changes in chromatin structure in responses to drugs of abuse found that acute administration of cocaine rapidly increased histone h4 acetylation on the immediate early genes c - fos and fosb in striatum , two genes known to play a critical role in cocaine - related behaviors . hie histone acetyltransferase cbp appears to be required for the drug - induced acetylation of the fosb promoter , and probably many other , yet to be identified genes as well . interestingly , despite several control gene promoters where acute cocaine does not affect histone acetylation , an acute cocaine dose increases total levels of histone h4 acetylation , and histone h3 phosphoacetylation in striatum , as measured by western blotting . these global increases in histone acetylation , which are also observed in response to environmental enrichment and tests of learning and memory , may be accounted for by high levels of acetylation on specific subsets of genes . this is likely , as global increases in histone k9 methylation , a repressive histone modification , are also observed after cocaine exposure and appear to occur on unique subsets of genes . the promoters of certain genes induced by chronic cocaine exposure are hyperacetylated for days to weeks after the last drug exposure ( figure 2 ) . for example , the expression of cdk5 ( cyclin - dependent kinase 5 ) , bdnf ( brain derived neurotrophic factor ) , npy ( neuropeptide y ) , and sirt1 and sirt2 ( two subtypes of sirtuins ) , among many other genes , were found to be upregulated after chronic cocaine administration and their gene promoters hyperacetylated , while egr-1 ( early growth response 1 ) was found to be downregulated and hypoacetylated after cocaine withdrawal . moreover , altered expression of each of these genes has been shown to contribute to the addiction behavioral phenotype . these findings suggest a role of histone acetylation in the maintenance of gene expression involved in drug addiction , including drug withdrawal and relapse . cocaine - induced alterations in chromatin structure in the nucleus accumbens ( nac ) , the ventral portion of striatum heavily implicated as a brain reward region , have been shown to regulate behavioral responses to drugs of abuse . pharmacological inhibition of hdacs in the nac , which increases histone acetylation in this brain region , significantly potentiates the locomotor - activating and rewarding responses to cocaine . , conversely , reducing histone acetylation by overexpressing certain hdacs , or knockdown of the hat , cbp , results in less sensitivity to cocaine . two reports have extended these findings in rat models of cocaine self - administration , where animals are trained to press levers to receive the drug . interestingly , delivery of the hdac inhibitor , sodium butyrate , potentiates drug - taking while delivery of the hdac inhibitor , trichostatin a , attenuates it . the explanation for these different observations is unclear , but it may involve experimental differences with the self - administration paradigm or the hdac inhibitor used . cocaine alters histone acetylation through many enzymes in the nac , but one particular hdac , hdac5 , responds uniquely to chronic cocaine administration , raising the interesting possibility that this hdac is involved in the behavioral transitions which occur between acute and chronic cocaine exposure ( eg , drug experimentation to compulsive drug use ) . chronic cocaine administration increases the phosphorylation of iidac5 and shuttles it out of the nucleus , permitting hyperacetylation of histones at target genes for hdacs ( figure 2 ) . this phosphorylation reaction may be mediated by ca / calmodulin - dependent protein kinase ii ( camkii ) , since ex vivo inhibition of camkii reduces the activity - induced phosphorylation of hdac5 . consistent with its regulation by cocaine , mice deficient for hdacs display normal rewarding responses to initial cocaine exposures , but become hypersensitive when treated with a chronic course of cocaine . thus , pharmacological and genetic manipulations that increase histone acetylation appear to potentiate behavioral responses to cocaine and suggest that altered histone acetylation may contribute to establishment of an addicted state . histone h3 phosphorylation and phosphoacetylation also appear to play key roles in drug - regulated behaviors . global levels of histone h3 phosphorylation at serine 10 are induced by acute cocaine in striatum , a process which requires the kinase mskl . , the function of msk1 is behaviorally important , as mice lacking this kinase have attenuated locomotor responses to cocaine . cocaine - induced inhibition of protein phosphatase-1 also plays an important role in ib phosphorylation in striatum ( figure 2 ) . dopamine d1 receptor activation alters the phosphorylation of dopamine - regulated and cyclic - amp - regulated phosphoprotein of 32kd ( darpp-32 ) at particular serine residues ; the protein then accumulates in the nucleus to inhibit protein phosphatase-1 from dephosphorylating histone h3 . the simultaneous activation of an h3 kinase and inhibition of an h3 phosphatase results in the robust increase in h3 phosphorylation after acute cocaine exposure . the genes at which histone phosphorylation is occurring in response to cocaine remain poorly defined with an exception of c - fos , where dramatic histone phosphorylation occurs in conjunction with acetylation ( phosphoacetylation ) . as mentioned earlier , global histone methylation of h3k9 is also regulated by cocaine and , in turn , alters behavioral responses to the drug . for example , inhibition of a particular h3k9 histone methyltransferase , kmt1c ( g9a ) , whose expression is regulated in the nac by chronic cocaine administration , potentiates behavioral responses to the drug . these findings are consistent with histone acetylation findings , since inhibition of h3k9 methylation would also be expected to enhance gene activity . together , these data suggest that , in general , increases in gene expression potentiate behavioral sensitivity to drugs of abuse . as well , advances are being made in identifying the individual gene promoters where chronic cocaine induces alterations in h3k9 methylation and thereby regulates gene expression in the nac . overall , these findings implicate changes in histone acetylation , phosphorylation , and methylation in mediating expression changes in specific sets of genes that are crucial for controlling behavioral responses to drugs of abuse . depression is a chronic disorder characterized by many debilitating symptoms including dysphoria , anhedonia , sleep disturbances , and weight changes . most people diagnosed with depression are prescribed some type of antidepressant medication , of which selective serotonin reuptake inhibitors ( ssris ) or mixed serotonin - norepinephrine reuptake inhibitors ( snris ) are the most common . unfortunately , less than 50% of patients exhibit a complete response to ssris , snris , or related antidepressants , thus leaving a substantial portion of depressed patients with a chronic syndrome for which few effective clinical alternatives are available . psychiatric research is thus focused on identifying new mechanisms that are involved in the pathogenesis and maintenance of depression , which may serve as novel targets for more effective therapeutics . one of the most challenging obstacles for depression research has been the development of an animal model that accurately recapitulates human depression . while no model can effectively model all aspects of human depression ( eg , suicide ) , some of the major symptoms such as anhedonia and sleep and weight disturbances , and their reversal by antidepressant treatment , can be studied in rodents . the pathogenesis of depressed - like states is typically modeled in rodents by chronic exposure to stress . one such model , chronic social defeat stress , involves the repeated exposure of an experimental mouse to a series of aggressive mice over 10 days . each day the stress begins as a brief physical encounter ( typically 5 to 10 minutes ) followed by a full day of sensory contact ( eg , smell , sight ) as the mice are separated by a screen . after 10 days of social defeat , the experimental mice develop a chronic syndrome ( lasting more than a month ) that is characterized by anhedonia , anxiety - like symptoms , weight loss , and loss of interest in social interaction . importantly , ssris or snris reverse most of these behavioral end points , making chronic social defeat stress an attractive model in which to study the molecular adaptations associated with a depressed - like state and those involved with antidepressant action . brain derived neurotrophic factor ( bdnf ) plays a critical role in the development of the social defeat phenotype and its reversal by antidepressant treatment . it was observed that bdnf in the hippocampus is downregulated for at least 1 month after chronic social defeat stress , and that chronic antidepressant treatment reversed this downregulation . a mechanism for this long - lasting regulation of gene expression was identified as methylation of h3k27 , a repressive histone modification , that remains hypermethylated on the bdnf promoter within hippocampus for at least a month after defeat stress . while chronic antidepressant treatment of mice exposed to chronic social defeat ameliorates many of the behavioral deficits and restores bdnf mrna to normal levels , h3k27 remains hypermethylated . the maintenance of h3k27 methyiation even after chronic antidepressant treatment suggests that bdnf expression might revert to a repressed state if drug administration were stopped . this novel epigenetic mechanism , which was proposed as a form of molecular scar , may describe a potential mechanism by which the symptoms of depressed patients reappear after cessation of antidepressant treatment , however , this remains speculative and further research is needed . the recovery of bdnf expression after antidepressant treatment is likely mediated by the antidepressantinduced increase in histone h3k4 methylation and h3 polyacetylation in hippocampus , which are associated with gene activation . interestingly , tranylcypromine , which inhibits monoamine oxidases and is used as an antidepressant , is actually a much stronger inhibitor of the histone h3k4 demethylase kmt1a ( formerly , lsd1 ) than it is of either monamine oxidase a or b. thus , it will be interesting to determine whether any of the antidepressant properties of tranylcypromine derive from its blockade of kmt1 a and the subsequent facilitation of h3k4 methylation . arguing against this interpretation is the knowledge that several structurally unrelated monoamine oxidase inhibitors , which have not been shown to inhibit histone demethylases , are still effective antidepressants . the increase in h3 acetylation by antidepressant treatment suggested that hdac inhibitors may also have antidepressant - like effects . indeed , in both the chronic social defeat model and in the forced swim test , hdac inhibitors demonstrated antidepressant - like prosperities . this was especially apparent when an i i d ac inhibitor was administered in addition to an ssri , fluoxetine . while these inhibitors target numerous hdac5 , one specific isoform , hdac5 , stood out because it was oppositely regulated by stress and antidepressant treatment . indeed , overexpression of hdac5 in the hippocampus blocks the behavioral effects of chronic antidepressant treatment , suggesting that increased histone acetylation on the bdnf promoter is a key mechanism to overcome the repressive effects of h3k27 methylation . another intriguing aspect of chronic social defeat stress is that the severity of the depression - like phenotype varies within a cohort of inbred ( ie , virtually genetically identical ) mice . it was observed that mice susceptible to defeat stress show significantly higher firing rates of dopaminergic neurons in the ventral tegmental area ( vta ) after stress exposure compared with resilient mice . these resilient mice had normal vta firing rates because of a stress - induced upregulation of potassium channels in this brain region . why do certain mice upregulate protective potassium channels in the vta while others fail to do this and become depressed ? perhaps an epigenetic mechanism is involved in altering the promoters of certain potassium channels to ultimately determine if the gene will be induced in response to chronic stress . these are important questions that may shed fundamentally new light onto an extraordinarily complex syndrome , and provide new avenues for the development of more effective antidepressants . another important epigenetic mechanism that may contribute to long - lasting changes in neural function and behavior is dna methylation . early insight into the role of dna methylation in behavior followed from studies of maternal care that clearly demonstrate an experience - dependent rather than genetic basis for how rats treat their offspring . rats that receive poor maternal care as pups grow up to become poor mothers to their pups . in addition to becoming poor mothers , these rats also develop long - lasting heightened anxiety and stress responses . meaney and colleagues identified a region of the glucocorticoid receptor ( gr ) gene , which was hypermethylated throughout adulthood in rats who received poor maternal care . treatment with an hdac inhibitor not only reduced dna methylation on the gr receptor gene but also improved anxiety and stress responses in these rats . more recently , these studies have been translated from rats into humans by studying the hippocampus of patients who committed suicide with or without a history of child abuse . in patients with a history of child abuse , it was observed that dna methylation on the gr gene promoter was significantly higher , while gr mrna expression was significantly lower than patients with no history of child abuse . while these data do not demonstrate causation , they are among the best evidence to date implicating epigenetic mechanisms in anxiety and stress and suggest that dna methylation at the gr gene promoter ( and probably other genes ) in both rats and humans may contribute to this phenomenon . taken together , these studies demonstrate that chromatin structure is an important substrate for long - lasting changes in behavioral responses to stress and antidepressant treatments . while the precise signaling mechanisms by which environmental stresses converge on chromatin are still under investigation ( eg , figure 2 ) , these early studies suggest the exciting possibility that pharmacological manipulation of chromatin remodeling pathways could be a novel approach to new antidepressant development . chromatin structure is emerging as a key substrate in the pathogenesis and maintenance of chronic psychiatric illnesses . this is important because novel therapeutics could target chromatin remodeling enzymes or chromatin itself to ultimately block or even reverse , for example , a chronically addicted or depressed state . ultimately though , the key function of chromatin remodeling is to alter the transcription or the transcriptional potential of genes which eventually affect neural function , so any study of chromatin regulation is , in theory , inexorably linked with the study of the underlying gene activity . while extremely exciting , epigenetic research in psychiatry is still in its infancy , and far more research is needed to identify both the dysregulated genes and chromatin modifications responsible for individual psychiatric diseases . fortunately , new advances in high- throughput sequencing are enabling such characterization of chromatin regulation and gene expression , genome - wide , at an incredible rate and resolution . armed with these and other new research tools , epigenetic research in psychiatry is progressing at a spectacular speed , and may soon prove to be a major avenue for novel therapeutics .

alterations in gene expression are implicated in the pathogenesis of several neuropsychiatrie disorders , including drug addiction and depression , increasing evidence indicates that changes in gene expression in neurons , in the context of animal models of addiction and depression , are mediated in part by epigenetic mechanisms that alter chromatin structure on specific gene promoters . this review discusses recent findings from behavioral , molecular , and bioinformatic approaches that are being used to understand the complex epigenetic regulation of gene expression in brain by drugs of abuse and by stress . these advances promise to open up new avenues for improved treatments of these disorders .

Epigenetic mechanisms Histone acetylation Histone phosphorylation Histone methylation DNA methylation Epigenetic mechanisms in drug addiction Epigenetic mechanisms in depression Concluding remarks

chromatin is the complex of dna , histories , and associated nonhistone proteins in the cell nucleus . dna wraps around histone octamers made up of two copies of histone h2a , h2b , h3 , and h4 , which then supercoil to form a highly condensed structure ( figure 1 ) . initially , it was thought that this elaborate chromatin structure only functioned to condense meters of dna into the microscopic cell nucleus , but it is now known to participate directly in gene regulation . because dna is tightly associated with histones and often embedded deep within chromatin supercoils , , cellular mechanisms exist to modify and remodel chromatin structure to allow for the coordinated expression of specific transcriptional programs and the silencing of others . such modifications typically occur on n - terminal histone tails and include acetylation , phosphorylation , methylation , or several other covalent modifications of histones , methylation of dna , and many others , with each modification either directly altering histone - dna interactions or serving as a mark that recruits specific proteins to positively or negatively regulate the underlying gene 's activity . as mentioned earlier , genetic mutations in many of these chromatin remodeling enzymes an especially important aspect of certain chromatin modifications is their apparent stability , as is seen with genetic imprinting or x - inactivation , where dna methylation contributes to lifelong gene silencing . however , despite the apparent stability of some epigenetic mechanisms in vivo , all types of chromatin modifications identified to date are potentially reversible and have specific enzymes or processes which mediate the addition or removal of each mark . acetylation of histone lysine residues reduces the electrostatic interaction between histone proteins and dna , which relaxes chromatin structure and improves access of transcriptional regulators to dna ( figure 1 ) . genome - wide studies indicate that high levels of histone acetylation in gene promoter regions are generally associated with higher gene activity , while low levels of acetylation correlating with reduced gene activity . many hats can also acetylate nonhistone proteins such as transcription factors ( eg , p53 ) , and some transcription factors ( eg , atf2 [ activating transcription factor 2 ] , clock ) even possess intrinsic hat activity that contributes to gene activation . , histone deacetylases ( hdacs ) , which remove acetyl groups from histones , are divided into four classes . class i hdacs ( eg , hdac1 , 2 , 3 , and 8) are ubiquitously expressed and likely mediate the majority of deacetylase activity within cells . while class ii hdacs can deacetylate histones , they are much less efficient enzymes than class i hdacs , and may also deacetylate other cellular substrates . , there is currently one class iv hdac , hdac11 , and it has characteristics of both class i and class ii enzymes . class iii iidacs ( also referred to as sirtuins ) are mechanistically distinct from the other hdacs , and have been implicated in the regulation of lifespan and metabolism . , one of the best - characterized histone phosphorylation sites is serine 10 on histone h3 ( h3s10).this modification stabilizes the hat , gcn5 , on associated gene promoters while antagonizing the repressive modification - methylation of lysine 9 on histone h3 ( h3k9 ) and its subsequent recruitment of hp1 ( heterochromatin protein 1 , see below ) . the mitogen - activated protein kinase , msk1 , and the dopamine and cyclic - amp regulated protein phosphatase inhibitor , darrp-32 , are elegant examples shown to regulate h3s10 phosphorylation in the adult brain in response to cocaine exposure . thus , histone phosphorylation likely plays an important role in the regulation of brain function . in gene promoter regions for example , trimethylation of it3k4 is highly associated with gene activation , whereas trimethylation of h3k9 or h3k27 is repressive . the repression caused by trimethylation of ii3k9 is mediated in part via the recruitment of corepressors , such as hp1 , as stated earlier . however , even this is an oversimplification , as methylated h3k9 is often found in the coding region downstream of a gene promoter and may be involved in transcriptional elongation . histone methyltransferases ( hmts ) add methyl groups to specific lysine residues of histones , and histone demelhylases ( hdms ) remove them ( figure 1 ) . proper balance of histone methylation has already been strongly implicated in normal brain function , as the hdm , kmt5c ( smcx ) , controls dendritic spine density and is mutated in patients with mental retardation . , dna methylation refers to the enzymatic methylation of cytosine bases , a fundamental cellular process required for development , tissue - specific gene expression , x - inactivation , and genetic imprinting , to name a few examples . dna methylation is thought to repress gene expression by interfering with the binding of transcription factors to their target sequences or by initiating the recruitment of corepressors . for example , the camp - response element ( cre ) contains a cytosine - guanine dinucleotide in the middle of its consensus sequence , which , when methylated , prevents the transcription factor cre - binding protein ( creb ) from binding . methylated dna can also recruit methyl - binding domain - containing proteins , such as mecp2 , which can then recruit and stabilize transcriptional corepressors such as hdacs on specific gene promoters . while there is a strong correlation between methylated dna and repressed gene activity , recent studies of mecp2 indicate it may also serve to activate gene activity under some circumstances , suggesting that the context in which dna methylation occurs is an important factor in its ultimate effect on transcription . there are three known enzymes which catalyze dna cytosine methylation : dnmt1 , dnmt3a , and dnmt3b . while the regulation of these enzymes in brain remains unclear , pharmacological inhibition of dna methylation in the brain in vivo results in rapid demethylation of specific gene targets and severe deficits in learning and memory . nevertheless , regulation of dna methylation by environmental stimuli remains an attractive mediator of long - lasting changes in transcription in adult neurons . acetylation of histone lysine residues reduces the electrostatic interaction between histone proteins and dna , which relaxes chromatin structure and improves access of transcriptional regulators to dna ( figure 1 ) . genome - wide studies indicate that high levels of histone acetylation in gene promoter regions are generally associated with higher gene activity , while low levels of acetylation correlating with reduced gene activity . many hats can also acetylate nonhistone proteins such as transcription factors ( eg , p53 ) , and some transcription factors ( eg , atf2 [ activating transcription factor 2 ] , clock ) even possess intrinsic hat activity that contributes to gene activation . , histone deacetylases ( hdacs ) , which remove acetyl groups from histones , are divided into four classes . class i hdacs ( eg , hdac1 , 2 , 3 , and 8) are ubiquitously expressed and likely mediate the majority of deacetylase activity within cells . , there is currently one class iv hdac , hdac11 , and it has characteristics of both class i and class ii enzymes . class iii iidacs ( also referred to as sirtuins ) are mechanistically distinct from the other hdacs , and have been implicated in the regulation of lifespan and metabolism . , one of the best - characterized histone phosphorylation sites is serine 10 on histone h3 ( h3s10).this modification stabilizes the hat , gcn5 , on associated gene promoters while antagonizing the repressive modification - methylation of lysine 9 on histone h3 ( h3k9 ) and its subsequent recruitment of hp1 ( heterochromatin protein 1 , see below ) . the mitogen - activated protein kinase , msk1 , and the dopamine and cyclic - amp regulated protein phosphatase inhibitor , darrp-32 , are elegant examples shown to regulate h3s10 phosphorylation in the adult brain in response to cocaine exposure . thus , histone phosphorylation likely plays an important role in the regulation of brain function . in gene promoter regions for example , trimethylation of it3k4 is highly associated with gene activation , whereas trimethylation of h3k9 or h3k27 is repressive . the repression caused by trimethylation of ii3k9 is mediated in part via the recruitment of corepressors , such as hp1 , as stated earlier . however , even this is an oversimplification , as methylated h3k9 is often found in the coding region downstream of a gene promoter and may be involved in transcriptional elongation . histone methyltransferases ( hmts ) add methyl groups to specific lysine residues of histones , and histone demelhylases ( hdms ) remove them ( figure 1 ) . proper balance of histone methylation has already been strongly implicated in normal brain function , as the hdm , kmt5c ( smcx ) , controls dendritic spine density and is mutated in patients with mental retardation . , dna methylation refers to the enzymatic methylation of cytosine bases , a fundamental cellular process required for development , tissue - specific gene expression , x - inactivation , and genetic imprinting , to name a few examples . for example , the camp - response element ( cre ) contains a cytosine - guanine dinucleotide in the middle of its consensus sequence , which , when methylated , prevents the transcription factor cre - binding protein ( creb ) from binding . methylated dna can also recruit methyl - binding domain - containing proteins , such as mecp2 , which can then recruit and stabilize transcriptional corepressors such as hdacs on specific gene promoters . while there is a strong correlation between methylated dna and repressed gene activity , recent studies of mecp2 indicate it may also serve to activate gene activity under some circumstances , suggesting that the context in which dna methylation occurs is an important factor in its ultimate effect on transcription . there are three known enzymes which catalyze dna cytosine methylation : dnmt1 , dnmt3a , and dnmt3b . while the regulation of these enzymes in brain remains unclear , pharmacological inhibition of dna methylation in the brain in vivo results in rapid demethylation of specific gene targets and severe deficits in learning and memory . nevertheless , regulation of dna methylation by environmental stimuli remains an attractive mediator of long - lasting changes in transcription in adult neurons . drug addiction is a chronic relapsing disorder where motivation to seek and take drugs of abuse becomes compulsive and pathological . the exciting new possibility that druginduced alterations in chromatin structure may contribute to long - lasting behavioral changes provides a new avenue for novel therapeutics that improve drug rehabilitation . the first studies to implicate changes in chromatin structure in responses to drugs of abuse found that acute administration of cocaine rapidly increased histone h4 acetylation on the immediate early genes c - fos and fosb in striatum , two genes known to play a critical role in cocaine - related behaviors . hie histone acetyltransferase cbp appears to be required for the drug - induced acetylation of the fosb promoter , and probably many other , yet to be identified genes as well . interestingly , despite several control gene promoters where acute cocaine does not affect histone acetylation , an acute cocaine dose increases total levels of histone h4 acetylation , and histone h3 phosphoacetylation in striatum , as measured by western blotting . these global increases in histone acetylation , which are also observed in response to environmental enrichment and tests of learning and memory , may be accounted for by high levels of acetylation on specific subsets of genes . this is likely , as global increases in histone k9 methylation , a repressive histone modification , are also observed after cocaine exposure and appear to occur on unique subsets of genes . for example , the expression of cdk5 ( cyclin - dependent kinase 5 ) , bdnf ( brain derived neurotrophic factor ) , npy ( neuropeptide y ) , and sirt1 and sirt2 ( two subtypes of sirtuins ) , among many other genes , were found to be upregulated after chronic cocaine administration and their gene promoters hyperacetylated , while egr-1 ( early growth response 1 ) was found to be downregulated and hypoacetylated after cocaine withdrawal . moreover , altered expression of each of these genes has been shown to contribute to the addiction behavioral phenotype . these findings suggest a role of histone acetylation in the maintenance of gene expression involved in drug addiction , including drug withdrawal and relapse . cocaine - induced alterations in chromatin structure in the nucleus accumbens ( nac ) , the ventral portion of striatum heavily implicated as a brain reward region , have been shown to regulate behavioral responses to drugs of abuse . pharmacological inhibition of hdacs in the nac , which increases histone acetylation in this brain region , significantly potentiates the locomotor - activating and rewarding responses to cocaine . two reports have extended these findings in rat models of cocaine self - administration , where animals are trained to press levers to receive the drug . cocaine alters histone acetylation through many enzymes in the nac , but one particular hdac , hdac5 , responds uniquely to chronic cocaine administration , raising the interesting possibility that this hdac is involved in the behavioral transitions which occur between acute and chronic cocaine exposure ( eg , drug experimentation to compulsive drug use ) . dopamine d1 receptor activation alters the phosphorylation of dopamine - regulated and cyclic - amp - regulated phosphoprotein of 32kd ( darpp-32 ) at particular serine residues ; the protein then accumulates in the nucleus to inhibit protein phosphatase-1 from dephosphorylating histone h3 . the simultaneous activation of an h3 kinase and inhibition of an h3 phosphatase results in the robust increase in h3 phosphorylation after acute cocaine exposure . as mentioned earlier , global histone methylation of h3k9 is also regulated by cocaine and , in turn , alters behavioral responses to the drug . for example , inhibition of a particular h3k9 histone methyltransferase , kmt1c ( g9a ) , whose expression is regulated in the nac by chronic cocaine administration , potentiates behavioral responses to the drug . together , these data suggest that , in general , increases in gene expression potentiate behavioral sensitivity to drugs of abuse . as well , advances are being made in identifying the individual gene promoters where chronic cocaine induces alterations in h3k9 methylation and thereby regulates gene expression in the nac . overall , these findings implicate changes in histone acetylation , phosphorylation , and methylation in mediating expression changes in specific sets of genes that are crucial for controlling behavioral responses to drugs of abuse . depression is a chronic disorder characterized by many debilitating symptoms including dysphoria , anhedonia , sleep disturbances , and weight changes . psychiatric research is thus focused on identifying new mechanisms that are involved in the pathogenesis and maintenance of depression , which may serve as novel targets for more effective therapeutics . while no model can effectively model all aspects of human depression ( eg , suicide ) , some of the major symptoms such as anhedonia and sleep and weight disturbances , and their reversal by antidepressant treatment , can be studied in rodents . the pathogenesis of depressed - like states is typically modeled in rodents by chronic exposure to stress . after 10 days of social defeat , the experimental mice develop a chronic syndrome ( lasting more than a month ) that is characterized by anhedonia , anxiety - like symptoms , weight loss , and loss of interest in social interaction . importantly , ssris or snris reverse most of these behavioral end points , making chronic social defeat stress an attractive model in which to study the molecular adaptations associated with a depressed - like state and those involved with antidepressant action . brain derived neurotrophic factor ( bdnf ) plays a critical role in the development of the social defeat phenotype and its reversal by antidepressant treatment . it was observed that bdnf in the hippocampus is downregulated for at least 1 month after chronic social defeat stress , and that chronic antidepressant treatment reversed this downregulation . a mechanism for this long - lasting regulation of gene expression was identified as methylation of h3k27 , a repressive histone modification , that remains hypermethylated on the bdnf promoter within hippocampus for at least a month after defeat stress . arguing against this interpretation is the knowledge that several structurally unrelated monoamine oxidase inhibitors , which have not been shown to inhibit histone demethylases , are still effective antidepressants . indeed , in both the chronic social defeat model and in the forced swim test , hdac inhibitors demonstrated antidepressant - like prosperities . while these inhibitors target numerous hdac5 , one specific isoform , hdac5 , stood out because it was oppositely regulated by stress and antidepressant treatment . indeed , overexpression of hdac5 in the hippocampus blocks the behavioral effects of chronic antidepressant treatment , suggesting that increased histone acetylation on the bdnf promoter is a key mechanism to overcome the repressive effects of h3k27 methylation . it was observed that mice susceptible to defeat stress show significantly higher firing rates of dopaminergic neurons in the ventral tegmental area ( vta ) after stress exposure compared with resilient mice . why do certain mice upregulate protective potassium channels in the vta while others fail to do this and become depressed ? these are important questions that may shed fundamentally new light onto an extraordinarily complex syndrome , and provide new avenues for the development of more effective antidepressants . another important epigenetic mechanism that may contribute to long - lasting changes in neural function and behavior is dna methylation . while these data do not demonstrate causation , they are among the best evidence to date implicating epigenetic mechanisms in anxiety and stress and suggest that dna methylation at the gr gene promoter ( and probably other genes ) in both rats and humans may contribute to this phenomenon . taken together , these studies demonstrate that chromatin structure is an important substrate for long - lasting changes in behavioral responses to stress and antidepressant treatments . chromatin structure is emerging as a key substrate in the pathogenesis and maintenance of chronic psychiatric illnesses . ultimately though , the key function of chromatin remodeling is to alter the transcription or the transcriptional potential of genes which eventually affect neural function , so any study of chromatin regulation is , in theory , inexorably linked with the study of the underlying gene activity . while extremely exciting , epigenetic research in psychiatry is still in its infancy , and far more research is needed to identify both the dysregulated genes and chromatin modifications responsible for individual psychiatric diseases . fortunately , new advances in high- throughput sequencing are enabling such characterization of chromatin regulation and gene expression , genome - wide , at an incredible rate and resolution . armed with these and other new research tools , epigenetic research in psychiatry is progressing at a spectacular speed , and may soon prove to be a major avenue for novel therapeutics .

tributyltin ( tbt ) is one of the most toxic compounds still used in antifouling paints for large commercial ships thereby distributed within the aquatic environment . its distribution and accumulation in aquatic organisms leads to severe effects and has already reduced the number of snail species in the near of sea lanes and harbours . moreover , the trophic transfer has been demonstrated , and the accumulation within the food chain up to the level of marine mammals has reached concentrations that might be biological relevant [ 36 ] . the most prominent biological effect investigated so far is the so - called imposex within sea snails and dogwhelks [ 1 , 7 , 8 ] , and this mechanism is used as biomonitoring tool for organotin compounds . despite the fact that a lot of studies have been carried out , the underlying molecular mechanism remains unclear [ 10 , 11 ] . it has been proposed that the inhibition of aromatase activity alters the ratio of the hormones inducing the development of imposex , the imposition of male sex characteristics on female snails [ 1 , 12 ] , but other studies came to other results [ 11 , 13 ] . as organotin compounds were still used and accumulate in the environment as well as in the food chain , the exposure of mammals and humans increases steadily . moreover , it has been described earlier that organotin compounds , especially tbt , have a clear immunotoxic effect in mammals [ 1416 ] , and this might be due to their exorbitant induction of apoptosis [ 17 , 18 ] . the effective concentration of tbt to induce apoptosis in the majority of treated cells is around or below 1 m and has been shown in vitro [ 14 , 1827 ] as well as in vivo [ 28 , 29 ] . the question whether the disturbance of the intracellular calcium homeostasis is responsible for the onset of apoptosis [ 22 , 27 , 3034 ] or the direct effect on mitochondrial functions is the first event [ 18 , 20 , 24 , 32 , 33 , 35 ] is under discussion for a long time . [ 18 , 23 ] have shown a decade ago that tbt induces apoptosis via the activation of caspases in various human cells , the link for this caspase activation was not yet found . the most obvious players have been discussed to be the increase in calcium concentration or the opening of the permeability pore of the mitochondria . but induction of apoptosis has been demonstrated for very low concentrations of tbt which do not induce calcium influx , and caspases are often inhibited by high calcium concentrations . some years ago , evidence arose that mitochondria - independent mechanisms contribute to the induction of apoptosis and possibly death receptors or direct caspase activation are involved in the tbt induced effect [ 3639 ] . it is now generally accepted that the programmed cell death can be physiologically induced via death receptors on the surface of the cells , activated by specific ligands that are strictly controlled for instance during development or inflammation leading to the formation of the so - called death - inducing signalling complex or disc . moreover , it has been shown that at least lymphoid cells can be discriminated into type i and type ii cells and only type ii cells are strongly dependent on functional mitochondria for their apoptotic machinery . jurkat t - lymphoblastoid cells are type ii cells and present a special tool for the investigation of mitochondrial - dependent cell death characteristics . additionally , genetic modifications of the disc within these cells enable a closer look at which point the sequence of events is started after tbt - treatment . in the present study , the mechanism of tbt - induced apoptosis has been investigated by the use of jurkat t - cells and two variants , caspase-8 and fadd - deficient jurkat cells , that provide a direct insight into the death - receptor - coupled mechanisms . the data presented here point to the involvement of initiator caspase activation , especially from caspase-10 , and are discussed in terms of the potential immunotoxic role of tbt in exposed mammals . when human immune cells were treated with tbt , changes of morphological as well as biochemical parameters of apoptosis can be observed . in all experiments done in this study , we used 1 m tbt , a concentration that induces apoptosis in the majority of the treated cells within 4 hours . after that , time membrane blebbing and the externalisation of phosphatidylserine ( ps ) occur and chromatin condensation could be observed in jurkat a3 t - cells ( figures 1(a ) and 1(b ) ) . chromatin condensation was shown by the use of the dna dye hoechst 33342 , and ps on the outer leaflet of the plasma membrane is detected with annexin v - fitc by flow cytometry ( facs ) and fluorescence microscopy ( figures 1(c)1(h ) ) . as demonstrated by two typical facs dot blots , more than 60% of the treated cells undergo apoptosis and exhibit green fluorescence at the plasma membrane without being necrotic as the counter staining with propidium iodide demonstrates clearly . looking closer to the different proteins that are involved in the apoptotic machinery , the complete sequence of events from initiator caspases down to death substrates is switched on . focussing at the level of initiator caspases , both , caspase-8 and caspase-10 , are cleaved and their active subunits can be detected by western blotting ( figure 1 , left ) . downstream the initiator caspases , the bid protein is an important linker to the mitochondrial pathway in type ii cells and this protein is cleaved after tbt treatment . from the multitude of caspases downstream of the mitochondria we tested for procaspase-9 , -7 , -6 , and -3 and found all these proteases cleaved . as one of the most prominent death substrates poly(adp - ribose ) polymerase ( parp ) has also been shown to be cleaved within this series of events ( figure 1 , left ) . moreover , we tested hepatocytes transfected with a fusion protein of cytochrome c / green fluorescent protein ( kindly provided by d. green , la jolla institute for allergy and immunology , san diego , usa ) and found the release of cytochrome c after tbt treatment ( data not shown ) . the extrinsic pathway upstream the mitochondria is further characterised by use of two genetically modified jurkat cell lines where one is caspase-8 deficient and the other is fadd adaptor protein deficient . furthermore , various caspase inhibitors were used to dissect their roles as possible starting point of the apoptotic sequence of events induced by tbt . firstly , caspase-8 deficient cells exhibit only a slight reduction of apoptosis in all three cell lines when incubated with 1 or 1.5 m tbt ( figure 2 ) , whereas fas - ligand - induced apoptosis was completely abolished ( not shown ) . without caspase-8 , the only measurable protection was found for ps externalisation that is reduced by one third ( figure 3 ) . secondly , fadd deficiency affords an improved protection against the effects caused by tbt exposure , especially at lower concentrations ( figure 2 , 1 m tbt ) . this is further corroborated by analysis of ps externalisation and the total caspase activity by a fluorescence assay in living cells . the externalisation of ps was reduced in the same order of magnitude as in the caspase-8-deficient cells ( figure 3 ) , but the caspatag assay demonstrates high protease activity in tbt - treated wild - type cells as well as in the caspase-8-deficient variant , whereas in fadd - deficient cells , this activity is obviously reduced ( figure 4 ) . the importance of caspases for organotin - provoked apoptosis has been investigated by use of several inhibitors . the overall caspase inhibitor zvad - fmk blocks totally all described effects that normally can be detected after tbt exposure ( data not shown ) . in this study , we used further specific inhibitors of caspases downstream as well as upstream of mitochondria . when caspase-9 and caspase-3 were inhibited as most potent elements of the caspase cascade downstream of the mitochondria , tbt - induced apoptosis is fully prevented in all three cell lines ( figure 2 ) . preincubation of the jurkat cells with zlehd - fmk ( caspase-9 inhibitor ) and zdevd - fmk ( caspase-3 inhibitor ) rescues all viable functions . nevertheless , a closer look at the western blots revealed often a slight reduction of those elements that were cleaved upstream of the mitochondria , especially bid , even though at slightly higher concentrations of tbt ( figure 2 , 1.5 m ) . while caspase-8 is activated in fact after tbt treatment of wild - type jurkat cells , caspase-8 deficient cells undergo apoptosis to a comparable extent . this result suggests that caspase-8 can not play a substantial role within this concert of effects after tbt treatment . therefore , we looked closer for caspase-10 , the second initiator caspase at the receptor level . when caspase-10 is inhibited by zaevd - fmk , ps externalisation ( figure 3 ) and overall caspase activity is drastically reduced in all cell lines investigated in this study ( figure 4 ) . next , we wanted to know if initiator caspases could be found in an activated disc and which ones . immunoprecipitations ( ip ) with an antibody against the fas - receptor coprecipitated caspase-8 ( data not shown ) , but this caspase has no substantial relevance for the tbt effect in jurkat cells as shown above . therefore , we tested jurkat cells for other death receptors and found additionally trail - r1 , trail - r2 , trail - r3 , trail - r4 , and tnf - r1 . as trail - r2 was the dominant form and trail - r3 and trail - r4 are decoy receptors , we used a trail - r2 antibody for our ips . with this antibody , we could precipitate both initiator caspases after 3 h of treatment with 1 m tbt ( figure 5(a ) ) . additionally , we detected the procaspases-8 and -10 in the untreated controls but to a much lesser extent and we never found the activated subunits . analysing the three different cell lines reveals the fact that caspase-10 could be found in all activated disc forms even though at different levels ( figure 5(b ) ) . a direct comparison of the effects of fas - ligand and tbt in all three cell lines pre - treated with and without the caspase-10 inhibitor zaevd - fmk provides a detailed insight into the different roles of the two initiator caspases in human jurkat t cells . fas - ligand treatment of the two deficient cell lines has no effect at all , and , thus , these data were not included in figure 6 . the jurkat a3 wild - type cells , however , were driven into apoptosis , and this effect is only to a minor degree diminished by the pretreatment with the caspase-10 inhibitor , and apoptosis still proceeds . tbt treatment , however , has approximately the same effect as fas - ligand in the absence of zaevd - fmk , but all consequences of this treatment were prevented in the presence of aevd . phosphatidylserine externalisation is reduced to nearly control levels ( figure 3 ) , and activation of caspases is strongly decreased in all three cell lines ( figure 4 ) . in addition , the cleavage of important caspases is prevented ( caspase-8 and caspase-3 , figure 6 ) , bid cleavage is drastically diminished , parp is completely rescued , and dna fragmentation does not proceed anymore ( figure 6 ) . trialkylated tin compounds , especially tbt , are distributed all over the environment , and were taken up by cells in vitro fast and effectively and their toxicity is a function of both concentration and duration of exposure . it has long been discussed that this cytotoxicity of organotin compounds might be the result of a massive alteration of the intracellular calcium concentration [ ca]i . various investigations demonstrated an increase of [ ca]i after exposure to a variety of trialkytins , and this effect should be responsible for their cytotoxicity , immunotoxicity , and neurotoxicity not only in mammalian [ 14 , 17 , 3032 ] but also in fish cell systems . but more and more evidence has been supplied that alteration of [ ca]i is not the major event in the nonacute cytotoxic scenario [ 43 , 44 ] . numerous studies have been carried out during the last two decades indicating the induction of apoptosis in various biological systems without elucidating the starting point of the involved molecular mechanism [ 14 , 1725 , 27 , 28 , 36 ] . as early as in 2001 , the first publication demonstrated a possible involvement of the death receptors , and this was confirmed a few years later . nevertheless , recently published data connect developmental abnormalities of fish larvae with the induction of apoptosis on the level of caspase 3 , and the initiating molecular mechanism by which tbt induces apoptosis is not described . thus , this study was carried out to enlighten the mechanism in more detail . there exist two different pathways for apoptosis that can be distinguished from each other , the extrinsic and the intrinsic pathway . the intrinsic pathway is dependent from proapoptotic events on the level of the mitochondria and is mostly affected by environmental chemicals or stress factors . thus , it seems to be obvious that toxic substances such as tbt exert their effect on mitochondria . a multitude of studies have shown that different parameters of mitochondria were altered after treatment of cells with tbt [ 18 , 32 , 37 ] . nonetheless , the induction of apoptosis could not be explained sufficiently by all these examinations because mitochondria - independent apoptosis has been described as well [ 37 , 47 ] and inhibition of the intrinsic pathway by bcl-2 overexpression protects only type ii cells but not type i cells from apoptosis although the mitochondrial membrane potential m is still high . moreover , it has been published earlier that various metal compounds may activate the extrinsic apoptotic pathway [ 3739 , 49 ] . a closer look on the formation of the death - inducing signalling complex ( disc ) reveals its formation within 1 to 3 h after treatment with tbt ( figure 5 ) . normally , in jurkat t cells , the disc consists out of the fas - receptor molecules to which the adaptor molecules fadd and initiator caspase-8 are bound . but the caspase-8-deficient jurkat cells showed no or only little reduction in apoptosis after tbt treatment , and solely fadd deficiency decreases substantially the apoptotic cell number although not all . so we looked for other elements as possible constituents of the disc . as it was published by several groups that not only caspase-8 but also caspase-10 can be recruited to death receptors [ 50 , 51 ] , apo2l / trail is able to activate both initiator caspases , and caspase-10 is described as important caspase in hct 116 colon carcinoma cells , we analysed the disc formation in more detail . as mentioned above , caspase-10 could be found in the disc and is co - precipitated by anti - trail - r2 antibody . these results were confirmed by measuring various caspase activities in lysates using the substrates ietd - pna ( caspase-8 ) , aevd - pna ( caspase-10 ) , and devd - pna ( caspase-3 ) , respectively . in lysates of tbt - treated cells , all caspases have been found to be active ( data not shown ) . thus , tbt leads not only to unspecific cleavage of caspases but directly to their activation . because caspase-8-deficient jurkat cells express lower amounts of caspase-10 compared to their parental cell line , these cells might be somewhat less sensitive to tbt as demonstrated here ( figure 3 ) . but determination of all apoptotic markers revealed a nearly unchanged sensitivity to tbt of jurkat cells lacking caspase-8 when treatment is prolonged to a minimum of 4 h. another set of experiments focuses on caspases in more detail . overall inhibition of caspases with zvad - fmk , an unspecific inhibitor of all cellular caspases , inhibits totally tbt - induced apoptosis in human neutrophils as well as in jurkat cells ( data not shown ) . a strong evidence for a specific role of the initiator caspase-10 comes from our experiments with its specific inhibitor zaevd - fmk . while fas - ligand - induced apoptosis is only slightly prevented after pre - incubation of the cells with aevd ( figure 6 ) , tbt - treatment has no effect at all , when caspase-10 was inactivated before . nevertheless , kischkel and coworkers described fadd as an obligatory adaptor for both initiator caspases to trail receptor ; thus , we expected the fadd - deficient cells to be protected against tbt - induced apoptosis . but this is the case only for lower concentrations of tbt up to 1 m . in this case , the prevention of fadd - deficient cells is apparent ( figure 2 ) , whereas slightly higher concentrations ( 1.5 m ) overcome this protective effect . this might be due to the fact that these higher concentrations directly affect , on the one hand , the intrinsic machinery of apoptosis or , on the other hand , tbt might be able to activate directly caspases as has been demonstrated earlier [ 36 , 47 ] . another evidence for caspase-10 dependency with no or only less involvement of fadd - adaptor protein has been described recently for another chemical but with the same set of jurkat cells . this group found the same total inhibition of all effects by the caspase-10 inhibitor zaevd - fmk and no reduction in caspase-8-deficient cells . moreover , fadd recruitment was not involved because the fadd - deficient jurkat cells exhibited dna fragmentation and other signs of apoptosis ; thus , these results are obviously congruent with the data presented here . furthermore , it has been published lately that caspase-10 may cleave specific substrates , as the proapoptotic protein bid , without being cleaved before into its active subunits . this may be the reason why type ii cells are more sensitive to bcl-2 overexpression than type i cells , as type ii cells are dependent on bid cleavage and the activation of the mitochondrial pathway . in our hands , the type i cell line skw has a higher level of caspase-10 expression , and this is cleaved at the disc in both variants , the wild type as well as in the bcl-2 overexpressing line ( data not shown ) . the question to what extent the extrinsic or the intrinsic pathway is responsible for the tbt - induced apoptosis in the absence of a functional fadd adaptor protein can be answered by the concentrations of tbt used within the experiments . lower concentrations not disturbing the lysosomal or mitochondrial systems are more or less totally dependent on the formation of an activated disc , whereas higher concentrations overcome this mechanism and stress the intracellular machinery via lysosomes or / and mitochondria leading to caspase - independent responses [ 5557 ] . the here described results indicate that tbt in principle activates initiator caspase-10 leading to bid cleavage and activation of the mitochondria inducing the downstream apoptotic machinery ( figure 7 ) . besides mammalian system cells from other species were affected by tbt as well . in trout blood cells , 15 m tbt induces apoptosis within 1 h , and , in gill tissue of the mussel mytilus galloprovincialis treated with 1 g / g bw tbt ( 3 m ) , apoptosis could be detected after 24 h incubation . the strongly discussed immunosuppressive properties of tbt in vivo might be the consequence of specific induction of cell death in immunocompetent cells . such a killing of lymphocytes by tbt can be observed as a loss in thymus weight or thymus atrophy [ 29 , 58 ] that debilitates the immune function of animals , making them vulnerable to infectious diseases [ 5963 ] . on the background of these findings , the deadline for banning tbt must be possibly reconsidered as not only sea snails but also open water mammals and humans might be affected and new regulatory strategies have to be discussed independent from market forces . whereas the international maritime organisation ( imo ) has banned tbt since 2008 , the european commission has forbidden its use after 1st of july 2010 in articles where the concentration in the article , or part thereof , is greater than the equivalent of 0.1% by weight of tin , but articles treated with such biocides may still be imported into the community . all cell culture reagents were purchased from life technologies ( eggenstein , germany ) , petri dishes and multiwell plates were obtained from nunc ( wiesbaden , germany ) . annexin - fitc is from bd pharmingen ( heidelberg , germany ) , pi and hoechst 33342 from sigma ( deisenhofen , germany ) , and the inhibitors of caspase-10 ( zaevd - fmk ; fmk009 ) , caspase-9 ( zlehd - fmk ; fmk008 ) , and caspase-3 ( zdevd - fmk ; fmk004 ) were from r&d systems ( wiesbaden , germany ) . 9762 ) , anti - caspase-7 ( cat no . 9492 ) and anti - caspase-9 ( cat no . 9502 ) were purchased from cell signaling technology ( frankfurt , germany ) , anti - caspase-3 ( cat no . 551242 clone 3 - 1 - 9 ) from bd pharmingen ( heidelberg , germany ) , anti - caspase-10/a ( cat no . m059 - 3 ) from mobitec ( mbl ) ( gttingen , germany ) , anti - trail - r2 ( dr5 ) ( cat no . pc392 ) from calbiochem ( darmstadt , germany ) , anti - bid ( cat no . af846 ) from r&d systems ( wiesbaden , germany ) , and anti - parp ( cat no . as secondary reagents , we used : horseradish - peroxidase- ( hrp- ) conjugated goat anti - mouse igg1 ( cat no . p 0447 ) from dakocytomation ( glostrup , denmark ) and hrp - conjugated donkey anti - rabbit ( cat no . the jurkat cell line a3 as well as the fadd and caspase-8-deficient cell lines were kindly provided by j. blenis ( harvard medical school , boston , usa ) and were maintained in rpmi 1640 supplemented with 10% fcs and 1 mm hepes . the cultures were grown with 100 u / ml penicillin and 100 g / ml streptomycin in a humidified atmosphere containing 5% co2 at 37c . during experiments , 10 mm glucose were additionally included in the incubation medium . for induction of apoptosis , 2 10 cells per ml medium were incubated with 1 l / ml of a 1 mm stock solution of tbt ( merck ) in ethanol to give the final concentration of 1 m . as a positive control , 100 ng fas - ligand plus 1 g enhancer ( alexis , san diego , usa ) per ml incubation medium were used . all controls were incubated with the same amount of vehicle ( 1 l ethanol / ml ) to exclude side effects of the solvent . 2 10 were disrupted in 500 l lysis buffer ( 20 mm edta , 1% np 40 , 50 mm tris / hcl , ph 7.5 ) . after centrifugation at 1600 g for 5 min , supernatants containing the apoptotic dna were transferred into eppendorf tubes . after addition of 1% sds , samples were treated for 2 h with rnase - a ( 5 g/l ) at 56c and subsequently for further 2 h with proteinase k ( 2.5 g/l ) . the dna was precipitated by the addition of 50 l of 10 m ammoniumacetate and 250 l ice - cold ethanol , stored overnight at 20c followed by centrifugation . the pellet was resuspended in 25 l of te - buffer ( 10 mm tris / hcl , 1 mm edta , ph 8.0 ) , and the dna - fragments were analysed by agarose gel electrophoresis ( 1.8% agarose containing ethidium bromide as dna stain ) , and pictures were taken with a mwg gel documentation system . the assay was carried out as described by the manufacturer ( intergen , heidelberg , germany ) . from each treated sample , 150 l containing 3 10 cells were washed with fresh medium and 10 l of 30x solution of the caspase substrate fam - vad - fmk were added to give the final concentration of 10 m . then , 1 ml of washing solution was added , the suspension was centrifuged at 400 g for 5 min at room temperature . the resulting cell pellet was washed once with washing buffer and finally resuspended in 400 l washing buffer including 1 l pi as counterstain for necrosis . cells were left for 15 min on ice , and then the microscopic pictures were taken . trail - associated caspase-10 and caspase-8 were immunoprecipitated as follows : 10 cells ( 2 10/ml ) were treated with 1 m tbt and then lysed in 500 l cell lysis buffer ( 30 mm tris / hcl , ph 7.5 , 150 mm nacl , 1 mm phenylmethylsulfonyl fluoride , 10 g / ml aprotinin and 10 g / ml leupeptin , 1% triton and 10% glycerol ) . the lysates were centrifuged at 14 000 g for 10 min at 4c . the supernatants were then incubated for 4 h with protein a - sepharose ( sigma , deisenhofen , germany ) and 1.5 g anti - trail - r2 . the beads were centrifuged at 7000 g for 6 min at 4c , washed once with the same amount of lysis buffer , and centrifuged again . after having removed the supernatants carefully , the pellets were resuspended in 100 l electrophoresis buffer and 15 l of each sample were analysed by sds - gel electrophoresis and western blotting . apoptotic and necrotic cells were determined either microscopically or by flow cytometry using recombinant annexin v conjugated to fitc and propidium iodide . the determination of apoptosis is based on the binding of annexin v - fitc on the phosphatidylserine exposed at the surface of apoptotic cells . for the assay , 1 10 cells were pelleted at 1500 g and resuspended in 100 l binding buffer ( 10 mm hepes , ph 7.4 , 140 mm nacl and 5 mm cacl2 ) containing 5 l annexin v - fitc and 100 ng pi . after 15 min of incubation at 4c in the dark , cells were diluted in 400 l binding buffer and immediately analysed by flow cytometry . fluorescence was analysed at 530 14 nm ( fitc ) and 610 10 nm ( pi ) and quantified with the cellquest prosoftware ( bd , heidelberg , germany ) . for microscopic assessment of apoptosis and necrosis , 6 10 cells were diluted 1 : 1 with 2x binding buffer , containing 1 l annexin v - fitc and 3 ng pi . after 15 min of incubation in the dark , cells were analysed with a 63x oil objective and a zeiss axiovert s100 microscope ( carl zeiss gmbh , jena , germany ) , connected to a hamamatsu ccd camera ( c4880 - 80 ) . fluorescence and differential interference contrast pictures were taken using an automation procedure and merged using openlab software ( improvision , coventry , uk ) . chromatin condensation was determined after staining of the cells with hoechst 33342 . in short , after treatment , 1 10 cells were washed with phosphate - buffered saline ( pbs ) incubated with a final concentration of 10 m hoechst 33342 for 10 min , washed again with pbs to reduce background fluorescence , and finally visualised with the same system described above with excitation at 364 15 nm and emission at 460 10 nm .

tributyltin ( tbt ) is one of the most toxic compounds produced by man and distributed in the environment . a multitude of toxic activities have been described , for example , immunotoxic , neurotoxic , and endocrine disruptive effects . moreover , it has been shown for many cell types that they undergo apoptosis after treatment with tbt and the cell death of immune cells could be the molecular background of its immunotoxic effect . as low as 200 nm up to 1 m of tbt induces all signs of apoptosis in jurkat t cells within 1 to 24 hrs of treatment . when compared to fas - ligand control stimulation , the same sequence of events occurs : membrane blebbing , phosphatidylserine externalisation , the activation of the death - inducing signalling complex , and the following sequence of cleavage processes . in genetically modified caspase-8-deficient jurkat cells , the apoptotic effects are only slightly reduced , whereas , in fadd - negative jurkat cells , the tbt effect is significantly diminished . we could show that caspase-10 is recruited by the trail - r2 receptor and apoptosis is totally prevented when caspase-10 is specifically inhibited in all three cell lines .

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

tributyltin ( tbt ) is one of the most toxic compounds still used in antifouling paints for large commercial ships thereby distributed within the aquatic environment . moreover , the trophic transfer has been demonstrated , and the accumulation within the food chain up to the level of marine mammals has reached concentrations that might be biological relevant [ 36 ] . despite the fact that a lot of studies have been carried out , the underlying molecular mechanism remains unclear [ 10 , 11 ] . it has been proposed that the inhibition of aromatase activity alters the ratio of the hormones inducing the development of imposex , the imposition of male sex characteristics on female snails [ 1 , 12 ] , but other studies came to other results [ 11 , 13 ] . as organotin compounds were still used and accumulate in the environment as well as in the food chain , the exposure of mammals and humans increases steadily . moreover , it has been described earlier that organotin compounds , especially tbt , have a clear immunotoxic effect in mammals [ 1416 ] , and this might be due to their exorbitant induction of apoptosis [ 17 , 18 ] . the effective concentration of tbt to induce apoptosis in the majority of treated cells is around or below 1 m and has been shown in vitro [ 14 , 1827 ] as well as in vivo [ 28 , 29 ] . the question whether the disturbance of the intracellular calcium homeostasis is responsible for the onset of apoptosis [ 22 , 27 , 3034 ] or the direct effect on mitochondrial functions is the first event [ 18 , 20 , 24 , 32 , 33 , 35 ] is under discussion for a long time . [ 18 , 23 ] have shown a decade ago that tbt induces apoptosis via the activation of caspases in various human cells , the link for this caspase activation was not yet found . the most obvious players have been discussed to be the increase in calcium concentration or the opening of the permeability pore of the mitochondria . but induction of apoptosis has been demonstrated for very low concentrations of tbt which do not induce calcium influx , and caspases are often inhibited by high calcium concentrations . some years ago , evidence arose that mitochondria - independent mechanisms contribute to the induction of apoptosis and possibly death receptors or direct caspase activation are involved in the tbt induced effect [ 3639 ] . it is now generally accepted that the programmed cell death can be physiologically induced via death receptors on the surface of the cells , activated by specific ligands that are strictly controlled for instance during development or inflammation leading to the formation of the so - called death - inducing signalling complex or disc . moreover , it has been shown that at least lymphoid cells can be discriminated into type i and type ii cells and only type ii cells are strongly dependent on functional mitochondria for their apoptotic machinery . jurkat t - lymphoblastoid cells are type ii cells and present a special tool for the investigation of mitochondrial - dependent cell death characteristics . additionally , genetic modifications of the disc within these cells enable a closer look at which point the sequence of events is started after tbt - treatment . in the present study , the mechanism of tbt - induced apoptosis has been investigated by the use of jurkat t - cells and two variants , caspase-8 and fadd - deficient jurkat cells , that provide a direct insight into the death - receptor - coupled mechanisms . the data presented here point to the involvement of initiator caspase activation , especially from caspase-10 , and are discussed in terms of the potential immunotoxic role of tbt in exposed mammals . when human immune cells were treated with tbt , changes of morphological as well as biochemical parameters of apoptosis can be observed . in all experiments done in this study , we used 1 m tbt , a concentration that induces apoptosis in the majority of the treated cells within 4 hours . after that , time membrane blebbing and the externalisation of phosphatidylserine ( ps ) occur and chromatin condensation could be observed in jurkat a3 t - cells ( figures 1(a ) and 1(b ) ) . chromatin condensation was shown by the use of the dna dye hoechst 33342 , and ps on the outer leaflet of the plasma membrane is detected with annexin v - fitc by flow cytometry ( facs ) and fluorescence microscopy ( figures 1(c)1(h ) ) . as demonstrated by two typical facs dot blots , more than 60% of the treated cells undergo apoptosis and exhibit green fluorescence at the plasma membrane without being necrotic as the counter staining with propidium iodide demonstrates clearly . looking closer to the different proteins that are involved in the apoptotic machinery , the complete sequence of events from initiator caspases down to death substrates is switched on . from the multitude of caspases downstream of the mitochondria we tested for procaspase-9 , -7 , -6 , and -3 and found all these proteases cleaved . as one of the most prominent death substrates poly(adp - ribose ) polymerase ( parp ) has also been shown to be cleaved within this series of events ( figure 1 , left ) . the extrinsic pathway upstream the mitochondria is further characterised by use of two genetically modified jurkat cell lines where one is caspase-8 deficient and the other is fadd adaptor protein deficient . furthermore , various caspase inhibitors were used to dissect their roles as possible starting point of the apoptotic sequence of events induced by tbt . firstly , caspase-8 deficient cells exhibit only a slight reduction of apoptosis in all three cell lines when incubated with 1 or 1.5 m tbt ( figure 2 ) , whereas fas - ligand - induced apoptosis was completely abolished ( not shown ) . the externalisation of ps was reduced in the same order of magnitude as in the caspase-8-deficient cells ( figure 3 ) , but the caspatag assay demonstrates high protease activity in tbt - treated wild - type cells as well as in the caspase-8-deficient variant , whereas in fadd - deficient cells , this activity is obviously reduced ( figure 4 ) . when caspase-9 and caspase-3 were inhibited as most potent elements of the caspase cascade downstream of the mitochondria , tbt - induced apoptosis is fully prevented in all three cell lines ( figure 2 ) . preincubation of the jurkat cells with zlehd - fmk ( caspase-9 inhibitor ) and zdevd - fmk ( caspase-3 inhibitor ) rescues all viable functions . nevertheless , a closer look at the western blots revealed often a slight reduction of those elements that were cleaved upstream of the mitochondria , especially bid , even though at slightly higher concentrations of tbt ( figure 2 , 1.5 m ) . while caspase-8 is activated in fact after tbt treatment of wild - type jurkat cells , caspase-8 deficient cells undergo apoptosis to a comparable extent . when caspase-10 is inhibited by zaevd - fmk , ps externalisation ( figure 3 ) and overall caspase activity is drastically reduced in all cell lines investigated in this study ( figure 4 ) . immunoprecipitations ( ip ) with an antibody against the fas - receptor coprecipitated caspase-8 ( data not shown ) , but this caspase has no substantial relevance for the tbt effect in jurkat cells as shown above . therefore , we tested jurkat cells for other death receptors and found additionally trail - r1 , trail - r2 , trail - r3 , trail - r4 , and tnf - r1 . as trail - r2 was the dominant form and trail - r3 and trail - r4 are decoy receptors , we used a trail - r2 antibody for our ips . with this antibody , we could precipitate both initiator caspases after 3 h of treatment with 1 m tbt ( figure 5(a ) ) . analysing the three different cell lines reveals the fact that caspase-10 could be found in all activated disc forms even though at different levels ( figure 5(b ) ) . a direct comparison of the effects of fas - ligand and tbt in all three cell lines pre - treated with and without the caspase-10 inhibitor zaevd - fmk provides a detailed insight into the different roles of the two initiator caspases in human jurkat t cells . fas - ligand treatment of the two deficient cell lines has no effect at all , and , thus , these data were not included in figure 6 . the jurkat a3 wild - type cells , however , were driven into apoptosis , and this effect is only to a minor degree diminished by the pretreatment with the caspase-10 inhibitor , and apoptosis still proceeds . tbt treatment , however , has approximately the same effect as fas - ligand in the absence of zaevd - fmk , but all consequences of this treatment were prevented in the presence of aevd . phosphatidylserine externalisation is reduced to nearly control levels ( figure 3 ) , and activation of caspases is strongly decreased in all three cell lines ( figure 4 ) . in addition , the cleavage of important caspases is prevented ( caspase-8 and caspase-3 , figure 6 ) , bid cleavage is drastically diminished , parp is completely rescued , and dna fragmentation does not proceed anymore ( figure 6 ) . trialkylated tin compounds , especially tbt , are distributed all over the environment , and were taken up by cells in vitro fast and effectively and their toxicity is a function of both concentration and duration of exposure . it has long been discussed that this cytotoxicity of organotin compounds might be the result of a massive alteration of the intracellular calcium concentration [ ca]i . but more and more evidence has been supplied that alteration of [ ca]i is not the major event in the nonacute cytotoxic scenario [ 43 , 44 ] . numerous studies have been carried out during the last two decades indicating the induction of apoptosis in various biological systems without elucidating the starting point of the involved molecular mechanism [ 14 , 1725 , 27 , 28 , 36 ] . as early as in 2001 , the first publication demonstrated a possible involvement of the death receptors , and this was confirmed a few years later . nevertheless , recently published data connect developmental abnormalities of fish larvae with the induction of apoptosis on the level of caspase 3 , and the initiating molecular mechanism by which tbt induces apoptosis is not described . there exist two different pathways for apoptosis that can be distinguished from each other , the extrinsic and the intrinsic pathway . a multitude of studies have shown that different parameters of mitochondria were altered after treatment of cells with tbt [ 18 , 32 , 37 ] . nonetheless , the induction of apoptosis could not be explained sufficiently by all these examinations because mitochondria - independent apoptosis has been described as well [ 37 , 47 ] and inhibition of the intrinsic pathway by bcl-2 overexpression protects only type ii cells but not type i cells from apoptosis although the mitochondrial membrane potential m is still high . moreover , it has been published earlier that various metal compounds may activate the extrinsic apoptotic pathway [ 3739 , 49 ] . a closer look on the formation of the death - inducing signalling complex ( disc ) reveals its formation within 1 to 3 h after treatment with tbt ( figure 5 ) . normally , in jurkat t cells , the disc consists out of the fas - receptor molecules to which the adaptor molecules fadd and initiator caspase-8 are bound . but the caspase-8-deficient jurkat cells showed no or only little reduction in apoptosis after tbt treatment , and solely fadd deficiency decreases substantially the apoptotic cell number although not all . as it was published by several groups that not only caspase-8 but also caspase-10 can be recruited to death receptors [ 50 , 51 ] , apo2l / trail is able to activate both initiator caspases , and caspase-10 is described as important caspase in hct 116 colon carcinoma cells , we analysed the disc formation in more detail . as mentioned above , caspase-10 could be found in the disc and is co - precipitated by anti - trail - r2 antibody . in lysates of tbt - treated cells , all caspases have been found to be active ( data not shown ) . because caspase-8-deficient jurkat cells express lower amounts of caspase-10 compared to their parental cell line , these cells might be somewhat less sensitive to tbt as demonstrated here ( figure 3 ) . overall inhibition of caspases with zvad - fmk , an unspecific inhibitor of all cellular caspases , inhibits totally tbt - induced apoptosis in human neutrophils as well as in jurkat cells ( data not shown ) . while fas - ligand - induced apoptosis is only slightly prevented after pre - incubation of the cells with aevd ( figure 6 ) , tbt - treatment has no effect at all , when caspase-10 was inactivated before . but this is the case only for lower concentrations of tbt up to 1 m . in this case , the prevention of fadd - deficient cells is apparent ( figure 2 ) , whereas slightly higher concentrations ( 1.5 m ) overcome this protective effect . this might be due to the fact that these higher concentrations directly affect , on the one hand , the intrinsic machinery of apoptosis or , on the other hand , tbt might be able to activate directly caspases as has been demonstrated earlier [ 36 , 47 ] . another evidence for caspase-10 dependency with no or only less involvement of fadd - adaptor protein has been described recently for another chemical but with the same set of jurkat cells . this group found the same total inhibition of all effects by the caspase-10 inhibitor zaevd - fmk and no reduction in caspase-8-deficient cells . moreover , fadd recruitment was not involved because the fadd - deficient jurkat cells exhibited dna fragmentation and other signs of apoptosis ; thus , these results are obviously congruent with the data presented here . furthermore , it has been published lately that caspase-10 may cleave specific substrates , as the proapoptotic protein bid , without being cleaved before into its active subunits . this may be the reason why type ii cells are more sensitive to bcl-2 overexpression than type i cells , as type ii cells are dependent on bid cleavage and the activation of the mitochondrial pathway . in our hands , the type i cell line skw has a higher level of caspase-10 expression , and this is cleaved at the disc in both variants , the wild type as well as in the bcl-2 overexpressing line ( data not shown ) . the question to what extent the extrinsic or the intrinsic pathway is responsible for the tbt - induced apoptosis in the absence of a functional fadd adaptor protein can be answered by the concentrations of tbt used within the experiments . the here described results indicate that tbt in principle activates initiator caspase-10 leading to bid cleavage and activation of the mitochondria inducing the downstream apoptotic machinery ( figure 7 ) . in trout blood cells , 15 m tbt induces apoptosis within 1 h , and , in gill tissue of the mussel mytilus galloprovincialis treated with 1 g / g bw tbt ( 3 m ) , apoptosis could be detected after 24 h incubation . the strongly discussed immunosuppressive properties of tbt in vivo might be the consequence of specific induction of cell death in immunocompetent cells . on the background of these findings , the deadline for banning tbt must be possibly reconsidered as not only sea snails but also open water mammals and humans might be affected and new regulatory strategies have to be discussed independent from market forces . whereas the international maritime organisation ( imo ) has banned tbt since 2008 , the european commission has forbidden its use after 1st of july 2010 in articles where the concentration in the article , or part thereof , is greater than the equivalent of 0.1% by weight of tin , but articles treated with such biocides may still be imported into the community . annexin - fitc is from bd pharmingen ( heidelberg , germany ) , pi and hoechst 33342 from sigma ( deisenhofen , germany ) , and the inhibitors of caspase-10 ( zaevd - fmk ; fmk009 ) , caspase-9 ( zlehd - fmk ; fmk008 ) , and caspase-3 ( zdevd - fmk ; fmk004 ) were from r&d systems ( wiesbaden , germany ) . m059 - 3 ) from mobitec ( mbl ) ( gttingen , germany ) , anti - trail - r2 ( dr5 ) ( cat no . for induction of apoptosis , 2 10 cells per ml medium were incubated with 1 l / ml of a 1 mm stock solution of tbt ( merck ) in ethanol to give the final concentration of 1 m . as a positive control , 100 ng fas - ligand plus 1 g enhancer ( alexis , san diego , usa ) per ml incubation medium were used . the pellet was resuspended in 25 l of te - buffer ( 10 mm tris / hcl , 1 mm edta , ph 8.0 ) , and the dna - fragments were analysed by agarose gel electrophoresis ( 1.8% agarose containing ethidium bromide as dna stain ) , and pictures were taken with a mwg gel documentation system . trail - associated caspase-10 and caspase-8 were immunoprecipitated as follows : 10 cells ( 2 10/ml ) were treated with 1 m tbt and then lysed in 500 l cell lysis buffer ( 30 mm tris / hcl , ph 7.5 , 150 mm nacl , 1 mm phenylmethylsulfonyl fluoride , 10 g / ml aprotinin and 10 g / ml leupeptin , 1% triton and 10% glycerol ) . the supernatants were then incubated for 4 h with protein a - sepharose ( sigma , deisenhofen , germany ) and 1.5 g anti - trail - r2 . the beads were centrifuged at 7000 g for 6 min at 4c , washed once with the same amount of lysis buffer , and centrifuged again . the determination of apoptosis is based on the binding of annexin v - fitc on the phosphatidylserine exposed at the surface of apoptotic cells . in short , after treatment , 1 10 cells were washed with phosphate - buffered saline ( pbs ) incubated with a final concentration of 10 m hoechst 33342 for 10 min , washed again with pbs to reduce background fluorescence , and finally visualised with the same system described above with excitation at 364 15 nm and emission at 460 10 nm .

numerous studies have revealed the immense diversity of microorganisms and have lent insight into their distributions within and across ecosystems ( ward et al . , 1990 ; vres , 2000 ; floyd et al . , 2005 ) . a number of recent studies have demonstrated that microorganisms have distinct patterns of distribution ( microbial biodiversity over space and time ) reminiscent of those of macroorganisms ( hughes martiny et al . , 2006 ) . biogeography concepts can be used to understand why organisms are distributed as they are within and across ecosystems and why stable , mixed communities exist ( green et al . , 2008 ) . on large regional scales , biogeographic patterns of microorganisms are more heavily influenced by edaphic parameters than by the factors that typically dictate the well documented biogeography of plants and animals , such as latitude and mean annual temperature ( fierer and jackson , 2006 ) . however , on the local scale , microbial biogeography is influenced by both edaphic and biotic parameters such as vegetation type , carbon availability , nutrient availability , and soil moisture . these patterns have been observed in terrestrial , wetland , and aquatic ecosystems ( crump et al . , 2004 ; dolan , 2005 ; fierer and jackson , 2006 ) . salt marsh ecosystems are excellent locations for the investigation of biogeographical patterns due to the small number of dominant macrophytes and to predictable environmental effectors such as tidal inundation and salinity . niche differentiation patterns are well - established as gradients of these effectors and develop in response to tidal flooding and bioturbation in salt marsh ecosystems . microbial communities express niche differentiation based on environmental gradients , often responding strongly to one or more specific environmental effectors in the ecosystem ( crump et al . for example , microbial diversity in estuarine tidal creeks is strongly influenced by salinity ( hollibaugh et al . , 2000 ; selje and simon , 2003 ; crump et al . , 2004 ) , which reflects both the direct impact of salinity on microbial communities and the influences of advective inputs of marine and freshwater microorganisms . niche - based theories of ecology help to explain the partitioning of limiting resources between competing species and the differentiation of niche space across all species within a community ( leibold and mcpeek , 2008 ) . particularly with reference to functional assemblages ( i.e. , assemblages of organisms carrying out specific ecological activities ) , such theories can give insight into the distribution of organisms throughout ecosystems and their ability to contribute significantly to ecosystem function in differing niche dimensions . this review will examine recent findings from north american salt marsh ecosystems , focusing specifically on the diverse assemblage of free - living diazotrophic ( nitrogen fixing ) organisms found there . the salt marsh diazotroph assemblage includes organisms that are quite specific for a given marsh microenvironment as well as for specific marsh plant hosts . this assemblage is also sufficiently stable in composition ( determined through field manipulation experiments discussed below in the section niche specialization ) to make analysis of geographic patterns feasible . coastal salt marshes are characteristically dominated by herbs , low shrubs and grasses bordering saline water bodies and are subjected to periodic flooding as a result of fluctuations ( tidal or non - tidal ) in the sea / ocean ( adam , 1990 ) . the marshes depend on the tidal cycle to maintain water regimes , flush peat and sediment with water and dissolved nutrients , and to maintain salinity gradients ( gardner , 1973 ; hemond and fifield , 1982 ; adam , 1990 ) . tidally dominated salt marshes are transitional between the mainly submerged sea grass communities and fully exposed terrestrial zones , and have received extensive study due to their importance as nursery grounds for commercially important fisheries species and as sources of carbon and nitrogen to nearshore waters ( long and long , 1983 ; schubauer and hopkinson , 1984 ; dame and kenny , 1986 ; dame et al . , 1986 ; morris and haskin , 1990 ; morris et al . , 1990 ; the coastal salt marshes of the south - eastern united states are typically dominated by halophyte perennial graminoids which have extensive underground rhizome systems ( odum , 1988 ) . the number of plant species that are numerically significant in coastal salt marshes is small ( schubauer and hopkinson , 1984 ) and a clear zonation of plant species that is based on edaphic environmental conditions , interspecies competition and minor differences in elevation is routinely observed ( bertness and ellison , 1987 ; adam , 1990 ; pennings and callaway , 1992 ) . key abiotic factors that influence plant zonation include sediment porewater salinity , soluble sulfide concentration , ph and redox potential ( bertness , 1991 ) . microbial activities have also been thought to influence the development of plant zonation , as expressed through their ability to affect the concentrations of nutrients and other solutes , particularly sulfide ( king et al . , 1982 ) . the low marsh , which is regularly flooded and where salinities are higher , is dominated by halophyte grasses such as spartina alterniflora and spartina patens . the high marsh typically has lower salinities , undergoes irregular flooding ( with frequency of flooding based on tidal heights and the elevation gradient ) , is closer to the terrestrial zone typically fringing the marsh , and is dominated by rushes and grasses having lower salt tolerance , such as juncus roemerianus . areas of disturbance in the marsh , such as those experiencing long - term waterlogged conditions or unusually high rates of evapotranspiration , are often dominated by successional species such as salicornia virginica ( pennings and callaway , 1992 ; levine et al . , 1998 ) . these plant zones are easily recognized in the field and some are monotypic with respect to the plant present . crab haul creek basin , north inlet , south carolina hosts a typical tidally dominated salt marsh with obvious plant zonation , fresh water input primarily from local rainfall , and nitrogen limitation . north inlet is a high salinity , relatively pristine estuary ( blood and vernberg , 1992 ) which serves as a benchmark system in the united states national estuarine research reserve . the crab haul creek basin has a relatively minor elevation gradient , just over 1 m from mean high tide to the highest points within the basin , with semi - diurnal tides averaging 1.5 m ( kjerfve , 1986 ) . this relatively flat landscape results in the edaphic parameter gradients that have influenced the plant zonation patterns observed in this system ( figures 1 and 2 ) . porewater salinity and sulfide concentration gradients , in particular , drive the development of macrophyte zonation along this elevation gradient . monotypic stands of short and tall growth forms of s. alterniflora occur in the low marsh , the successional species s. virginica in the waterlogged region of the midmarsh ( as well as in a mixed stand with the short growth form s. alterniflora ) , and j. roemerianus in the high marsh and in patches of slightly higher elevation within the low marsh zone of short growth form s. alterniflora . this pattern of plant zonation has been stable for decades ( e.g. , dame and kenny , 1986 ; morris and haskin , 1990 ) . differences in site elevation above mean sea level ( amsl ) and distance of each sampling location from the forest edge . site designations : juncus roemerianus stand ( js ) ; salicornia virginica ( sv ) ; mixed plant zone of co - occurring s. virginica and short form spartina alterniflora ( ss ) ; short form s. alterniflora ( s ) ; patch of j. roemerianus in the s zone ( jp ) ; and tall form s. alterniflora ( t ) . reproduced with kind permission from davis et al . ( 2011 ) , figure 1 . typical marsh zonation including vegetation zonation and gradients of influential abiotic parameters as observed in crab haul creek basin , north inlet . productivity in salt marshes has important impacts on coastal processes , and salt marshes are among the most productive ecosystems known ( keefe , 1972 ; turner , 1976 ; morris and haskin , 1990 ) . the average annual production ranges from 167 to 938 g m year , however this value varies greatly with temporal and local conditions ( kirwan et al . , salt marshes are often nitrogen - limited as combined nitrogen compounds ( nitrate , nitrite and ammonia ) can be rapidly depleted and sources of new nitrogen , such as n2 fixation , must be utilized ( howarth et al . , 1988 ) . phosphorus can also be a limiting factor for productivity of salt marshes and a seasonal switch is sometimes observed between nitrogen limitation and phosphorus limitation ( valiela and teal , 1974 ; howarth et al . , 1988 ; van wijnen and bakker , 1999 ) . estuaries that are not subject to high levels of anthropogenic nutrient inputs are particularly susceptible to the effects of nutrient limitation . nitrogen limitation has been demonstrated in these relatively pristine systems through nutrient addition experiments ( valiela and teal , 1974 ; marinucci et al . , 1983 ; hopkinson and schubauer , 1984 ; morris , 1991 ; dai and wiegert , 1997 ; van wijnen and bakker , 1999 ; bagwell and lovell , 2000a ; piceno and lovell , 2000a , b ) and through ecosystem - level mass balance considerations ( hopkinson and schubauer , 1984 ; hopkinson , 1988 ; morris , 1995 ) . biological nitrogen fixation in salt marshes is thought to be more important than in most other ecosystems due to its significant contribution of n to the nitrogen budget in these systems ( capone and carpenter , 1982 ) . only organisms of the domains bacteria and archaea are capable of the reduction of gaseous dinitrogen to ammonia ( nitrogen fixation or diazotrophy ; postgate , 1998 ) . ammonium levels in the sediment porewater are much higher than in salt marsh surface waters , demonstrating that the sediments are a net source of nitrogen for the system ( mendelssohn , 1979 ; morris and dacey , 1984 ; sundareshwar et al . , 2003 ) . the diazotrophs in the sediments of salt marshes are thus the main source of the considerable contribution of fixed nitrogen from salt marshes to the global nitrogen cycle . this was confirmed by n2 enrichment studies using microcosms of sediment from a california estuary where nitrogen fixation was the primary method of n transfer to vegetation and other primary producers ( moseman - valtierra et al . , 2010 ) . diazotrophs associated with salt marsh grasses are present in high numbers , are most active on the roots of these plants ( patriquin , 1978 ; mcclung et al . , 1983 ; whiting et al . , 1986 ; yoch and whiting , 1986 ) and are supported by root exudates , as well as by products of plant decomposition ( whiting et al . , 1986 ; diazotrophs associated with salt marsh macrophytes , particularly the cordgrasses ( spartina spp . ) , have been studied extensively and the remarkable diversity of the salt marsh diazotroph assemblages documented ( bagwell et al . , 1998 , 2001 , 2002 ; , 2000 , 2001a , b ; piceno and lovell , 2000a , b ; bergholz et al . , 2001 ; , 2009 , 2010 ; gamble et al . , 2010 ; cramer et al . , 2011 ; davis et al . , 2011 ) bacterial diazotroph assemblages associated with salt marsh grasses are primarily composed of organisms taxonomically related to the - , - , - , - , and -proteobacteria ( bagwell et al . , 1998 , 2001 , 2002 ; piceno et al . , 1999 ; bagwell and lovell , 2000a ; lovell et al . , 2000 , 2001a , b ; piceno and lovell , 2000a , b ; burke et al . , 2002 , 2003 ; brown et al . , 2003 ; moisander et al . , 2005 ; archaeal diazotrophs have not been extensively studied in most ecosystems ( zehr et al . , 2003 ) and have yet to be examined in salt marshes . salt marsh diazotroph assemblages consist of mainly novel , uncultivated organisms whose physiologies have not yet been determined . this is a common observation of diazotroph assemblages across natural ecosystems and the diversity of these assemblages in combination with their potentially very different physiologies has inspired many studies ( zehr et al . culture - dependent studies have also revealed diverse culturable assemblages , largely composed of proteobacteria ( bagwell et al . , 1998 , 2001 ; bagwell and lovell , 2000a ; bergholz et al . these assemblages are capable of utilizing a host of carbon compounds found in plant root exudates as determined in studies on isolates from washed roots ( bagwell et al . , 2001 ; bergholz et al . , 2001 ) , and of surviving substantial ranges of environmental conditions such as ph and salinity ( cramer et al . , 2011 ) . the development of nitrogenase gene specific polymerase chain reaction ( pcr ) primers has allowed the investigation of salt marsh diazotroph assemblages via culture - independent methods ( zehr and mcreynolds , 1989 ; ueda et al . , 1995 ; olson et al . , 1998 ; , 1999 ; widmer et al . , 1999 ; lovell et al . , 2000 ; welsh et al . , 2007 ) . briefly , the nitrogenase enzyme is composed of two multi - subunit metalloproteins ( reviewed in postgate , 1998 ; zehr et al . , 2003 ) . component i contains the active site for n2 reduction and is composed of two heterodimers encoded by the nifd and nifk genes . component ii couples atp hydrolysis to inter - protein electron transfer and is composed of two identical subunits encoded by the nifh gene . primers specific for the nifh gene have been used extensively in culture - independent studies of diazotroph assemblages across many ecosystems resulting in a robust database of gene sequences from uncultivated microorganisms ( zehr et al . , 2003 ) . primers specific for nifd ( ueda et al . , 1995 ) have also been developed but have not been used as broadly or as frequently as the nifh - specific primers ( zehr et al . , 2003 ) . the diazotroph assemblages associated with salt marsh macrophytes have been investigated through pcr - amplification using nifh - specific primers and the nifh amplicons have in turn been analyzed using methods such as denaturing gradient gel electrophoresis ( dgge ; bagwell et al . , 1998 , 2001 , 2002 ; piceno et al . , 1999 ; bagwell and lovell , 2000a ; lovell et al . , 2000 , 2001a , b ; 2003 ; welsh et al . , 2007 , 2010 ; gamble et al . , 2010 ; davis et al . , 2011 ) , restriction fragment length polymorphism ( rflp ; burke et al . , 2002 , 2003 ) , and terminal restriction fragment length polymorphism ( t - rflp ; moseman - valtierra et al . , 2009 , 2010 ) . pcr - amplification of reverse - transcribed mrna extracts has also been used to a lesser extent ( brown et al . , 2003 ) . in the north inlet estuary , diazotrophs have been investigated via culture - dependent methods as well as dgge ( bagwell et al . , 1998 , 2001 , 2002 ; , 2000 , 2001a , b ; piceno and lovell , 2000a , b ; brown et al . , 2003 ; gamble et al . , 2010 ; davis et al . , 2011 ) , reverse sample genome probing ( bagwell and lovell , 2000a ) , clone library construction and screening ( lovell et al . , 2008 ) and sequencing of reverse - transcribed mrna extracts ( brown et al . , 2003 ) . the composition of the diazotroph assemblages of this ecosystem are similar to those recovered from other salt marsh ecosystems ( e.g. , moisander et al . , 2005 ) and other estuaries ( e.g. , affourtit et al . , 2001 ; due to the number of studies that have been done in this area , detailed phylogenetic and statistical analyses can be conducted on the nifh sequence data that have been obtained . assemblages from the north inlet estuary have proven to be stable , ubiquitous , functionally versatile , distributed in predictable patterns across the marsh landscape and are characterized by their content of novel and undescribed taxa ( gamble et al . , 2010 ; davis et al . , 2011 ) . culture - dependent and culture - independent analyses of diazotroph assemblages associated with the rhizospheres of salt marsh macrophytes of north inlet have demonstrated the occurrence of plant host specific organisms . the colonization of plant species by specific microbial populations has been repeatedly observed in terrestrial ecosystems ( reviewed in berg and smalla , 2009 ) . soil quality and soil type heavily influences the root colonizing microorganisms , however it has been proven repeatedly that plant species also exerts a direct influence ( berg and smalla , 2009 ) . the biogeography of soil microbial communities on a large scale ( i.e. , continental ) is controlled primarily by soil abiotic parameters , particularly soil ph ( fierer and jackson , 2006 ) , and it is assumed that this premise holds true for salt marsh sediment microbial communities . however , as previously mentioned , parameters such as vegetation type , carbon and nutrient availability , and soil moisture have a more direct influence on the diversity of the local microbial community ( fierer and jackson , 2006 ) . plant type and the root exudates that individual plant species produce affect the relative abundance and diversity of indigenous microbial communities at the root and in the rhizosphere ( berg and smalla , 2009 ) resulting in plant host specific phylotypes . larocque et al . ( 2004 ) examined diazotrophs isolated from j. roemerianus rhizoplanes from two habitats differing in abiotic parameters . the two monotypic juncus stands were in different marsh zones having different salinities ( 29 and 40 parts per thousand ) , different porewater chemistries and experiencing different extents and durations of tidal inundation ( larocque et al . , 2004 ) . the higher salinity juncus habitat is regularly inundated during daily tidal cycles , whereas the lower salinity stand is in the high marsh and not as frequently inundated . diazotrophs from both habitats were analyzed in parallel using the same protocols ( including nifh pcr - amplification ) . the presence of nifh was confirmed in each isolate and isolates were grouped based on their carbon substrate utilization patterns . three major substrate utilization clusters were identified and each cluster contained isolates from both monotypic stands . dgge analysis of rhizoplane ( sediment free roots ) and rhizosphere ( sediments influenced by the roots ) nifh amplicons from both juncus stands confirmed the presence of host specific diazotrophs diazotrophs that were present in both assemblages despite differences in salinity , sulfide , and ph . the use of both culture - dependent and independent methods in this study reduced the impact of biases that could have been introduced had only data from a single method been examined . ( 2001 ) isolated diazotrophs from the rhizoplanes of s. patens growing in two physically different habitats of north inlet estuary , along with diazotrophs from other macrophytes . the diazotrophs were grouped based on substrate utilization and strains from s. patens formed two distinct clusters each containing strains from both habitats . of the 16 physiologically different groups , only one strain isolated from s. patens was similar to diazotroph strains isolated from other macrophytes in the same salt marsh ecosystem . diazotroph strains from s. virginica were different from strains from other plants growing in other marsh zones , but were more similar to strains from host plants growing in the same marsh zone ( bagwell et al . , 2001 ) . as described above and in figure 2 , marsh zones are defined by edaphic parameters ( bertness and ellison , 1987 ; adam , 1990 ; pennings and callaway , 1992 ) . diazotrophs isolated from plant hosts in the same zone are presumed to be heavily influenced by the edaphic parameters of the marsh zone . nifh dgge analysis of diazotroph assemblages recovered from the rhizosphere and rhizoplane of host plants in north inlet also confirm the plant host specificity observed in the culture - dependent studies ( gamble et al . assemblages from s. alterniflora growing in two differing tidal drainage systems within north inlet estuary showed seasonal variability but no significant site - influenced variability ( gamble et al . , 2010 ) . the host plants were the same in both locations ( short and tall growth forms of s. alterniflora ) and the subtle changes in edaphic parameters with changes in seasons shaped the seasonally responsive segments of the diazotroph assemblages in these two locations . assemblages from the rhizospheres of plant hosts in the crab haul creek basin that exhibit zonation due to elevation differences , contained host specific diazotrophs as well as diazotrophs having broad distributions across the landscape ( davis et al . , 2011 ) . as described above , plant zones contain specific macrophytes due to the differences in marsh environments . a clear response of diazotroph assemblages to plant host zonation and seasonal changes in edaphic parameters was observed from principal component analysis ( pca ) of the recovered nifh amplicons ( gamble et al . , 2010 ; davis et al . , 2011 ) . ( 2011 ) confirmed the seasonal responses ( figure 3 ) and plant host specificity ( figure 4 ) observed by gamble et al . principal components analysis results for dates june ( ss , jp , s , and t ) and september 2000 ( sv , ss , s , and t ) . circles denote significance ( p < 0.001 ) for clustering . for june 2000 axis 1 represents 28.4% of the variance and axis 2 represents 14.7% of the variance . for september 2000 axis 1 represents 31.5% of the variance and axis 2 represents 11.5% of the variance . principal components analysis results for vegetation zones juncus roemerianus stand and tall form spartina alterniflora on all dates . . for j. roemerianus stand axis 1 represents 31.2% of the variance and axis 2 represents 20.8% of the variance . for tall form s. alterniflora axis 1 represents 25.8% of the variance and axis 2 represents 15.5% of the variance . reproduced with kind permission from davis et al . ( 2011 ) , supplementary figure 1a . to confirm the results from pca analysis , mothur ( schloss et al . , 2009 ) was used to conduct parsimony tests on neighbor - joining trees created using nifh sequences ( 400420 bp ) . these sequences were recovered from dgge banding patterns of rhizoplane and rhizosphere samples from all plant zones except the mixed zone of co - occurring s. virginica and short form s. alterniflora ( davis , 2010 ) . included in the phylogenetic analysis were plant zones ( with designations ) : j. roemerianus stand ( js ) , s. virginica ( sv ) , short form s. alterniflora ( ss ) , patch of j. roemerianus in the ss zone ( jp ) , and tall form s. alterniflora ( ts ) . using a significance threshold of p < 0.01 it was confirmed that diazotroph assemblages in the same marsh zone ( based on edaphic parameters ) had similar structures ( table 1 ) . phylogenetic analyses of the sequences recovered from these assemblages also revealed clusters of plant host specific sequences that were considered identical ( sensu venter et al . , 2004 ; see also lovell et al . , 2008 ) throughout the nifh phylogenetic trees ( figure 5 ; table a2 in appendix , 19 clusters in the high marsh ; figures 6 and 7 ; table a4 in appendix , 39 clusters in the low marsh ) . results from parsimony tests ( mothur ) of neighbor - joining trees containing diazotroph sequences recovered from band stabs of dgge gels of pcr - amplified nifh amplicons from rhizoplane ( roots ) or rhizosphere ( sediments ) of different plant zones . * * * significance threshold ( using bonferroni corrections ) = 0.05/5 = 0.01 . all pairs yielding a parsimony significance < 0.01 have significantly different structures from each other . key : jpx , j. roemerianus patch in short growth form s. alterniflora ; ssx , short growth form s. alterniflora ; xxr , rhizoplane ; xxs , rhizosphere . phylogenetic analysis of all nifh sequences from dgge bands of rhizoplane ( r ) and rhizosphere ( s ) samples from the high marsh plant zones ( nucleotide sequences , neighbor - joining , jukes cantor correction , complete deletion of gaps and missing data , 1000 bootstrap replicates ) . js , juncus roemerianus stand ; sv , salicornia virginica . phylogenetic analysis of all nifh sequences from dgge bands of rhizoplane ( r ) and rhizosphere ( s ) samples from the low marsh plant zones ( nucleotide sequences , neighbor - joining , jukes cantor correction , complete deletion of gaps and missing data , 1000 bootstrap replicates ) . abbreviations : jp , j. roemerianus patch ; ss , short form s. alterniflora ; ts , tall form s. alterniflora . continuation of figure 6 phylogenetic analysis of all nifh sequences recovered from the low marsh plant zones ( nucleotide sequences , neighbor - joining , jukes cantor correction , complete deletion of gaps and missing data , 1000 bootstrap replicates ) . abbreviations : jp , j. roemerianus patch ; ss , short form s. alterniflora ; ts , tall form s. alterniflora . multiple analyses of datasets obtained in several different studies provide strong evidence of plant host specific diazotroph assemblages associated with the rhizospheres of salt marsh plants . these organisms have been detected through both culture - dependent and culture - independent methods , in different sites throughout north inlet estuary , and over the course of more than a decade . it is very clear that salt marsh diazotrophs have a distinct biogeography driven by both biotic ( host plant ) and abiotic environmental parameters ( i.e. , salinity , tidal inundation , elevation , etc . ) . the results of these studies also indicate substantial resilience of some members of the diazotroph assemblages that are capable of withstanding wide ranges of environmental conditions and are present in multiple marsh zones . studies of diazotroph assemblages associated with the salt marsh plants in north inlet have demonstrated a diverse yet stable assemblage . bagwell and lovell ( 2000b ) conducted a series of field fertilization experiments and through reverse sample genome probing revealed a highly resilient , niche specialized diazotroph assemblage . the assemblage responded to environmental manipulations through increases and decreases in quantities of specific taxa , but not through loss of taxa ( bagwell and lovell , 2000b ) . dgge analysis of diazotroph assemblages in long- and short - term fertilization experiments ( fertilized with 16.3 g n m nh4no3 and 18.0 g p m p2o5 ) confirmed the stability of these assemblages ( piceno and lovell , 2000a , b ) . the results of these experiments indicate that salt marsh diazotrophs are quite competitive with non - diazotrophs and are able to maintain their populations despite the energy expense of nitrogen fixation . the resilience of diazotrophs associated with salt marsh plants has also been observed during periods of extreme environmental conditions ( davis , 2010 ) . in stark contrast to the diversity of diazotrophs associated with the rhizospheres of salt marsh plants , nifh sequences recovered from standing dead s. alterniflora biomass from north inlet and sapelo island ( ga , usa ) salt marshes revealed a diazotroph assemblage with markedly lower diversity ( lovell et al . , 2001b ) . these assemblages can produce significant levels of diazotrophy ( based on rates of nitrogen fixation ) despite the highly mineralized and frequently dry microenvironments they occupy ( newell et al . the analysis methods ( including nifh - specific pcr ) , which were also utilized in other studies , revealed that approximately 90% of nifh sequences from these assemblages were similar to those from -proteobacteria . this demonstrates that the methods of nifh sequence recovery employed in these studies are sound , as the -proteobacteria are poorly represented in most salt marsh rhizosphere diazotroph assemblages ( lovell et al . , 2000 , 2008 ; gamble et al . , 2010 ; davis et al . , 2011 ) . for example , figures 57 show the small number of nifh sequences that are similar to those from -proteobacteria ( * -labeled clusters in tables a1 and a4 in appendix ) in comparison to the large number of recovered sequences related to those from the -proteobacteria . the majority of the -proteobacteria - like salt marsh sequences have been recovered from the high marsh , particularly in the j. roemerianus plant zone . in addition , approximately half of the -proteobacteria - like sequences that were recovered from the low marsh were from the patch of j. roemerianus growing in the short form s. alterniflora zone . ( 2010 ) revealed that changes in edaphic parameters associated with plant senescence , tidal intrusion , and nutrient cycling produced different responses from the seasonally ( growing season / summer vs. plant senescence / winter ) responsive groups within the diazotroph assemblages ( gamble et al . , 2010 ) . a seasonally responsive -proteobacteria clade was detected during the spartina growing season while , an anaerobic clade was only detected during plant senescence . some clades were also recovered throughout all seasons , indicating a resilient portion of the assemblage . these diazotrophs are capable of competing for resources and maintaining populations over seasonal changes in both biotic and abiotic environmental variables . ( 2011 ) demonstrated broadly distributed ( and mostly novel ) , frequently detected ( fd ) diazotroph sequences that did not respond detectably to seasonal changes ( davis et al . , these sequences were not detected in studies employing the same methods to examine diazotrophic assemblages associated with standing dead spartina ( lovell et al . sequences were also identified ( via statistical tests in pca ) as contributing significantly to the structure of the diazotroph assemblages ( lv sequences from bands producing long eigenvectors in euclidean bi - plot analysis of pca results ; for full explanation see davis et al . , phylogenetic analysis revealed several groups of fd and lv sequences that were virtually identical ( sensu venter et al . , 2004 ; see also lovell et al . , 2008 ) to each other and to previously recovered seasonally non - responsive ( detected throughout all seasons ) sequences from gamble et al . they have been recovered from multiple sites in north inlet estuary and differ from those recovered from other ecosystems . most importantly , these organisms have maintained ubiquity and strong influence on ecosystem function in an ecosystem characterized by distinct plant zones resulting from varying edaphic parameters . it has been determined in terrestrial ecosystems that free - living diazotrophs are functionally significant ( brgmann et al . , 2004 ; hsu and buckley , 2009 ) . it is reasonable to assume , based on the results of these studies , that these organisms are equally important in salt marsh ecosystems . phylogenetic analysis of sequences recovered from high marsh and low marsh zones ( figures 57 ) also confirm the presence of diazotrophs specific to marsh zones . throughout the phylograms are clades containing virtually identical sequences recovered from host plants in the same marsh zone ( plant host labeled clusters in bolded font in figures 57 , and sequences listed in tables a2 and a4 in appendix ) , indicating niche specificity of these diazotrophs for the edaphic parameters present in these zones . these data have given insight into the distributions of diazotroph populations throughout the nitrogen - limited salt marsh ecosystem of north inlet , sc , usa . these diazotrophs are specialized for the niches within this ecosystem , whether the niches are defined by the microenvironment of the plant host , or by marsh zone , or by both . they maintain populations that are readily detectable by methods employed in multiple studies ( particularly nifh based pcr and dgge fingerprinting ) and influence ecosystem function as well as diazotroph assemblage structure . studies of this salt marsh ecosystem have revealed that the structure of a functional microbial assemblage can be predicted , based on specific environmental parameters . plant hosts and edaphic factors that vary in response to elevation are the key parameters that predict the biogeography of these organisms . on a continental scale , the biogeography of microbial communities in terrestrial ecosystems is predicted by abiotic parameters , particularly soil ph , rather than by geographic distance or vegetation type ( fierer and jackson , 2006 ) . however , on a local scale , environmental parameters and vegetation type more heavily influence the diversity of microbial populations . these patterns have been observed in this estuarine system and can be used to predict biogeographical patterns of diazotroph assemblages associated with salt marsh grass rhizospheres . diazotroph assemblages of salt marsh ecosystems are also capable of resisting relatively large changes in environmental parameters to maintain detectable and functionally important populations ( davis , 2010 ) and can be understood in the context of biogeographical concepts that have been applied to macroorganisms . strong evidence of plant host specificity and niche specialization in these assemblages has been compiled in this review ; however the methods used to obtain the data to support these hypotheses are not perfect . limitations of dgge include the co - migration of dna fragments which will result in a loss of information ( gelsomino et al . , 1999 ; also , the detection of nifh gene sequences by pcr does not confirm the expression of this gene . methods of quantification such as quantitative reverse transcriptase - pcr would be useful as this would identify the dominant diazotrophic phylotypes that actively fix nitrogen and could help to quantify the sizes of their populations . continuing difficulties in the cultivation of these organisms results in a very limited understanding of their physiology , with much of that understanding inferred from known physiologies of related organisms . finally , the evolutionary distances separating most salt marsh diazotrophs from their nearest characterized relatives limits even inference of ecologically significant characteristics of many of these organisms . a great deal remains to be done in identifying the taxa that contribute substantially to diazotrophy in salt marsh ecosystems , though a small start has been made ( brown et al . greater knowledge of these organisms may allow us to refine our cultivation strategies and finally isolate the key organisms into pure culture for characterization . perhaps metagenomic approaches could be employed , but the great diversity of distinct strains within even well studied lineages may limit the gains from these methods as chimeric genomic fragments may be encountered . clearly , many challenges remain to be overcome . based on current climate models , coastal salt marshes are in danger of being completely submerged by the end of the century ( nicholls et al . , 2007 ; craft et al . , 2009 currently , it is expected that the ecogeomorphic feedbacks of these dynamic coastal ecosystems [ i.e. , rapid vegetation growth during years of low and high sea level ( morris et al . , 2002 ) ; sediment trapping , organic matter accretion and limiting erosion ( fagherazzi et al . , 2004 ) ] that have allowed the survival of intertidal wetlands over time will continue to do so . however , the ever increasing number of completely submerged wetlands indicates that the current rate of sea level rise may exceed the abilities of ecogeomorphic feedbacks to preserve tidal ecosystems , particularly salt marshes ( kirwan et al . , 2010 ) . continuous submersion of coastal salt marshes will result in losses of vegetation ( penland et al . , 2001 ; hartig et al . , 2002 ; kearnery et al . , 2002 ; kirwan et al . , 2010 ) and consequently loss of microorganisms restricted to a particular plant host or microenvironment ( berg and smalla , 2009 ) . loss of microorganisms critical to ecosystem function would clearly be detrimental to the ecosystem that is dependent on their activity and interactions with macrophytes ( primary producers ) . these events will eventually interrupt food webs and littoral biogeochemical cycles affecting the quality of estuarine water due to eutrophication and increasing the export of particulates ( kearnery et al . , 2002 ) . the ability to predict biogeographical distributions of microorganisms as critical to ecosystem function as diazotrophs are to salt marshes will be advantageous to ecosystem modelers , especially those involved in determining the effects of global climate change . these data clearly show that the distribution of diazotrophs is non - random and is driven directly or indirectly by edaphic parameters in their habitat . the assemblages contain microorganisms that appear to be restricted in their distributions , as well as broadly distributed microorganisms . each group is clearly engaged in ecosystem function as evidenced by their resilience and stability within the assemblages . however , even with this apparent ability to prosper over wide ranges of environmental parameters , there are limits to their survival . the flooding of coastal wetlands as sea level continues to rise could result in large losses from these assemblages . the biogeographical patterns observed in north inlet estuary can be applied to tidally dominated coastal wetlands when the edaphic parameters of the system in question are taken into consideration . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

salt marshes located on the east coast of temperate north america are highly productive , typically nitrogen - limited , and support diverse assemblages of free - living nitrogen fixing ( diazotrophic ) bacteria . this article reviews and analyzes data from north inlet estuary ( sc , usa ) , addressing diazotroph assemblage structure and the influence of plant host and environmental conditions on the assemblage . the north inlet estuary is a salt marsh ecosystem in which anthropogenic influences are minimal and the distributions of diazotrophs are governed by the natural biota and dynamics of the system . denaturing gradient gel electrophoresis fingerprinting and phylogenetic analyses of recovered sequences demonstrated that the distributions of some diazotrophs reflect plant host specificity and that diazotroph assemblages distributed across marsh gradients are also heavily influenced by edaphic conditions . broadly distributed diazotrophs that are capable of maintaining populations under all environmental conditions spanning such gradients are also present in these assemblages . statistical analyses indicate that the structures of diazotroph assemblages in different vegetation zones are significantly ( p < 0.01 ) different . new data presented here demonstrate the heterogeneity of salt marsh rhizosphere microenvironments , and corroborate previous findings from different plant hosts growing at several locations within this estuary . the data from these collected works support the hypothesis that the biogeography of microorganisms is non - random and these biogeographic patterns are predictable .

Introduction Salt Marsh Ecosystems Diazotroph Assemblages Associated with Salt Marsh Grasses Plant Host Specificity Niche Specialization Concluding Remarks Conflict of Interest Statement

on large regional scales , biogeographic patterns of microorganisms are more heavily influenced by edaphic parameters than by the factors that typically dictate the well documented biogeography of plants and animals , such as latitude and mean annual temperature ( fierer and jackson , 2006 ) . however , on the local scale , microbial biogeography is influenced by both edaphic and biotic parameters such as vegetation type , carbon availability , nutrient availability , and soil moisture . niche differentiation patterns are well - established as gradients of these effectors and develop in response to tidal flooding and bioturbation in salt marsh ecosystems . , 2004 ) , which reflects both the direct impact of salinity on microbial communities and the influences of advective inputs of marine and freshwater microorganisms . , assemblages of organisms carrying out specific ecological activities ) , such theories can give insight into the distribution of organisms throughout ecosystems and their ability to contribute significantly to ecosystem function in differing niche dimensions . this review will examine recent findings from north american salt marsh ecosystems , focusing specifically on the diverse assemblage of free - living diazotrophic ( nitrogen fixing ) organisms found there . the salt marsh diazotroph assemblage includes organisms that are quite specific for a given marsh microenvironment as well as for specific marsh plant hosts . coastal salt marshes are characteristically dominated by herbs , low shrubs and grasses bordering saline water bodies and are subjected to periodic flooding as a result of fluctuations ( tidal or non - tidal ) in the sea / ocean ( adam , 1990 ) . the marshes depend on the tidal cycle to maintain water regimes , flush peat and sediment with water and dissolved nutrients , and to maintain salinity gradients ( gardner , 1973 ; hemond and fifield , 1982 ; adam , 1990 ) . tidally dominated salt marshes are transitional between the mainly submerged sea grass communities and fully exposed terrestrial zones , and have received extensive study due to their importance as nursery grounds for commercially important fisheries species and as sources of carbon and nitrogen to nearshore waters ( long and long , 1983 ; schubauer and hopkinson , 1984 ; dame and kenny , 1986 ; dame et al . , 1990 ; the coastal salt marshes of the south - eastern united states are typically dominated by halophyte perennial graminoids which have extensive underground rhizome systems ( odum , 1988 ) . the number of plant species that are numerically significant in coastal salt marshes is small ( schubauer and hopkinson , 1984 ) and a clear zonation of plant species that is based on edaphic environmental conditions , interspecies competition and minor differences in elevation is routinely observed ( bertness and ellison , 1987 ; adam , 1990 ; pennings and callaway , 1992 ) . the high marsh typically has lower salinities , undergoes irregular flooding ( with frequency of flooding based on tidal heights and the elevation gradient ) , is closer to the terrestrial zone typically fringing the marsh , and is dominated by rushes and grasses having lower salt tolerance , such as juncus roemerianus . crab haul creek basin , north inlet , south carolina hosts a typical tidally dominated salt marsh with obvious plant zonation , fresh water input primarily from local rainfall , and nitrogen limitation . north inlet is a high salinity , relatively pristine estuary ( blood and vernberg , 1992 ) which serves as a benchmark system in the united states national estuarine research reserve . monotypic stands of short and tall growth forms of s. alterniflora occur in the low marsh , the successional species s. virginica in the waterlogged region of the midmarsh ( as well as in a mixed stand with the short growth form s. alterniflora ) , and j. roemerianus in the high marsh and in patches of slightly higher elevation within the low marsh zone of short growth form s. alterniflora . productivity in salt marshes has important impacts on coastal processes , and salt marshes are among the most productive ecosystems known ( keefe , 1972 ; turner , 1976 ; morris and haskin , 1990 ) . , salt marshes are often nitrogen - limited as combined nitrogen compounds ( nitrate , nitrite and ammonia ) can be rapidly depleted and sources of new nitrogen , such as n2 fixation , must be utilized ( howarth et al . phosphorus can also be a limiting factor for productivity of salt marshes and a seasonal switch is sometimes observed between nitrogen limitation and phosphorus limitation ( valiela and teal , 1974 ; howarth et al . biological nitrogen fixation in salt marshes is thought to be more important than in most other ecosystems due to its significant contribution of n to the nitrogen budget in these systems ( capone and carpenter , 1982 ) . only organisms of the domains bacteria and archaea are capable of the reduction of gaseous dinitrogen to ammonia ( nitrogen fixation or diazotrophy ; postgate , 1998 ) . ammonium levels in the sediment porewater are much higher than in salt marsh surface waters , demonstrating that the sediments are a net source of nitrogen for the system ( mendelssohn , 1979 ; morris and dacey , 1984 ; sundareshwar et al . the diazotrophs in the sediments of salt marshes are thus the main source of the considerable contribution of fixed nitrogen from salt marshes to the global nitrogen cycle . diazotrophs associated with salt marsh grasses are present in high numbers , are most active on the roots of these plants ( patriquin , 1978 ; mcclung et al . , have been studied extensively and the remarkable diversity of the salt marsh diazotroph assemblages documented ( bagwell et al . , 2011 ) bacterial diazotroph assemblages associated with salt marsh grasses are primarily composed of organisms taxonomically related to the - , - , - , - , and -proteobacteria ( bagwell et al . salt marsh diazotroph assemblages consist of mainly novel , uncultivated organisms whose physiologies have not yet been determined . this is a common observation of diazotroph assemblages across natural ecosystems and the diversity of these assemblages in combination with their potentially very different physiologies has inspired many studies ( zehr et al . these assemblages are capable of utilizing a host of carbon compounds found in plant root exudates as determined in studies on isolates from washed roots ( bagwell et al . , 2001 ) , and of surviving substantial ranges of environmental conditions such as ph and salinity ( cramer et al . the development of nitrogenase gene specific polymerase chain reaction ( pcr ) primers has allowed the investigation of salt marsh diazotroph assemblages via culture - independent methods ( zehr and mcreynolds , 1989 ; ueda et al . primers specific for the nifh gene have been used extensively in culture - independent studies of diazotroph assemblages across many ecosystems resulting in a robust database of gene sequences from uncultivated microorganisms ( zehr et al . the diazotroph assemblages associated with salt marsh macrophytes have been investigated through pcr - amplification using nifh - specific primers and the nifh amplicons have in turn been analyzed using methods such as denaturing gradient gel electrophoresis ( dgge ; bagwell et al . , 2002 , 2003 ) , and terminal restriction fragment length polymorphism ( t - rflp ; moseman - valtierra et al . in the north inlet estuary , diazotrophs have been investigated via culture - dependent methods as well as dgge ( bagwell et al . the composition of the diazotroph assemblages of this ecosystem are similar to those recovered from other salt marsh ecosystems ( e.g. , 2001 ; due to the number of studies that have been done in this area , detailed phylogenetic and statistical analyses can be conducted on the nifh sequence data that have been obtained . assemblages from the north inlet estuary have proven to be stable , ubiquitous , functionally versatile , distributed in predictable patterns across the marsh landscape and are characterized by their content of novel and undescribed taxa ( gamble et al . culture - dependent and culture - independent analyses of diazotroph assemblages associated with the rhizospheres of salt marsh macrophytes of north inlet have demonstrated the occurrence of plant host specific organisms . the biogeography of soil microbial communities on a large scale ( i.e. , continental ) is controlled primarily by soil abiotic parameters , particularly soil ph ( fierer and jackson , 2006 ) , and it is assumed that this premise holds true for salt marsh sediment microbial communities . however , as previously mentioned , parameters such as vegetation type , carbon and nutrient availability , and soil moisture have a more direct influence on the diversity of the local microbial community ( fierer and jackson , 2006 ) . plant type and the root exudates that individual plant species produce affect the relative abundance and diversity of indigenous microbial communities at the root and in the rhizosphere ( berg and smalla , 2009 ) resulting in plant host specific phylotypes . the two monotypic juncus stands were in different marsh zones having different salinities ( 29 and 40 parts per thousand ) , different porewater chemistries and experiencing different extents and durations of tidal inundation ( larocque et al . dgge analysis of rhizoplane ( sediment free roots ) and rhizosphere ( sediments influenced by the roots ) nifh amplicons from both juncus stands confirmed the presence of host specific diazotrophs diazotrophs that were present in both assemblages despite differences in salinity , sulfide , and ph . ( 2001 ) isolated diazotrophs from the rhizoplanes of s. patens growing in two physically different habitats of north inlet estuary , along with diazotrophs from other macrophytes . of the 16 physiologically different groups , only one strain isolated from s. patens was similar to diazotroph strains isolated from other macrophytes in the same salt marsh ecosystem . as described above and in figure 2 , marsh zones are defined by edaphic parameters ( bertness and ellison , 1987 ; adam , 1990 ; pennings and callaway , 1992 ) . diazotrophs isolated from plant hosts in the same zone are presumed to be heavily influenced by the edaphic parameters of the marsh zone . nifh dgge analysis of diazotroph assemblages recovered from the rhizosphere and rhizoplane of host plants in north inlet also confirm the plant host specificity observed in the culture - dependent studies ( gamble et al . assemblages from s. alterniflora growing in two differing tidal drainage systems within north inlet estuary showed seasonal variability but no significant site - influenced variability ( gamble et al . the host plants were the same in both locations ( short and tall growth forms of s. alterniflora ) and the subtle changes in edaphic parameters with changes in seasons shaped the seasonally responsive segments of the diazotroph assemblages in these two locations . assemblages from the rhizospheres of plant hosts in the crab haul creek basin that exhibit zonation due to elevation differences , contained host specific diazotrophs as well as diazotrophs having broad distributions across the landscape ( davis et al . a clear response of diazotroph assemblages to plant host zonation and seasonal changes in edaphic parameters was observed from principal component analysis ( pca ) of the recovered nifh amplicons ( gamble et al . ( 2011 ) confirmed the seasonal responses ( figure 3 ) and plant host specificity ( figure 4 ) observed by gamble et al . circles denote significance ( p < 0.001 ) for clustering . included in the phylogenetic analysis were plant zones ( with designations ) : j. roemerianus stand ( js ) , s. virginica ( sv ) , short form s. alterniflora ( ss ) , patch of j. roemerianus in the ss zone ( jp ) , and tall form s. alterniflora ( ts ) . using a significance threshold of p < 0.01 it was confirmed that diazotroph assemblages in the same marsh zone ( based on edaphic parameters ) had similar structures ( table 1 ) . phylogenetic analyses of the sequences recovered from these assemblages also revealed clusters of plant host specific sequences that were considered identical ( sensu venter et al . all pairs yielding a parsimony significance < 0.01 have significantly different structures from each other . multiple analyses of datasets obtained in several different studies provide strong evidence of plant host specific diazotroph assemblages associated with the rhizospheres of salt marsh plants . these organisms have been detected through both culture - dependent and culture - independent methods , in different sites throughout north inlet estuary , and over the course of more than a decade . the results of these studies also indicate substantial resilience of some members of the diazotroph assemblages that are capable of withstanding wide ranges of environmental conditions and are present in multiple marsh zones . studies of diazotroph assemblages associated with the salt marsh plants in north inlet have demonstrated a diverse yet stable assemblage . dgge analysis of diazotroph assemblages in long- and short - term fertilization experiments ( fertilized with 16.3 g n m nh4no3 and 18.0 g p m p2o5 ) confirmed the stability of these assemblages ( piceno and lovell , 2000a , b ) . the results of these experiments indicate that salt marsh diazotrophs are quite competitive with non - diazotrophs and are able to maintain their populations despite the energy expense of nitrogen fixation . the resilience of diazotrophs associated with salt marsh plants has also been observed during periods of extreme environmental conditions ( davis , 2010 ) . in stark contrast to the diversity of diazotrophs associated with the rhizospheres of salt marsh plants , nifh sequences recovered from standing dead s. alterniflora biomass from north inlet and sapelo island ( ga , usa ) salt marshes revealed a diazotroph assemblage with markedly lower diversity ( lovell et al . the analysis methods ( including nifh - specific pcr ) , which were also utilized in other studies , revealed that approximately 90% of nifh sequences from these assemblages were similar to those from -proteobacteria . this demonstrates that the methods of nifh sequence recovery employed in these studies are sound , as the -proteobacteria are poorly represented in most salt marsh rhizosphere diazotroph assemblages ( lovell et al . for example , figures 57 show the small number of nifh sequences that are similar to those from -proteobacteria ( * -labeled clusters in tables a1 and a4 in appendix ) in comparison to the large number of recovered sequences related to those from the -proteobacteria . ( 2010 ) revealed that changes in edaphic parameters associated with plant senescence , tidal intrusion , and nutrient cycling produced different responses from the seasonally ( growing season / summer vs. plant senescence / winter ) responsive groups within the diazotroph assemblages ( gamble et al . some clades were also recovered throughout all seasons , indicating a resilient portion of the assemblage . these diazotrophs are capable of competing for resources and maintaining populations over seasonal changes in both biotic and abiotic environmental variables . ( 2011 ) demonstrated broadly distributed ( and mostly novel ) , frequently detected ( fd ) diazotroph sequences that did not respond detectably to seasonal changes ( davis et al . sequences were also identified ( via statistical tests in pca ) as contributing significantly to the structure of the diazotroph assemblages ( lv sequences from bands producing long eigenvectors in euclidean bi - plot analysis of pca results ; for full explanation see davis et al . they have been recovered from multiple sites in north inlet estuary and differ from those recovered from other ecosystems . it has been determined in terrestrial ecosystems that free - living diazotrophs are functionally significant ( brgmann et al . it is reasonable to assume , based on the results of these studies , that these organisms are equally important in salt marsh ecosystems . throughout the phylograms are clades containing virtually identical sequences recovered from host plants in the same marsh zone ( plant host labeled clusters in bolded font in figures 57 , and sequences listed in tables a2 and a4 in appendix ) , indicating niche specificity of these diazotrophs for the edaphic parameters present in these zones . these data have given insight into the distributions of diazotroph populations throughout the nitrogen - limited salt marsh ecosystem of north inlet , sc , usa . these diazotrophs are specialized for the niches within this ecosystem , whether the niches are defined by the microenvironment of the plant host , or by marsh zone , or by both . they maintain populations that are readily detectable by methods employed in multiple studies ( particularly nifh based pcr and dgge fingerprinting ) and influence ecosystem function as well as diazotroph assemblage structure . studies of this salt marsh ecosystem have revealed that the structure of a functional microbial assemblage can be predicted , based on specific environmental parameters . plant hosts and edaphic factors that vary in response to elevation are the key parameters that predict the biogeography of these organisms . on a continental scale , the biogeography of microbial communities in terrestrial ecosystems is predicted by abiotic parameters , particularly soil ph , rather than by geographic distance or vegetation type ( fierer and jackson , 2006 ) . these patterns have been observed in this estuarine system and can be used to predict biogeographical patterns of diazotroph assemblages associated with salt marsh grass rhizospheres . diazotroph assemblages of salt marsh ecosystems are also capable of resisting relatively large changes in environmental parameters to maintain detectable and functionally important populations ( davis , 2010 ) and can be understood in the context of biogeographical concepts that have been applied to macroorganisms . strong evidence of plant host specificity and niche specialization in these assemblages has been compiled in this review ; however the methods used to obtain the data to support these hypotheses are not perfect . based on current climate models , coastal salt marshes are in danger of being completely submerged by the end of the century ( nicholls et al . however , the ever increasing number of completely submerged wetlands indicates that the current rate of sea level rise may exceed the abilities of ecogeomorphic feedbacks to preserve tidal ecosystems , particularly salt marshes ( kirwan et al . , 2010 ) and consequently loss of microorganisms restricted to a particular plant host or microenvironment ( berg and smalla , 2009 ) . the ability to predict biogeographical distributions of microorganisms as critical to ecosystem function as diazotrophs are to salt marshes will be advantageous to ecosystem modelers , especially those involved in determining the effects of global climate change . these data clearly show that the distribution of diazotrophs is non - random and is driven directly or indirectly by edaphic parameters in their habitat . the biogeographical patterns observed in north inlet estuary can be applied to tidally dominated coastal wetlands when the edaphic parameters of the system in question are taken into consideration .

endoplasmic reticulum inner mitochondrial membrane inositol 1 , 4 , 5-trisphosphate receptor membrane - associated membrane mitochondrial ca uniporter mitochondrial permeability transition pore mechanistic target of rapamycin complex 2 outer mitochondrial membrane rna - dependent protein kinase ( pkr)-like er kinase promyelocytic leukemia phosphatase and tensin homolog deleted on chromosome 10 reactive oxygen species voltage - dependent anion channel over the past 15 years , alternative subcellular districts have been described as pivotal sites of action for several oncogenes and oncosuppressors . although the core apoptotic machinery is tightly controlled at the transcriptional level , a series of post - translational mechanisms , such as translocation to different intracellular compartments , phosphorylation events , and protein protein interactions , represent important aspects of regulation of the apoptotic pathway . although the nucleus has been identified as the main target of different oncogenes , such as c - myc , and tumor suppressors , such as p53 , it is now clear that coordination of the apoptotic process occurs at multiple cellular levels , far removed from the biochemical scheme of one protein these unconventional pathways probably act in synergy with transcription - dependent pathways , and in some pathological contexts they function not only to support the nuclear mechanisms but can also be considered the primary molecular route . among the different non - nuclear activities , mitochondria are the major site of localization for oncogenes and oncosuppressors because of their central role as integrators and transducers for proapoptotic signals . however , protein targeting to mitochondria generally requires the presence of specific import signals ; therefore , a large number of proteins can not easily enter mitochondria but instead exert their effects in the surrounding zone , especially in the contact areas between the endoplasmic reticulum ( er ) and mitochondria . membrane - bound organelles exchange metabolic signals and information through the formation of specific membrane contact sites , and the er - mitochondria interface represents one such connection . the mitochondria and er join together at multiple contact sites , forming a specified subcellular fraction that is currently termed mitochondria - associated membranes ( mam ) . the er and mitochondria not only physically couple but also establish a tight communication that plays a crucial role in several processes , such as lipid trafficking , calcium ( ca)-transfer , inflammation , and apoptosis . nevertheless , several regulatory factors are shared by the 2 organelles , leading to the establishment of a molecular platform to receive and decode a wide range of inputs , including apoptotic signals . during cellular stress , er - mitochondria connections lead to the prompt activation of caspase - dependent and caspase - independent cell death effector mechanisms based on the capacity of the 2 organelles to sense and react to a multiple array of danger signals . thus , the mam can be conceived as a physical and functional scaffold for the primary response to alterations of cellular homeostasis , and it is therefore logical that oncogenes and tumor suppressors can localize at or move to mam upon cellular stress . this review will address the structure of mam , the main apoptotic - related functions of mam ( with a focus on reactive oxygen species ( ros ) and ca exchange ) and the proteins that regulate the cell death pathways through their localization to the er - mitochondria interface ( table 1 ) . mam , mitochondria - associated er membranes ; mt , mitochondria ; pam , plasma membrane associated membranes ; er , endoplasmic reticulum ; nu , nucleus ; cyt , cytosol ; pm , plasma membraneproteinlocalizationfunctions at mammam interactorsreferencesp66shcmam , pam , cyt , mtcytosolic adaptor protein involved in cellular response to oxidative stressunknownaktcyt , nu , mamserine / threonine protein kinase . inhibition of ca release from er ; antiapoptotic functionsbad , bax , hk2 , ip3r , pacs2 , pten , pml , mtorc2bcl-2nu , er , mt , cyt , maminduction of ca leakage from er ; antiapoptotic functionsbad , bcl - xl , ip3rbcl - xlnu , cyt , mtinduction of ca leakage from er ; antiapoptotic functionsbad , bcl-2 , ip3rbadcyt , mt , erproapoptotic functionsbcl-2 , bcl - xlbaxcyt , mt , erproapoptotic functionsakt , bcl-2hk2mtglucose phosphorylation ; antiapoptotic functionsakt , vdac1ptencyt , mt , mam , numost commonly lost or mutated tumor suppressor in human cancers ; negative regulator of akt , regulation of ca2 + release via ip3r3 ; proapoptotic functionsakt , ip3r , pp2apmlmam , er , nuimplicated in the pathogenesis of leukemia and other cancers ; negative regulator of akt ; proapoptotic functionsakt , ip3r , pp2amtorc2 ( mtor complex 2)er , mt , mamserine / threonine protein kinase ; akt activator ; control of mam integrity ; regulation of ca uptake ; regulation of mitochondrial bioenergetics ; antiapoptotic functions.akt , pacs-2sig1-rmam / ermolecular chaperone stabilizing the conformation of proteins at the mam ; promotes cellular survival ; antiapoptotic functionsgrp78/bip , ( ip3r3)grp78/biper , mt , cyt , mam , nuer chaperone - folding and assembly of membrane or secreted proteins ; stabilizes ip3r3 at mamsig1-r , clucluer , mt , cytstress - induced chaperone ; antiapoptotic functionsgrp78 sig1-rmcl-1mt , nu , er , maminduction of ca leakage from er ; antiapoptotic functionsbok , ip3rk - ras4bpm , er , mt , cytregulation of bcl - xl activity ; antiapoptotic functionsip3r , bcl - xlh - ras12vmam , pam , mt , cyt , regulation of ca signaling ; antiapoptotic functionscaveolin-1k - rasmam , pam , mt , cytinhibition of ca release from er and reduction of er calevels ; antiapoptotic functionsip3rvmiamt , maminhibition of apoptosisbaxhbxomm , cyt , nuinduction of mitochondrial fragmentation and mitophagy ; induction of dysfunction of permeability transition pore ( ptp ) complexvdac3enterovirus 2b proteiner , golgi - derived vesiclesregulation of ca homeostasis ; antiapoptotic functionsunknownbokgolgi , er , mam , cytupstream of bax and bak in control of the transmission of er / mam - derived apoptotic signals toward mitochondria ; proapoptotic activitymcl-1 , ip3rero1-mam , erkey controller of oxidative folding and er redox homeostasis ; enriched at mam and regulates ca fluxesip3r , pac-1fis1mam , mtformation of a tripartite protein complex with procaspase-8 and bap31 ; induction of apoptosis.bap31bap31,mam , er , ommformation of a tripartite protein complex with procaspase-8 and fis1 ; induction of apoptosis.fis1 , caspase-8perkmam , erinvolved in folded protein response during er stress ; physically increases contacts between mitochondria and er summary of the most important oncogenes and oncosuppressors discussed in this review . mam , mitochondria - associated er membranes ; mt , mitochondria ; pam , plasma membrane associated membranes ; er , endoplasmic reticulum ; nu , nucleus ; cyt , cytosol ; pm , plasma membrane it has been demonstrated that , in order to function properly , mitochondria need to communicate with other organelles and intracellular structures . such communication between the er and mitochondria can occur at close contact sites between organelles , even without the direct fusion of interacting membranes ( fig . studies on the specific interactions between mitochondria ( which form an efficient calcium ion buffer ) and the er ( the main intracellular calcium store ) were initiated by copeland and dalton in the late 1950s . these pioneer studies were performed in cells of the pseudobranch gland of teleost fish and showed that part of the er exists in association with mitochondria . approximately 10 years later , other groups visualized these contacts in the rat liver and the onion stem by electron microscopy . initial evaluation of the extent of the mitochondrial involvement in such interactions gave surprisingly high values , indicating that approximately 80 % of the mitochondrion interacted with the er . in contrast , later studies showed that only 520 % of the mitochondrial surface interacts with the er . initially , studies with electron microscopy tomography estimated the distance between the er and mitochondria as 100 nm , but later studies by achleitner et al . indicated that this distance varied , and was in the range of only 1060 nm . more recent data further reduced this distance to 1025 nm , which allows proteins from the er to associate directly with proteins and lipids present at the outer surface of the mitochondrial membrane . protocols describing the isolation of er - mitochondrial contacts indicated that these interactions are strong enough to be preserved upon subcellular fractionation . the subcellular fraction that was enriched for the contact sites between mitochondria and er was named the mitochondria - associated membrane ( mam ) fraction . a detailed protocol to isolate the mam fraction was first described by jean vance in the early 1990s . since then , the isolation procedure has been improved and adapted to isolate the mam fraction from different organs , tissues , and various cell lines as well as from yeast . the isolated mam fraction is composed of membrane shreds from both the er and outer mitochondrial membrane ( omm ) that were in close contact at the time of subcellular fractionation . more recently , the mam fraction has also been regarded as an intracellular lipid raft of detergent - resistant domains of the er . the contact sites between mitochondria and the er are dynamically formed as a direct result of a stochastic apposition of er with mitochondria and are dependent on intracellular signaling . the variety of roles played by the mam fraction , as described in the literature , is related to their unique lipid and protein composition . studies performed in the last decade revealed the molecular components of the mam fraction , demonstrating that it contains several proteins ( more than 75 according to raturi and simmen ) and is crucial for many cellular processes , including protein sorting , inflammation , er stress , ca handling , lipid synthesis , trafficking , and apoptosis . the localization of some proteins in the mam fraction and the extent of their enrichment are still under debate because their connection to the mam fraction is unclear . bcl-2 counteracts bax bak activities both at mitochondria and er sides of the mam . akt phosphorylates ip3r , reducing ca release from the er . at the mitochondria , akt promotes the association between hk-2 and vdac . bap31 , caspase 8 , and fis1 form a platform to transduce the cell death signals between the er and mitochondria . bak , bcl-2-antagonist killer ; bap31 , b - cell receptor - associated protein 31 ; bax , bcl-2-associated x protein ; bcl-2 , b - cell cll / lymphoma 2 ; bcl - xl , b - cell lymphoma - extra large protein ; bok , bcl-2-related ovarian killer ; clu , clusterin ; c sub , c subunit of mitochondrial atp synthase ; cyt . c , cytochrome c ; er , endoplasmic reticulum ; ero1- , er oxidoreductin-1 ; fis1 , fission 1 homolog ; grp75 , glucose regulated protein 75 ; grp78 , glucose regulated protein 78 ; hk2 , hexokinase 2 ; ip3r , inositol 1,4,5 trisphosphate receptor ; mcl-1 , myeloid cell leukemia sequence 1 ; mcu , mitochondrial calcium uniporter ; mptp , mitochondrial permeability transition pore ; mtorc2 , mechanistic target of rapamycin complex 2 ; perk , rna - dependent protein kinase ( pkr)-like er kinase ; pml , promyelocytic leukemia protein ; pro - casp . 8 , pro - caspase 8 ; pten , phosphatase and tensin homolog deleted on chromosome 10 ; serca , sarco / endoplasmatic reticulum ca atpase ; sig1-r , sigma 1 receptor ; vdac , voltage - dependent anion channel . oncogenes and oncosuppressors at mitochondria - associated membranes . oncogenes and tumor suppressors acting at the er bcl-2 counteracts bax bak activities both at mitochondria and er sides of the mam . akt phosphorylates ip3r , reducing ca release from the er . at the mitochondria , akt promotes the association between hk-2 and vdac . bap31 , caspase 8 , and fis1 form a platform to transduce the cell death signals between the er and mitochondria . bak , bcl-2-antagonist killer ; bap31 , b - cell receptor - associated protein 31 ; bax , bcl-2-associated x protein ; bcl-2 , b - cell cll / lymphoma 2 ; bcl - xl , b - cell lymphoma - extra large protein ; bok , bcl-2-related ovarian killer ; clu , clusterin ; c sub , c subunit of mitochondrial atp synthase ; cyt . c , cytochrome c ; er , endoplasmic reticulum ; ero1- , er oxidoreductin-1 ; fis1 , fission 1 homolog ; grp75 , glucose regulated protein 75 ; grp78 , glucose regulated protein 78 ; hk2 , hexokinase 2 ; ip3r , inositol 1,4,5 trisphosphate receptor ; mcl-1 , myeloid cell leukemia sequence 1 ; mcu , mitochondrial calcium uniporter ; mptp , mitochondrial permeability transition pore ; mtorc2 , mechanistic target of rapamycin complex 2 ; perk , rna - dependent protein kinase ( pkr)-like er kinase ; pml , promyelocytic leukemia protein ; pro - casp . 8 , pro - caspase 8 ; pten , phosphatase and tensin homolog deleted on chromosome 10 ; serca , sarco / endoplasmatic reticulum ca atpase ; sig1-r , sigma 1 receptor ; vdac , voltage - dependent anion channel originally , the mam fraction was described as the location of lipid synthesis and trafficking between the er and mitochondrial membranes based on the presence of long - chain fatty acid - coa ligase type 4 ( facl4 ) and phosphatidylserine synthase-1 ( pss-1 ) enzymes . the close apposition of mitochondria to the er also explains the selective transmission of physiological and pathological ca and ros signals directly from the er to the mitochondria . the mam also contains ca signaling elements of both organelles , thus supporting the central role of er / mitochondria crosstalk in signal transduction . therefore , the er - mitochondria contact sites can be considered specialized microdomains for the transfer of ca signals . ca ions released from the er by inositol 1,4,5-trisphosphate receptors ( ip3rs ) cross the freely permeable omm through voltage - dependent anion channels ( vdacs ) , reach the inner mitochondrial membrane ( imm ) , and accumulate in the matrix via the mitochondrial ca uniporter ( mcu ) complex . close apposition between the er and mitochondria ensures the formation of microdomains at high [ ca ] that overcome the low apparent ca affinity of the mcu . hence , at the " ca hotspot " stage , the local [ ca ] is > 10 m , allowing rapid ca transduction to the matrix despite the low ca affinity of the uniporter pore ( fig . , the mam chaperone glucose regulated protein 75 ( grp75 ) mediates the interaction between the ip3r and vdac , structurally linking the ca efflux system at the er with the channels at the omm to favor positive regulation of mitochondrial ca uptake . interestingly , vdac1 , but not vdac2 and vdac3 , interacts with ip3r , sustaining transmission of the low - amplitude apoptotic ca signals to mitochondria . although ca exchange between the er and mitochondria serves as a regulator of cellular bioenergetics , accumulation of ca can trigger opening of the mitochondrial permeability transition pore ( mptp ) , leading to release of proapoptotic factors , such as cytochrome c , into the cytosol . the molecular nature of the mptp is still controversial , but recent evidence suggests the involvement of new structural components in pore formation , in particular the c subunit of mitochondrial atp synthase . conversely , the molecular composition of the mcu complex has been determined and its importance in the regulation of cell death pathways has been described in many cellular environments . notably , silencing of a core component of the mcu complex , the regulatory subunit mitochondrial calcium uptake 1 ( micu1 ) , exposes the mitochondria to drastic ca accumulation at basal conditions , produces ros , and triggers the apoptotic process . the er and mitochondria are 2 of the major sites for ros production inside the cell . exchange of ros takes place at the mam , and this ros trafficking has a wide relevance in many pathological contexts , especially during er stress ( as discussed under " mam as a strategic platform for oncosuppressor - dependent cell death " ) . therefore , many regulators of the oxidative state of the cell are located at the mam . p66shc , a cytosolic adaptor protein involved in a cellular response to oxidative stress , has recently been identified at mitochondria - er association sites . there is a positive relationship between the level of p66shc and the rate of cell proliferation in prostate cancer cells . this relationship is particularly relevant to steroid - induced signaling and elevated levels of ros , which act as secondary messengers in cancer cells . the upregulation of ros production by androgen or estrogens is accompanied by an increase in p66shc , thereby promoting cell proliferation in the aforementioned types of cancer cells . the association of p66shc with mitochondrial ros production has also been repeatedly documented in our previous studies . the level of p66shc in the mam fraction increases in an age - dependent manner and correlates with mitochondrial ros production , which has also been found to increase with age . as cited in the aforementioned studies , ca and ros transfer is the main apoptotic - related function of mam . by localizing at er - mitochondria membranes , oncogenes and oncosuppressors survival signals involving akt activation include both the caspase cascade and transcriptional control of apoptosis . upon growth factor stimulation , phosphorylated akt translocates to the nucleus from the cytoplasm via an activation process . through a phosphorylation cascade , akt promotes nuclear exclusion ( cytoplasmic retention ) of transcription factors of the forkhead family , thereby preventing transcription of the proapoptotic genes fas ligand , bim , trail , and tradd . additionally , phosphorylation of mdm2 by akt is necessary for localization of mdm2 to the nucleus , where it associates with p53 to target its ubiquitination . moreover , akt - dependent nf-b nuclear translocation promotes the transcription of antiapoptotic genes , such as bfl1 , ciap1 , and ciap2 . in addition to its transcriptional activity , akt has also been physically and functionally linked to both the er and the mitochondria . akt - dependent phosphorylation of the proapoptotic bh3-only protein bad causes its dissociation from the bcl-2/bcl - xl complex at the outer mitochondrial membrane , inhibiting its cell death functions . similar to its relationship with bad , phosphorylation of bax by akt results in inhibition of the apoptotic features of bax , such as oligomerization , insertion into mitochondrial membrane , and the formation of large pores to allow the release of proapoptotic factors . akt also phosphorylates hexokinase 2 ( hk2 ) to promote its association with the mam protein vdac1 . this association not only affects the metabolic state of the cell by increasing efficiency and rate of the glycolytic pathway , but also protects cells from apoptosis . vdac1 binding seems to promote the closed state of the channel , preventing ca - dependent opening of the mptp and release of the proapoptotic protein cytochrome c. furthermore , at the er side of mam , akt phosphorylates all ip3r isoforms , inhibits ca release from er , and protects cells from apoptosis . our group showed that akt inhibits ca fluxes and apoptosis in the er by preferentially phosphorylating the type 3 ip3r ( ip3r3 ) . ip3r3 is mainly localized to the mam , suggesting a pivotal role of the proto - oncogene akt at the er - mitochondria interface . these results provided evidence for the hypothesis of individuation at mam of both negative regulators of akt ( the tumor suppressors pten and pml , see section below ) and the akt activator mechanistic target of rapamycin complex 2 ( mtorc2 ) . previous observations suggest that mtorc2 can interact with both the er and the mitochondria . indeed , mtorc2 resides at the mam , where it regulates the phosphorylation state of ip3r3 and ca release from the er . mtorc2 controls mam integrity , at least in part , via akt - dependent phosphorylation of phosphofurin acidic cluster sorting protein 2 ( pacs2 ) , as suggested by the observation that pacs2 is a substrate of akt and is required for mam integrity . moreover , mtorc2 controls mitochondrial functions and physiology in an akt - dependent manner through hk2 phosphorylation . thus , mtorc2 localization to mam can be considered the ideal link to the multiple apoptotic - related functions of akt , underlining the crucial role of the proto - oncogene at er mitochondria contact sites . as in the case of akt , strategic positioning at the mam and regulation of ca fluxes bcl-2 , the patriarch of the family , is highly enriched at the mam , where it functions at both the er and the mitochondrial side to exert its antiapoptotic function . at the mitochondria , bcl-2 binds bax / bak , preventing their oligomerization and inhibiting bax / bak pore formation ( for a review , see ) . at the er , bcl-2 modulates ca transfer by decreasing net influx into the er through increased ca leakage rather than reduced ca release . increased ca leakage results in reduced ca transfer to mitochondria and inhibits apoptosis . nevertheless , bax / bak double knockout cells were shown to have a reduced steady state of er ca , and hence are protected from a variety of apoptotic challenges . however , an akt - like function ( i.e. , inhibition of er ca release ) , rather than augmented ca leakage , has also been suggested for bcl-2 , as the antiapoptotic ca effect of bcl-2 might be due to direct interaction of its bh4 domain with ip3r . in either case , bcl-2 promotes survival in multiple cellular environments by limiting ca transfer from the er to the mitochondria . interestingly , bcl-2 expression , but not stabilization , is significantly regulated by the mam protein sigma-1 receptor ( sig1-r ) . as a molecular chaperone , sig1-r interacts with many effectors at mam , but not with bcl-2 . conversely , sig1-r transcriptionally controls the expression of bcl-2 by regulating the ros / nf-b pathway . of note , the sig1-r - mediated downregulation of bcl-2 mrna is abolished by ros scavengers and by the inhibition of nf-b . thus , sig1-r affects cell survival through the regulation of bcl-2 levels , revealing that increased mam activities during stress conditions can directly impact the response at nuclear level . sig-1r is a molecular chaperone that stabilizes the conformation of proteins at the mam , such as ip3r3 or the er stress sensor ire1 . sig1-r has been implicated in several human diseases , and one of its most important functions is its robust cellular protective effect . sig1-r agonists have been shown to promote cellular survival by preventing oxidative stress caused by multiple pathological conditions . moreover , sig1-r plays a crucial role in the control of er - mitochondrial interorganelle ca signaling . sig1-r at the mam forms a complex with grp78 ( also known as bip ) to regulate ca homeostasis between the er and the mitochondria through ip3r . grp78 is primarily located in the er lumen , but under er stress a significant pool of grp78 is localized in different subcellular compartments , such as the cytosol and mitochondria . a recent study reported the association between grp78 and clusterin ( clu ) , a stress - induced multifunctional secreted and cytoplasmic molecular chaperone , under er stress conditions . this interaction elicits clu redistribution on the mitochondria , promoting survival of prostate cancer cells during treatment stress . at er - mitochondria contact sites , bcl - xl interacts with ip3rs and sensitizes them to low ip3 concentrations , thus reducing er ca concentrations , stimulating mitochondrial energy , and preserving survival . the same molecular mechanism is shared by myeloid cell leukemia sequence 1 ( bcl-2-related ) ( mcl-1 ) , providing additional evidence for the crucial role of ca leakage in the prosurvival functions of the antiapoptotic bcl-2 family subgroup . the localization of bcl - xl at er and mitochondria indicates its distinct roles in cell death and ca homeostasis . using cell lines derived from er- and mitochondria - targeted bcl - xl chimeras that are deficient for bcl - xl , li and co - workers showed that er - targeted bcl - xl is required to restore ca homeostasis in knockout cells , whereas mitochondrial localization alone is sufficient to provide protection . recently , the bcl - xl activity at er has been linked to a specific form of the ras oncoprotein , k - ras4b . phosphorylation of k - ras4b by protein kinase c promotes its translocation from the plasma membrane to the er and omm , which is associated with induction of the cell death pathway . k - ras4b associates with ip3r , limiting the ability of bcl - xl to sensitize ip3r to the activity of its ligand ip3 and thereby abolishing its typical antiapoptotic role . interestingly , our group has shown that oncogenic h - ras ( h - ras12v ) is localized at both mam and plasma membrane - associated membranes , suggesting a cooperation between the plasma membrane , the er , and the mitochondria that is essential for ca signaling and the maintenance of ca homeostasis in cancer progression . moreover , oncogenic k - ras inhibits ca release from er , reduces er ca levels , and suppresses ca influx to the mitochondria in colon cancer cell lines . thus , multiple forms of ras act at the er mitochondria interface to manipulate ca transfer , which in turn contributes to the prosurvival properties of ras that are associated with the oncogenic phenotype . in addition to its interaction with cancer - related proteins , the oncogenic functions of mam extend to some viruses . human cytomegalovirus encodes multiple antiapoptotic proteins , including viral mitochondrion - localized inhibitor of apoptosis ( vmia ) . vmia resides at the mam , where it re - targets proapoptotic bax to the er / mam lipid drafts to induce bax poly - ubiquitination , proteasome degradation , and consequent inhibition of apoptosis . human hepatitis b virus ( hbv ) is associated with chronic liver disease and with the development of hepatocellular carcinoma . hbv encodes the regulatory protein hbx , which localizes at the omm and interacts with the mam protein vdac3 . hbv / hbx induces mitochondrial fragmentation and mitophagy ( the selective autophagic - removal of damaged mitochondria ) , leading to apoptosis attenuation and most likely viral persistence . lastly , enteroviruses , such as coxsackievirus , poliovirus , and echovirus , confer an antiapoptotic state that not only suppresses the host defense mechanisms but is also protective against cell death induced by pharmacological treatments . the enterovirus 2b protein is localized to the surface of the er- and golgi - derived membrane vesicles where viral replication takes place . expression of 2b protein decreases the steady - state ca levels of both the er and golgi , reducing the mitochondrial ca content and suppressing caspase activation and apoptotic cell death induced by various stimuli . in recent years it has been demonstrated that many oncosuppressor proteins are localized to the er and at mam ( fig . , different antiapoptotic members of the bcl-2 family appear to play an important role in modulating ca - dependent apoptotic signals within the mitochondria at the er and mam side . in contrast , various proapoptotic members of the same family exert an opposite effect on the er ca stores , and thus on the amplitude of ca signals reaching the key effector during apoptosis , the mitochondria . in the first phase of bax protein overexpression , before the catastrophic changes in mitochondrial and er morphology and other intracellular parameters , there is an increase in er [ ca ] levels . this higher er [ ca ] content correlates with an increase in mitochondrial ca loading after activation by stimuli causing the release of ca from the er ca stores . as discussed above , these results agree with findings from bax / bak knockout embryonic fibroblasts , in which a dramatic reduction in [ ca]er , was observed . bcl-2 and bax / bak proteins primarily target the ip3r type 1 to affect [ ca]er . indeed , downregulation of ip3r1 counteracted the reduction of [ ca]er in cells from bax / bak knockout animals , which is consistent with the correlation between low expression of ip3r and inhibition of apoptosis . these data indicate that bax / bak directly counteract the effect of bcl-2 on ca signaling . recently , another proapoptotic member of the bcl-2 family , bcl-2-related ovarian killer ( bok ) has recently been described as localized to the er and the mam . bok has been shown to selectively interact with mcl-1 and bfl-1/a1 , but not with bcl-2 or bcl - xl . although bok shares high sequence similarity with bax and bak , bok is not a surrogate of these proteins and is unable to compensate for the combined loss of bax and bak in triggering mptp opening and cell death via apoptosis . in contrast , bok - induced apoptosis is almost blunted in the absence of bax or bak , and cells lacking mcl-1 are significantly more sensitive to bok - induced apoptosis than control cells . these observations suggest that bok may function upstream of bax and bak in the control of the transmission of er / mam - derived apoptotic signals toward mitochondria . furthermore , it has been recently shown that bok can interact with ip3r channels , affecting ip3r levels by protecting the proteins from proteolytic degradation without modifying their ability to release er calcium stores . however , the role of bok in regulation of the apoptotic process remains to be elucidated , especially considering recent results obtained in vivo that describe a minimal impact of loss of bok in mice . indeed , bok deficiency in lymphoid and myeloid cells fails to confer any protection against a wide range of apoptotic stimuli and loss of bok does not accelerate lymphoma development in c - myc - overexpressing transgenic mice . involvement of the mam domain is also important in cell death independent of the bcl-2 family members . indeed , procaspase - activating compound-1 ( pac-1 ) ( a small molecule that converts procaspase-3 to caspase-3 in cancer cells in vitro and in vivo ) does not require bax and bak but is dependent on the engagement of mam through er oxidoreductin-1 ( ero1- ) . ero1- , a key controller of oxidative folding and er redox homeostasis , is enriched at mam and regulates ca fluxes . the efficacy of pac-1 in activating caspase-3 requires cytochrome c release from the mitochondria , which is induced by mitochondrial ca overload and an increase in mitochondrial ros through er stress that is mediated by p53 upregulated modulator of apoptosis ( puma ) and ero1-. er stress induces er ca release that is preferentially transferred into the mitochondria because pac-1 treatment leads to an increase in the number of mam via the upregulation of ero1-. additionally , the mam is an important site for the recruitment and processing of procaspase-8 to caspase-8 . the proteins participating in this complex are b - cell - receptor - associated protein 31 ( bap31 ) at the er and fission 1 homolog ( fis1 ) at the mitochondria . the physical interaction between bap31 and fis1 provides a tethering force between the er and the mitochondria , thus facilitating the mam structure . during the apoptotic program bap31 this processing occurs at the mam with the formation of a tripartite protein complex between procaspase-8 , bap31 , and fis1 . the key role of the caspase - activated bap31fis1 complex at the mam is to transduce the cell death signals between the er and mitochondria . indeed , bap31 cleavage ( i.e. , activation ) results in a downstream increase in cytosolic [ ca ] caused by er ca release , leading to mitochondrial ca uptake . this dyshomeostasis of mitochondrial [ ca ] will trigger ptp opening , causing release of the mitochondrial cofactors into the cytosol to complete the apoptotic process . thus , the mam appears to be a hotspot domain for decoding different intracellular signals of stress . in light of this role , it is not surprising that the p66shc protein ( an important ros sensor involved in apoptosis ) and the rna - dependent protein kinase ( pkr)-like er kinase ( perk ) ( the er stress sensor of the unfolded protein response ) are present at the mam . the perk protein is particularly enriched at the mam , where it promotes efficient crosstalk between the er and the mitochondria based on the transfer of ca and ros . perk appears to be crucial for tethering the er to the mitochondria and thus for mam integrity . indeed , cells deficient in perk display a fragmented er structure , causing impairment in the ca signaling from the er to the mitochondria that is required for efficient apoptosis . moreover , destabilization of mam in the absence of perk prevents the rapid transfer of ros from the er to the mitochondria that is required for the insertion of bax into the outer mitochondrial membrane and release of cytochrome c from mitochondria via oxidation of the phospholipid cardiolipin . in recent years , the increased interest in mams , in particular their emerging role in controlling cell death , has led to in - depth analysis of other tumor suppressors that share this intracellular localization . the promyelocytic leukemia ( pml ) protein , which is encoded by a tumor suppressor gene implicated in the pathogenesis of leukemia and other cancers , displays both nuclear and cytosolic distribution . at the nucleus , pml forms multiprotein nuclear structures called pml - nuclear bodies ( pml - nbs ) . in the cytosol studies in knockout mice and cells revealed an essential pleiotropic role for pml in multiple p53-dependent and -independent apoptotic pathways . as a result , pml - null mice and cells are protected from apoptosis triggered by a number of stimuli . we were able to demonstrate that the er / mam localization of pml is essential for the apoptotic pathway and orchestrating ca homeostasis and , eventually , for cell death . in particular , pml modulates ip3r type 3 activities by promoting the formation of a multiprotein complex containing ip3r type 3 , akt , and the protein phosphatase pp2a . in the absence of pml , pp2a is unable to localize to the mam and thus unable to prevent the ip3r3 phosphorylation mediated by akt . as described above , hyperphosphorylation of ip3r3 inhibits ca transfer from the er to mitochondria , thereby inhibiting the apoptotic process . the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 ( pten ) also localizes to mam . pten is one of the most commonly lost or mutated tumor suppressors in human cancers , and germline mutations of pten have been found in cancer predisposition syndromes . pten is a phosphatase that has both lipid and dual - specificity protein phosphatase activity . its growth - attenuating activity has primarily been ascribed to the dephosphorylation of plasma membrane - localized pip3 . however , emerging evidence demonstrates that additional pten - dependent mechanisms are implicated in tumor suppression . indeed , pten at the mam regulates er ca release via ip3r3 in a protein phosphatase - dependent manner that counteracts akt phosphorylation of the ip3r3 . the final result of the action of pten on ip3rs is thus the maintenance of sustained activity of the ip3r3 during the apoptotic stimulation and , consequently , enhanced transfer of ca from the er to mitochondria . lastly , consistent with the importance of sustained ca transfer from the er to the mitochondria for the induction of apoptosis , it has been demonstrated that overexpression of the sarco / endoplasmatic reticulum ca atpase ( serca ) , a protein that is enriched at the mam , causes er [ ca ] overload , which increases spontaneous and induced apoptosis , favoring er - mitochondria ca transfer and leading to a breakdown of mitochondrial function . in conclusion , data emerging from recent literature highlight the mam as an important hub for the control and integration of apoptosis operated by different oncosuppressors via different signal transduction mechanisms that in many cases share the same ca signal key effectors . the mam can be conceived as hotspot sites for ca homeostasis , and any perturbation of the fine regulation of ca signaling can induce tumor suppressors to switch from a survival role to a cell death mechanism . it has been demonstrated that , in order to function properly , mitochondria need to communicate with other organelles and intracellular structures . such communication between the er and mitochondria can occur at close contact sites between organelles , even without the direct fusion of interacting membranes ( fig . 1 ) . studies on the specific interactions between mitochondria ( which form an efficient calcium ion buffer ) and the er ( the main intracellular calcium store ) were initiated by copeland and dalton in the late 1950s . these pioneer studies were performed in cells of the pseudobranch gland of teleost fish and showed that part of the er exists in association with mitochondria . approximately 10 years later , other groups visualized these contacts in the rat liver and the onion stem by electron microscopy . initial evaluation of the extent of the mitochondrial involvement in such interactions gave surprisingly high values , indicating that approximately 80 % of the mitochondrion interacted with the er . in contrast , later studies showed that only 520 % of the mitochondrial surface interacts with the er . initially , studies with electron microscopy tomography estimated the distance between the er and mitochondria as 100 nm , but later studies by achleitner et al . indicated that this distance varied , and was in the range of only 1060 nm . more recent data further reduced this distance to 1025 nm , which allows proteins from the er to associate directly with proteins and lipids present at the outer surface of the mitochondrial membrane . protocols describing the isolation of er - mitochondrial contacts indicated that these interactions are strong enough to be preserved upon subcellular fractionation . the subcellular fraction that was enriched for the contact sites between mitochondria and er was named the a detailed protocol to isolate the mam fraction was first described by jean vance in the early 1990s . since then , the isolation procedure has been improved and adapted to isolate the mam fraction from different organs , tissues , and various cell lines as well as from yeast . the isolated mam fraction is composed of membrane shreds from both the er and outer mitochondrial membrane ( omm ) that were in close contact at the time of subcellular fractionation . more recently , the mam fraction has also been regarded as an intracellular lipid raft of detergent - resistant domains of the er . the contact sites between mitochondria and the er are dynamically formed as a direct result of a stochastic apposition of er with mitochondria and are dependent on intracellular signaling . thus , mam composition is transient and can be changing at any given time . the variety of roles played by the mam fraction , as described in the literature , is related to their unique lipid and protein composition . studies performed in the last decade revealed the molecular components of the mam fraction , demonstrating that it contains several proteins ( more than 75 according to raturi and simmen ) and is crucial for many cellular processes , including protein sorting , inflammation , er stress , ca handling , lipid synthesis , trafficking , and apoptosis . however , the localization of some proteins in the mam fraction and the extent of their enrichment are still under debate because their connection to the mam fraction is unclear . bcl-2 counteracts bax bak activities both at mitochondria and er sides of the mam . akt phosphorylates ip3r , reducing ca release from the er . at the mitochondria , akt promotes the association between hk-2 and vdac . bap31 , caspase 8 , and fis1 form a platform to transduce the cell death signals between the er and mitochondria . bak , bcl-2-antagonist killer ; bap31 , b - cell receptor - associated protein 31 ; bax , bcl-2-associated x protein ; bcl-2 , b - cell cll / lymphoma 2 ; bcl - xl , b - cell lymphoma - extra large protein ; bok , bcl-2-related ovarian killer ; clu , clusterin ; c sub , c subunit of mitochondrial atp synthase ; cyt . c , cytochrome c ; er , endoplasmic reticulum ; ero1- , er oxidoreductin-1 ; fis1 , fission 1 homolog ; grp75 , glucose regulated protein 75 ; grp78 , glucose regulated protein 78 ; hk2 , hexokinase 2 ; ip3r , inositol 1,4,5 trisphosphate receptor ; mcl-1 , myeloid cell leukemia sequence 1 ; mcu , mitochondrial calcium uniporter ; mptp , mitochondrial permeability transition pore ; mtorc2 , mechanistic target of rapamycin complex 2 ; perk , rna - dependent protein kinase ( pkr)-like er kinase ; pml , promyelocytic leukemia protein ; pro - casp . 8 , pro - caspase 8 ; pten , phosphatase and tensin homolog deleted on chromosome 10 ; serca , sarco / endoplasmatic reticulum ca atpase ; sig1-r , sigma 1 receptor ; vdac , voltage - dependent anion channel . oncogenes and oncosuppressors at mitochondria - associated membranes . oncogenes and tumor suppressors acting at the er mitochondria interface are shown as red hexagons and green octagons , respectively . bcl-2 counteracts bax bak activities both at mitochondria and er sides of the mam . akt phosphorylates ip3r , reducing ca release from the er . at the mitochondria , akt promotes the association between hk-2 and vdac . bap31 , caspase 8 , and fis1 form a platform to transduce the cell death signals between the er and mitochondria . bak , bcl-2-antagonist killer ; bap31 , b - cell receptor - associated protein 31 ; bax , bcl-2-associated x protein ; bcl-2 , b - cell cll / lymphoma 2 ; bcl - xl , b - cell lymphoma - extra large protein ; bok , bcl-2-related ovarian killer ; clu , clusterin ; c sub , c subunit of mitochondrial atp synthase ; cyt . c , cytochrome c ; er , endoplasmic reticulum ; ero1- , er oxidoreductin-1 ; fis1 , fission 1 homolog ; grp75 , glucose regulated protein 75 ; grp78 , glucose regulated protein 78 ; hk2 , hexokinase 2 ; ip3r , inositol 1,4,5 trisphosphate receptor ; mcl-1 , myeloid cell leukemia sequence 1 ; mcu , mitochondrial calcium uniporter ; mptp , mitochondrial permeability transition pore ; mtorc2 , mechanistic target of rapamycin complex 2 ; perk , rna - dependent protein kinase ( pkr)-like er kinase ; pml , promyelocytic leukemia protein ; pro - casp . 8 , pro - caspase 8 ; pten , phosphatase and tensin homolog deleted on chromosome 10 ; serca , sarco / endoplasmatic reticulum ca atpase ; sig1-r , sigma 1 receptor ; vdac , voltage - dependent anion channel . originally , the mam fraction was described as the location of lipid synthesis and trafficking between the er and mitochondrial membranes based on the presence of long - chain fatty acid - coa ligase type 4 ( facl4 ) and phosphatidylserine synthase-1 ( pss-1 ) enzymes . the close apposition of mitochondria to the er also explains the selective transmission of physiological and pathological ca and ros signals directly from the er to the mitochondria . the mam also contains ca signaling elements of both organelles , thus supporting the central role of er / mitochondria crosstalk in signal transduction . therefore , the er - mitochondria contact sites can be considered specialized microdomains for the transfer of ca signals . ca ions released from the er by inositol 1,4,5-trisphosphate receptors ( ip3rs ) cross the freely permeable omm through voltage - dependent anion channels ( vdacs ) , reach the inner mitochondrial membrane ( imm ) , and accumulate in the matrix via the mitochondrial ca uniporter ( mcu ) complex . close apposition between the er and mitochondria ensures the formation of microdomains at high [ ca ] that overcome the low apparent ca affinity of the mcu . hence , at the " ca hotspot " stage , the local [ ca ] is > 10 m , allowing rapid ca transduction to the matrix despite the low ca affinity of the uniporter pore ( fig . 1 ) . at the molecular level , the mam chaperone glucose regulated protein 75 ( grp75 ) mediates the interaction between the ip3r and vdac , structurally linking the ca efflux system at the er with the channels at the omm to favor positive regulation of mitochondrial ca uptake . interestingly , vdac1 , but not vdac2 and vdac3 , interacts with ip3r , sustaining transmission of the low - amplitude apoptotic ca signals to mitochondria . although ca exchange between the er and mitochondria serves as a regulator of cellular bioenergetics , accumulation of ca can trigger opening of the mitochondrial permeability transition pore ( mptp ) , leading to release of proapoptotic factors , such as cytochrome c , into the cytosol . the molecular nature of the mptp is still controversial , but recent evidence suggests the involvement of new structural components in pore formation , in particular the c subunit of mitochondrial atp synthase . conversely , the molecular composition of the mcu complex has been determined and its importance in the regulation of cell death pathways has been described in many cellular environments . notably , silencing of a core component of the mcu complex , the regulatory subunit mitochondrial calcium uptake 1 ( micu1 ) , exposes the mitochondria to drastic ca accumulation at basal conditions , produces ros , and triggers the apoptotic process . the er and mitochondria are 2 of the major sites for ros production inside the cell . exchange of ros takes place at the mam , and this ros trafficking has a wide relevance in many pathological contexts , especially during er stress ( as discussed under " mam as a strategic platform for oncosuppressor - dependent cell death " ) . therefore , many regulators of the oxidative state of the cell are located at the mam . p66shc , a cytosolic adaptor protein involved in a cellular response to oxidative stress , has recently been identified at mitochondria - er association sites . there is a positive relationship between the level of p66shc and the rate of cell proliferation in prostate cancer cells . this relationship is particularly relevant to steroid - induced signaling and elevated levels of ros , which act as secondary messengers in cancer cells . the upregulation of ros production by androgen or estrogens is accompanied by an increase in p66shc , thereby promoting cell proliferation in the aforementioned types of cancer cells . the association of p66shc with mitochondrial ros production has also been repeatedly documented in our previous studies . the level of p66shc in the mam fraction increases in an age - dependent manner and correlates with mitochondrial ros production , which has also been found to increase with age . as cited in the aforementioned studies , ca and ros transfer is the main apoptotic - related function of mam . by localizing at er - mitochondria membranes , oncogenes and oncosuppressors survival signals involving akt activation include both the caspase cascade and transcriptional control of apoptosis . upon growth factor stimulation , phosphorylated akt translocates to the nucleus from the cytoplasm via an activation process . through a phosphorylation cascade , akt promotes nuclear exclusion ( cytoplasmic retention ) of transcription factors of the forkhead family , thereby preventing transcription of the proapoptotic genes fas ligand , bim , trail , and tradd . additionally , phosphorylation of mdm2 by akt is necessary for localization of mdm2 to the nucleus , where it associates with p53 to target its ubiquitination . moreover , akt - dependent nf-b nuclear translocation promotes the transcription of antiapoptotic genes , such as bfl1 , ciap1 , and ciap2 . in addition to its transcriptional activity , akt has also been physically and functionally linked to both the er and the mitochondria . akt - dependent phosphorylation of the proapoptotic bh3-only protein bad causes its dissociation from the bcl-2/bcl - xl complex at the outer mitochondrial membrane , inhibiting its cell death functions . similar to its relationship with bad , phosphorylation of bax by akt results in inhibition of the apoptotic features of bax , such as oligomerization , insertion into mitochondrial membrane , and the formation of large pores to allow the release of proapoptotic factors . akt also phosphorylates hexokinase 2 ( hk2 ) to promote its association with the mam protein vdac1 . this association not only affects the metabolic state of the cell by increasing efficiency and rate of the glycolytic pathway , but also protects cells from apoptosis . vdac1 binding seems to promote the closed state of the channel , preventing ca - dependent opening of the mptp and release of the proapoptotic protein cytochrome c. furthermore , at the er side of mam , akt phosphorylates all ip3r isoforms , inhibits ca release from er , and protects cells from apoptosis . our group showed that akt inhibits ca fluxes and apoptosis in the er by preferentially phosphorylating the type 3 ip3r ( ip3r3 ) . ip3r3 is mainly localized to the mam , suggesting a pivotal role of the proto - oncogene akt at the er - mitochondria interface . these results provided evidence for the hypothesis of individuation at mam of both negative regulators of akt ( the tumor suppressors pten and pml , see section below ) and the akt activator mechanistic target of rapamycin complex 2 ( mtorc2 ) . indeed , mtorc2 resides at the mam , where it regulates the phosphorylation state of ip3r3 and ca release from the er . mtorc2 deficiency , as well as akt downregulation , causes mam disruption . mtorc2 controls mam integrity , at least in part , via akt - dependent phosphorylation of phosphofurin acidic cluster sorting protein 2 ( pacs2 ) , as suggested by the observation that pacs2 is a substrate of akt and is required for mam integrity . moreover , mtorc2 controls mitochondrial functions and physiology in an akt - dependent manner through hk2 phosphorylation . thus , mtorc2 localization to mam can be considered the ideal link to the multiple apoptotic - related functions of akt , underlining the crucial role of the proto - oncogene at er mitochondria contact sites . as in the case of akt , strategic positioning at the mam and regulation of ca fluxes bcl-2 , the patriarch of the family , is highly enriched at the mam , where it functions at both the er and the mitochondrial side to exert its antiapoptotic function . at the mitochondria , bcl-2 binds bax / bak , preventing their oligomerization and inhibiting bax / bak pore formation ( for a review , see ) . at the er , bcl-2 modulates ca transfer by decreasing net influx into the er through increased ca leakage rather than reduced ca release . increased ca leakage results in reduced ca transfer to mitochondria and inhibits apoptosis . nevertheless , bax / bak double knockout cells were shown to have a reduced steady state of er ca , and hence are protected from a variety of apoptotic challenges . however , an akt - like function ( i.e. , inhibition of er ca release ) , rather than augmented ca leakage , has also been suggested for bcl-2 , as the antiapoptotic ca effect of bcl-2 might be due to direct interaction of its bh4 domain with ip3r . in either case , bcl-2 promotes survival in multiple cellular environments by limiting ca transfer from the er to the mitochondria . interestingly , bcl-2 expression , but not stabilization , is significantly regulated by the mam protein sigma-1 receptor ( sig1-r ) . as a molecular chaperone , sig1-r interacts with many effectors at mam , but not with bcl-2 . conversely , sig1-r transcriptionally controls the expression of bcl-2 by regulating the ros / nf-b pathway . of note , the sig1-r - mediated downregulation of bcl-2 mrna is abolished by ros scavengers and by the inhibition of nf-b . thus , sig1-r affects cell survival through the regulation of bcl-2 levels , revealing that increased mam activities during stress conditions can directly impact the response at nuclear level . sig-1r is a molecular chaperone that stabilizes the conformation of proteins at the mam , such as ip3r3 or the er stress sensor ire1 . sig1-r has been implicated in several human diseases , and one of its most important functions is its robust cellular protective effect . sig1-r agonists have been shown to promote cellular survival by preventing oxidative stress caused by multiple pathological conditions . moreover , sig1-r plays a crucial role in the control of er - mitochondrial interorganelle ca signaling . sig1-r at the mam forms a complex with grp78 ( also known as bip ) to regulate ca homeostasis between the er and the mitochondria through ip3r . grp78 is primarily located in the er lumen , but under er stress a significant pool of grp78 is localized in different subcellular compartments , such as the cytosol and mitochondria . a recent study reported the association between grp78 and clusterin ( clu ) , a stress - induced multifunctional secreted and cytoplasmic molecular chaperone , under er stress conditions . this interaction elicits clu redistribution on the mitochondria , promoting survival of prostate cancer cells during treatment stress . at er - mitochondria contact sites , bcl - xl interacts with ip3rs and sensitizes them to low ip3 concentrations , thus reducing er ca concentrations , stimulating mitochondrial energy , and preserving survival . the same molecular mechanism is shared by myeloid cell leukemia sequence 1 ( bcl-2-related ) ( mcl-1 ) , providing additional evidence for the crucial role of ca leakage in the prosurvival functions of the antiapoptotic bcl-2 family subgroup . the localization of bcl - xl at er and mitochondria indicates its distinct roles in cell death and ca homeostasis . using cell lines derived from er- and mitochondria - targeted bcl - xl chimeras that are deficient for bcl - xl , li and co - workers showed that er - targeted bcl - xl is required to restore ca homeostasis in knockout cells , whereas mitochondrial localization alone is sufficient to provide protection . recently , the bcl - xl activity at er has been linked to a specific form of the ras oncoprotein , k - ras4b . phosphorylation of k - ras4b by protein kinase c promotes its translocation from the plasma membrane to the er and omm , which is associated with induction of the cell death pathway . k - ras4b associates with ip3r , limiting the ability of bcl - xl to sensitize ip3r to the activity of its ligand ip3 and thereby abolishing its typical antiapoptotic role . interestingly , our group has shown that oncogenic h - ras ( h - ras12v ) is localized at both mam and plasma membrane - associated membranes , suggesting a cooperation between the plasma membrane , the er , and the mitochondria that is essential for ca signaling and the maintenance of ca homeostasis in cancer progression . moreover , oncogenic k - ras inhibits ca release from er , reduces er ca levels , and suppresses ca influx to the mitochondria in colon cancer cell lines . thus , multiple forms of ras act at the er mitochondria interface to manipulate ca transfer , which in turn contributes to the prosurvival properties of ras that are associated with the oncogenic phenotype . in addition to its interaction with cancer - related proteins , the oncogenic functions of mam extend to some viruses . human cytomegalovirus encodes multiple antiapoptotic proteins , including viral mitochondrion - localized inhibitor of apoptosis ( vmia ) . vmia resides at the mam , where it re - targets proapoptotic bax to the er / mam lipid drafts to induce bax poly - ubiquitination , proteasome degradation , and consequent inhibition of apoptosis . human hepatitis b virus ( hbv ) is associated with chronic liver disease and with the development of hepatocellular carcinoma . hbv encodes the regulatory protein hbx , which localizes at the omm and interacts with the mam protein vdac3 . hbv / hbx induces mitochondrial fragmentation and mitophagy ( the selective autophagic - removal of damaged mitochondria ) , leading to apoptosis attenuation and most likely viral persistence . lastly , enteroviruses , such as coxsackievirus , poliovirus , and echovirus , confer an antiapoptotic state that not only suppresses the host defense mechanisms but is also protective against cell death induced by pharmacological treatments . the enterovirus 2b protein is localized to the surface of the er- and golgi - derived membrane vesicles where viral replication takes place . expression of 2b protein decreases the steady - state ca levels of both the er and golgi , reducing the mitochondrial ca content and suppressing caspase activation and apoptotic cell death induced by various stimuli . in recent years it has been demonstrated that many oncosuppressor proteins are localized to the er and at mam ( fig . , different antiapoptotic members of the bcl-2 family appear to play an important role in modulating ca - dependent apoptotic signals within the mitochondria at the er and mam side . in contrast , various proapoptotic members of the same family exert an opposite effect on the er ca stores , and thus on the amplitude of ca signals reaching the key effector during apoptosis , the mitochondria . in the first phase of bax protein overexpression , before the catastrophic changes in mitochondrial and er morphology and other intracellular parameters , there is an increase in er [ ca ] levels . this higher er [ ca ] content correlates with an increase in mitochondrial ca loading after activation by stimuli causing the release of ca from the er ca stores . as discussed above , these results agree with findings from bax / bak knockout embryonic fibroblasts , in which a dramatic reduction in [ ca]er , was observed . bcl-2 and bax / bak proteins primarily target the ip3r type 1 to affect [ ca]er . indeed , downregulation of ip3r1 counteracted the reduction of [ ca]er in cells from bax / bak knockout animals , which is consistent with the correlation between low expression of ip3r and inhibition of apoptosis . these data indicate that bax / bak directly counteract the effect of bcl-2 on ca signaling . recently , another proapoptotic member of the bcl-2 family , bcl-2-related ovarian killer ( bok ) has recently been described as localized to the er and the mam . bok has been shown to selectively interact with mcl-1 and bfl-1/a1 , but not with bcl-2 or bcl - xl . although bok shares high sequence similarity with bax and bak , bok is not a surrogate of these proteins and is unable to compensate for the combined loss of bax and bak in triggering mptp opening and cell death via apoptosis . in contrast , bok - induced apoptosis is almost blunted in the absence of bax or bak , and cells lacking mcl-1 are significantly more sensitive to bok - induced apoptosis than control cells . these observations suggest that bok may function upstream of bax and bak in the control of the transmission of er / mam - derived apoptotic signals toward mitochondria . furthermore , it has been recently shown that bok can interact with ip3r channels , affecting ip3r levels by protecting the proteins from proteolytic degradation without modifying their ability to release er calcium stores . however , the role of bok in regulation of the apoptotic process remains to be elucidated , especially considering recent results obtained in vivo that describe a minimal impact of loss of bok in mice . indeed , bok deficiency in lymphoid and myeloid cells fails to confer any protection against a wide range of apoptotic stimuli and loss of bok does not accelerate lymphoma development in c - myc - overexpressing transgenic mice . involvement of the mam domain is also important in cell death independent of the bcl-2 family members . indeed , procaspase - activating compound-1 ( pac-1 ) ( a small molecule that converts procaspase-3 to caspase-3 in cancer cells in vitro and in vivo ) does not require bax and bak but is dependent on the engagement of mam through er oxidoreductin-1 ( ero1- ) . ero1- , a key controller of oxidative folding and er redox homeostasis , is enriched at mam and regulates ca fluxes . the efficacy of pac-1 in activating caspase-3 requires cytochrome c release from the mitochondria , which is induced by mitochondrial ca overload and an increase in mitochondrial ros through er stress that is mediated by p53 upregulated modulator of apoptosis ( puma ) and ero1-. er stress induces er ca release that is preferentially transferred into the mitochondria because pac-1 treatment leads to an increase in the number of mam via the upregulation of ero1-. additionally , the mam is an important site for the recruitment and processing of procaspase-8 to caspase-8 . the proteins participating in this complex are b - cell - receptor - associated protein 31 ( bap31 ) at the er and fission 1 homolog ( fis1 ) at the mitochondria . the physical interaction between bap31 and fis1 provides a tethering force between the er and the mitochondria , thus facilitating the mam structure . during the apoptotic program bap31 this processing occurs at the mam with the formation of a tripartite protein complex between procaspase-8 , bap31 , and fis1 . the key role of the caspase - activated bap31fis1 complex at the mam is to transduce the cell death signals between the er and mitochondria . indeed , bap31 cleavage ( i.e. , activation ) results in a downstream increase in cytosolic [ ca ] caused by er ca release , leading to mitochondrial ca uptake . this dyshomeostasis of mitochondrial [ ca ] will trigger ptp opening , causing release of the mitochondrial cofactors into the cytosol to complete the apoptotic process . thus , the mam appears to be a hotspot domain for decoding different intracellular signals of stress . in light of this role , it is not surprising that the p66shc protein ( an important ros sensor involved in apoptosis ) and the rna - dependent protein kinase ( pkr)-like er kinase ( perk ) ( the er stress sensor of the unfolded protein response ) are present at the mam . the perk protein is particularly enriched at the mam , where it promotes efficient crosstalk between the er and the mitochondria based on the transfer of ca and ros . perk appears to be crucial for tethering the er to the mitochondria and thus for mam integrity . indeed , cells deficient in perk display a fragmented er structure , causing impairment in the ca signaling from the er to the mitochondria that is required for efficient apoptosis . moreover , destabilization of mam in the absence of perk prevents the rapid transfer of ros from the er to the mitochondria that is required for the insertion of bax into the outer mitochondrial membrane and release of cytochrome c from mitochondria via oxidation of the phospholipid cardiolipin . in recent years , the increased interest in mams , in particular their emerging role in controlling cell death , has led to in - depth analysis of other tumor suppressors that share this intracellular localization . the promyelocytic leukemia ( pml ) protein , which is encoded by a tumor suppressor gene implicated in the pathogenesis of leukemia and other cancers , displays both nuclear and cytosolic distribution . at the nucleus , pml forms multiprotein nuclear structures called pml - nuclear bodies ( pml - nbs ) . in the cytosol studies in knockout mice and cells revealed an essential pleiotropic role for pml in multiple p53-dependent and -independent apoptotic pathways . as a result , pml - null mice and cells are protected from apoptosis triggered by a number of stimuli . we were able to demonstrate that the er / mam localization of pml is essential for the apoptotic pathway and orchestrating ca homeostasis and , eventually , for cell death . in particular , pml modulates ip3r type 3 activities by promoting the formation of a multiprotein complex containing ip3r type 3 , akt , and the protein phosphatase pp2a . in the absence of pml , pp2a is unable to localize to the mam and thus unable to prevent the ip3r3 phosphorylation mediated by akt . as described above , hyperphosphorylation of ip3r3 inhibits ca transfer from the er to mitochondria , thereby inhibiting the apoptotic process . the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 ( pten ) also localizes to mam . pten is one of the most commonly lost or mutated tumor suppressors in human cancers , and germline mutations of pten have been found in cancer predisposition syndromes . pten is a phosphatase that has both lipid and dual - specificity protein phosphatase activity . its growth - attenuating activity has primarily been ascribed to the dephosphorylation of plasma membrane - localized pip3 . however , emerging evidence demonstrates that additional pten - dependent mechanisms are implicated in tumor suppression . indeed , pten at the mam regulates er ca release via ip3r3 in a protein phosphatase - dependent manner that counteracts akt phosphorylation of the ip3r3 . the final result of the action of pten on ip3rs is thus the maintenance of sustained activity of the ip3r3 during the apoptotic stimulation and , consequently , enhanced transfer of ca from the er to mitochondria . lastly , consistent with the importance of sustained ca transfer from the er to the mitochondria for the induction of apoptosis , it has been demonstrated that overexpression of the sarco / endoplasmatic reticulum ca atpase ( serca ) , a protein that is enriched at the mam , causes er [ ca ] overload , which increases spontaneous and induced apoptosis , favoring er - mitochondria ca transfer and leading to a breakdown of mitochondrial function . in conclusion , data emerging from recent literature highlight the mam as an important hub for the control and integration of apoptosis operated by different oncosuppressors via different signal transduction mechanisms that in many cases share the same ca signal key effectors . the mam can be conceived as hotspot sites for ca homeostasis , and any perturbation of the fine regulation of ca signaling can induce tumor suppressors to switch from a survival role to a cell death mechanism . the large body of knowledge reviewed here depicts mam as a regulatory scaffold for the control of cell death . many factors that localize to the nucleus modulate apoptosis at a transcriptional level but can also reside or translocate to the mam under stress conditions . evidence for this role is the reciprocal transmission of different signals between the er and mitochondria through physical contact . additionally , mam may be considered the primary platform for the detection of intracellular danger . we have also reported that manipulation of ca fluxes represents the primary method of action for several oncogenes and tumor suppressors located at the mam . ip3r channels , including ip3rs present at mam , represent the key targets of different oncogenic and oncosuppressive proteins . indeed , regulation of ca flux through ip3r is also mediated by ros ; superoxide anions cause oxidation of the ip3r and sensitization of ca release to promote cytoplasmic ca oscillations and mitochondrial uptake . in addition to ca input , the mam provides specialized contact sites for transmitting ros - mediated signals . in this regard , the role of the mam protein sig1-r as an interorganelle signaling modulator has recently been reported , providing a new mechanism whereby mam controls cell fate by conveying the ros message from the mitochondria to the nucleus . characterization of mam as the main decoder of intracellular danger signals implicates a role for mam dynamics in several physiopathological scenarios .

the different mechanisms employed by proto - oncogenes and tumor suppressors to regulate cell death pathways are strictly linked to their localization . in addition to the canonical control of apoptosis at a transcriptional / nuclear level , intracellular zones are emerging as pivotal sites for the activities of several proapoptotic and antiapoptotic factors . here , we review the function of the endoplasmic reticulum - mitochondria interface as a primary platform for decoding danger signals as well as a structural accommodation for several regulator or effector proteins .

Abbreviations Introduction MAM structure Oncogenic function of MAM MAM as a strategic platform for oncosuppressor-dependent cell death Conclusion Disclosure of Potential Conflicts of Interest

endoplasmic reticulum inner mitochondrial membrane inositol 1 , 4 , 5-trisphosphate receptor membrane - associated membrane mitochondrial ca uniporter mitochondrial permeability transition pore mechanistic target of rapamycin complex 2 outer mitochondrial membrane rna - dependent protein kinase ( pkr)-like er kinase promyelocytic leukemia phosphatase and tensin homolog deleted on chromosome 10 reactive oxygen species voltage - dependent anion channel over the past 15 years , alternative subcellular districts have been described as pivotal sites of action for several oncogenes and oncosuppressors . although the core apoptotic machinery is tightly controlled at the transcriptional level , a series of post - translational mechanisms , such as translocation to different intracellular compartments , phosphorylation events , and protein protein interactions , represent important aspects of regulation of the apoptotic pathway . although the nucleus has been identified as the main target of different oncogenes , such as c - myc , and tumor suppressors , such as p53 , it is now clear that coordination of the apoptotic process occurs at multiple cellular levels , far removed from the biochemical scheme of one protein these unconventional pathways probably act in synergy with transcription - dependent pathways , and in some pathological contexts they function not only to support the nuclear mechanisms but can also be considered the primary molecular route . among the different non - nuclear activities , mitochondria are the major site of localization for oncogenes and oncosuppressors because of their central role as integrators and transducers for proapoptotic signals . membrane - bound organelles exchange metabolic signals and information through the formation of specific membrane contact sites , and the er - mitochondria interface represents one such connection . during cellular stress , er - mitochondria connections lead to the prompt activation of caspase - dependent and caspase - independent cell death effector mechanisms based on the capacity of the 2 organelles to sense and react to a multiple array of danger signals . thus , the mam can be conceived as a physical and functional scaffold for the primary response to alterations of cellular homeostasis , and it is therefore logical that oncogenes and tumor suppressors can localize at or move to mam upon cellular stress . this review will address the structure of mam , the main apoptotic - related functions of mam ( with a focus on reactive oxygen species ( ros ) and ca exchange ) and the proteins that regulate the cell death pathways through their localization to the er - mitochondria interface ( table 1 ) . inhibition of ca release from er ; antiapoptotic functionsbad , bax , hk2 , ip3r , pacs2 , pten , pml , mtorc2bcl-2nu , er , mt , cyt , maminduction of ca leakage from er ; antiapoptotic functionsbad , bcl - xl , ip3rbcl - xlnu , cyt , mtinduction of ca leakage from er ; antiapoptotic functionsbad , bcl-2 , ip3rbadcyt , mt , erproapoptotic functionsbcl-2 , bcl - xlbaxcyt , mt , erproapoptotic functionsakt , bcl-2hk2mtglucose phosphorylation ; antiapoptotic functionsakt , vdac1ptencyt , mt , mam , numost commonly lost or mutated tumor suppressor in human cancers ; negative regulator of akt , regulation of ca2 + release via ip3r3 ; proapoptotic functionsakt , ip3r , pp2apmlmam , er , nuimplicated in the pathogenesis of leukemia and other cancers ; negative regulator of akt ; proapoptotic functionsakt , ip3r , pp2amtorc2 ( mtor complex 2)er , mt , mamserine / threonine protein kinase ; akt activator ; control of mam integrity ; regulation of ca uptake ; regulation of mitochondrial bioenergetics ; antiapoptotic functions.akt , pacs-2sig1-rmam / ermolecular chaperone stabilizing the conformation of proteins at the mam ; promotes cellular survival ; antiapoptotic functionsgrp78/bip , ( ip3r3)grp78/biper , mt , cyt , mam , nuer chaperone - folding and assembly of membrane or secreted proteins ; stabilizes ip3r3 at mamsig1-r , clucluer , mt , cytstress - induced chaperone ; antiapoptotic functionsgrp78 sig1-rmcl-1mt , nu , er , maminduction of ca leakage from er ; antiapoptotic functionsbok , ip3rk - ras4bpm , er , mt , cytregulation of bcl - xl activity ; antiapoptotic functionsip3r , bcl - xlh - ras12vmam , pam , mt , cyt , regulation of ca signaling ; antiapoptotic functionscaveolin-1k - rasmam , pam , mt , cytinhibition of ca release from er and reduction of er calevels ; antiapoptotic functionsip3rvmiamt , maminhibition of apoptosisbaxhbxomm , cyt , nuinduction of mitochondrial fragmentation and mitophagy ; induction of dysfunction of permeability transition pore ( ptp ) complexvdac3enterovirus 2b proteiner , golgi - derived vesiclesregulation of ca homeostasis ; antiapoptotic functionsunknownbokgolgi , er , mam , cytupstream of bax and bak in control of the transmission of er / mam - derived apoptotic signals toward mitochondria ; proapoptotic activitymcl-1 , ip3rero1-mam , erkey controller of oxidative folding and er redox homeostasis ; enriched at mam and regulates ca fluxesip3r , pac-1fis1mam , mtformation of a tripartite protein complex with procaspase-8 and bap31 ; induction of apoptosis.bap31bap31,mam , er , ommformation of a tripartite protein complex with procaspase-8 and fis1 ; induction of apoptosis.fis1 , caspase-8perkmam , erinvolved in folded protein response during er stress ; physically increases contacts between mitochondria and er summary of the most important oncogenes and oncosuppressors discussed in this review . since then , the isolation procedure has been improved and adapted to isolate the mam fraction from different organs , tissues , and various cell lines as well as from yeast . oncogenes and tumor suppressors acting at the er bcl-2 counteracts bax bak activities both at mitochondria and er sides of the mam . therefore , the er - mitochondria contact sites can be considered specialized microdomains for the transfer of ca signals . hence , at the " ca hotspot " stage , the local [ ca ] is > 10 m , allowing rapid ca transduction to the matrix despite the low ca affinity of the uniporter pore ( fig . although ca exchange between the er and mitochondria serves as a regulator of cellular bioenergetics , accumulation of ca can trigger opening of the mitochondrial permeability transition pore ( mptp ) , leading to release of proapoptotic factors , such as cytochrome c , into the cytosol . conversely , the molecular composition of the mcu complex has been determined and its importance in the regulation of cell death pathways has been described in many cellular environments . the er and mitochondria are 2 of the major sites for ros production inside the cell . exchange of ros takes place at the mam , and this ros trafficking has a wide relevance in many pathological contexts , especially during er stress ( as discussed under " mam as a strategic platform for oncosuppressor - dependent cell death " ) . by localizing at er - mitochondria membranes , oncogenes and oncosuppressors survival signals involving akt activation include both the caspase cascade and transcriptional control of apoptosis . in addition to its transcriptional activity , akt has also been physically and functionally linked to both the er and the mitochondria . akt - dependent phosphorylation of the proapoptotic bh3-only protein bad causes its dissociation from the bcl-2/bcl - xl complex at the outer mitochondrial membrane , inhibiting its cell death functions . ip3r3 is mainly localized to the mam , suggesting a pivotal role of the proto - oncogene akt at the er - mitochondria interface . these results provided evidence for the hypothesis of individuation at mam of both negative regulators of akt ( the tumor suppressors pten and pml , see section below ) and the akt activator mechanistic target of rapamycin complex 2 ( mtorc2 ) . thus , mtorc2 localization to mam can be considered the ideal link to the multiple apoptotic - related functions of akt , underlining the crucial role of the proto - oncogene at er mitochondria contact sites . the same molecular mechanism is shared by myeloid cell leukemia sequence 1 ( bcl-2-related ) ( mcl-1 ) , providing additional evidence for the crucial role of ca leakage in the prosurvival functions of the antiapoptotic bcl-2 family subgroup . recently , the bcl - xl activity at er has been linked to a specific form of the ras oncoprotein , k - ras4b . phosphorylation of k - ras4b by protein kinase c promotes its translocation from the plasma membrane to the er and omm , which is associated with induction of the cell death pathway . thus , multiple forms of ras act at the er mitochondria interface to manipulate ca transfer , which in turn contributes to the prosurvival properties of ras that are associated with the oncogenic phenotype . in addition to its interaction with cancer - related proteins , the oncogenic functions of mam extend to some viruses . vmia resides at the mam , where it re - targets proapoptotic bax to the er / mam lipid drafts to induce bax poly - ubiquitination , proteasome degradation , and consequent inhibition of apoptosis . the enterovirus 2b protein is localized to the surface of the er- and golgi - derived membrane vesicles where viral replication takes place . recently , another proapoptotic member of the bcl-2 family , bcl-2-related ovarian killer ( bok ) has recently been described as localized to the er and the mam . although bok shares high sequence similarity with bax and bak , bok is not a surrogate of these proteins and is unable to compensate for the combined loss of bax and bak in triggering mptp opening and cell death via apoptosis . the key role of the caspase - activated bap31fis1 complex at the mam is to transduce the cell death signals between the er and mitochondria . moreover , destabilization of mam in the absence of perk prevents the rapid transfer of ros from the er to the mitochondria that is required for the insertion of bax into the outer mitochondrial membrane and release of cytochrome c from mitochondria via oxidation of the phospholipid cardiolipin . in recent years , the increased interest in mams , in particular their emerging role in controlling cell death , has led to in - depth analysis of other tumor suppressors that share this intracellular localization . we were able to demonstrate that the er / mam localization of pml is essential for the apoptotic pathway and orchestrating ca homeostasis and , eventually , for cell death . pten is one of the most commonly lost or mutated tumor suppressors in human cancers , and germline mutations of pten have been found in cancer predisposition syndromes . lastly , consistent with the importance of sustained ca transfer from the er to the mitochondria for the induction of apoptosis , it has been demonstrated that overexpression of the sarco / endoplasmatic reticulum ca atpase ( serca ) , a protein that is enriched at the mam , causes er [ ca ] overload , which increases spontaneous and induced apoptosis , favoring er - mitochondria ca transfer and leading to a breakdown of mitochondrial function . in conclusion , data emerging from recent literature highlight the mam as an important hub for the control and integration of apoptosis operated by different oncosuppressors via different signal transduction mechanisms that in many cases share the same ca signal key effectors . the mam can be conceived as hotspot sites for ca homeostasis , and any perturbation of the fine regulation of ca signaling can induce tumor suppressors to switch from a survival role to a cell death mechanism . oncogenes and tumor suppressors acting at the er mitochondria interface are shown as red hexagons and green octagons , respectively . therefore , the er - mitochondria contact sites can be considered specialized microdomains for the transfer of ca signals . hence , at the " ca hotspot " stage , the local [ ca ] is > 10 m , allowing rapid ca transduction to the matrix despite the low ca affinity of the uniporter pore ( fig . conversely , the molecular composition of the mcu complex has been determined and its importance in the regulation of cell death pathways has been described in many cellular environments . the er and mitochondria are 2 of the major sites for ros production inside the cell . exchange of ros takes place at the mam , and this ros trafficking has a wide relevance in many pathological contexts , especially during er stress ( as discussed under " mam as a strategic platform for oncosuppressor - dependent cell death " ) . by localizing at er - mitochondria membranes , oncogenes and oncosuppressors survival signals involving akt activation include both the caspase cascade and transcriptional control of apoptosis . in addition to its transcriptional activity , akt has also been physically and functionally linked to both the er and the mitochondria . akt - dependent phosphorylation of the proapoptotic bh3-only protein bad causes its dissociation from the bcl-2/bcl - xl complex at the outer mitochondrial membrane , inhibiting its cell death functions . ip3r3 is mainly localized to the mam , suggesting a pivotal role of the proto - oncogene akt at the er - mitochondria interface . these results provided evidence for the hypothesis of individuation at mam of both negative regulators of akt ( the tumor suppressors pten and pml , see section below ) and the akt activator mechanistic target of rapamycin complex 2 ( mtorc2 ) . thus , mtorc2 localization to mam can be considered the ideal link to the multiple apoptotic - related functions of akt , underlining the crucial role of the proto - oncogene at er mitochondria contact sites . the same molecular mechanism is shared by myeloid cell leukemia sequence 1 ( bcl-2-related ) ( mcl-1 ) , providing additional evidence for the crucial role of ca leakage in the prosurvival functions of the antiapoptotic bcl-2 family subgroup . recently , the bcl - xl activity at er has been linked to a specific form of the ras oncoprotein , k - ras4b . phosphorylation of k - ras4b by protein kinase c promotes its translocation from the plasma membrane to the er and omm , which is associated with induction of the cell death pathway . thus , multiple forms of ras act at the er mitochondria interface to manipulate ca transfer , which in turn contributes to the prosurvival properties of ras that are associated with the oncogenic phenotype . vmia resides at the mam , where it re - targets proapoptotic bax to the er / mam lipid drafts to induce bax poly - ubiquitination , proteasome degradation , and consequent inhibition of apoptosis . the enterovirus 2b protein is localized to the surface of the er- and golgi - derived membrane vesicles where viral replication takes place . recently , another proapoptotic member of the bcl-2 family , bcl-2-related ovarian killer ( bok ) has recently been described as localized to the er and the mam . these observations suggest that bok may function upstream of bax and bak in the control of the transmission of er / mam - derived apoptotic signals toward mitochondria . the key role of the caspase - activated bap31fis1 complex at the mam is to transduce the cell death signals between the er and mitochondria . moreover , destabilization of mam in the absence of perk prevents the rapid transfer of ros from the er to the mitochondria that is required for the insertion of bax into the outer mitochondrial membrane and release of cytochrome c from mitochondria via oxidation of the phospholipid cardiolipin . in recent years , the increased interest in mams , in particular their emerging role in controlling cell death , has led to in - depth analysis of other tumor suppressors that share this intracellular localization . we were able to demonstrate that the er / mam localization of pml is essential for the apoptotic pathway and orchestrating ca homeostasis and , eventually , for cell death . pten is one of the most commonly lost or mutated tumor suppressors in human cancers , and germline mutations of pten have been found in cancer predisposition syndromes . lastly , consistent with the importance of sustained ca transfer from the er to the mitochondria for the induction of apoptosis , it has been demonstrated that overexpression of the sarco / endoplasmatic reticulum ca atpase ( serca ) , a protein that is enriched at the mam , causes er [ ca ] overload , which increases spontaneous and induced apoptosis , favoring er - mitochondria ca transfer and leading to a breakdown of mitochondrial function . in conclusion , data emerging from recent literature highlight the mam as an important hub for the control and integration of apoptosis operated by different oncosuppressors via different signal transduction mechanisms that in many cases share the same ca signal key effectors . the mam can be conceived as hotspot sites for ca homeostasis , and any perturbation of the fine regulation of ca signaling can induce tumor suppressors to switch from a survival role to a cell death mechanism . the large body of knowledge reviewed here depicts mam as a regulatory scaffold for the control of cell death . many factors that localize to the nucleus modulate apoptosis at a transcriptional level but can also reside or translocate to the mam under stress conditions . additionally , mam may be considered the primary platform for the detection of intracellular danger . we have also reported that manipulation of ca fluxes represents the primary method of action for several oncogenes and tumor suppressors located at the mam . in addition to ca input , the mam provides specialized contact sites for transmitting ros - mediated signals . in this regard , the role of the mam protein sig1-r as an interorganelle signaling modulator has recently been reported , providing a new mechanism whereby mam controls cell fate by conveying the ros message from the mitochondria to the nucleus .

toll - like receptors ( tlrs ) are pattern recognition receptors of the innate immune system that recognize specific pathogen - associated molecular patterns ( pamps ) conserved among microorganisms . there are currently twelve known tlrs in mammals , which identify common constituents of invading pathogens including double - stranded and single - stranded rna , unmethylated cpg dna , bacterial lipopolysaccharide ( lps ) , lipoproteins , and flagellin . upon interaction with their ligands , tlrs signal through adapter proteins , most commonly myeloid differentiation primary response gene 88 ( myd88 ) , though tlr 3 alternatively signals via the tir - domain containing adaptor inducing interferon- ( trif ) adapter . these adapters recruit other molecules to initiate signaling cascades ultimately leading to the production of proinflammatory cytokines and type i ifn . these tlr responses are important in the functioning of both the innate and adaptive arms of immunity [ 4 , 5 ] . although tlrs are important for antimicrobial immunity , they have also been implicated in the pathogenesis of autoimmune diseases . for example , tlrs 2 and 4 have been identified as factors involved in the onset of type i diabetes mellitus [ 69 ] , and tlrs 16 are expressed by rheumatoid arthritis ( ra ) synovial fibroblasts and are thought to provoke joint inflammation in ra [ 10 , 11 ] . moreover , the nucleic acid binding tlrs 7 and 9 have been connected to both human and mouse models of systemic lupus erythematosus ( sle ) [ 1217 ] . because of these links between tlrs and autoimmunity , a great deal of work has been directed toward understanding how these receptors act in disease progression . two major possibilities arise in describing how tlrs might work in autoimmunity ; either they are stimulated by exogenous antigens , like viral ssrna , which then stimulate resident immune cells , or the tlrs recognize endogenous self - antigens to initiate and propagate inflammation and autoimmunity . the accumulation of evidence pointing towards tlrs in autoimmunity has opened the door for potential therapeutic interventions directed towards the modulation of toll - like receptors and their signaling pathways . since tlrs are normally responsive to microbial pathogens , there has been some speculation as to the application of tlr agonists as vaccine adjuvants to stimulate more robust immune responses [ 18 , 19 ] . on the other side of the spectrum , for autoimmunity , disease can result from aberrant hyperactive signaling ; therefore , the application of inhibitory , or antagonistic , tlr therapeutics is of considerable interest . a few inhibitors have been developed already and show some promise for potential human therapeutics . the focus of this paper is to summarize the present literature documenting how modulation of toll - like receptor pathways may be used as potential methods of treatment for autoimmunity , particularly sle . tlrs are localized to either the cell surface or endosomes of several cell types , most notably of antigen - presenting cells ( apcs ) such as dendritic cells [ 20 , 21 ] and b cells . under normal circumstances , tlrs aid in the identification and removal of materials that may be detrimental to the host organism ; these are usually of bacterial , viral , fungal , or protozoan origin [ 1 , 23 ] . because tlrs recognize common molecular motifs instead of specific peptide sequences , they maintain capacity for recognition of a broad repertoire of microbes . we know , with relative certainty , what most of the tlrs recognize and that the end result is inflammation , but how does this occur ? the cascade by which tlrs induce an inflammatory environment varies depending on the particular tlr that is stimulated . since we are more concerned with sle , we will concentrate on the signaling that takes place upon activation of nucleic acid - binding tlrs 3 , 7/8 , and 9 . ligand binding is thought to induce a conformational change resulting in interaction or close juxtaposition of the two cytosolic toll / il-1 receptor ( tir ) domains , thus providing an interface for adaptor protein binding and subsequent signal transduction . as described by several groups , myd88 is the most common of these adaptors and has been shown to be involved in signaling through all tlrs except tlr 3 [ 2427 ] . this adapter protein provides a scaffold for further interaction with il-1r - associated kinase ( irak ) 1 and 4 to propagate the signal to downstream effectors via phosphorylation . these activated kinases recruit tumor necrosis factor receptor - associated factor ( traf ) 6 , which is an e3 ubiquitin ligase required for activation of nfb by freeing it from its inhibitor , ib . in addition to this , interferon regulatory factors ( irfs ) 5 and 7 are also recruited to the myd88/irak / traf6 complex , where they can become phosphorylated and activated . ultimately , the activation of transcription factors nfb and irfs 5 and 7 results in their translocation into the nucleus where they initiate gene transcription and production of proinflammatory cytokines and type i ifn ( figure 1 ) [ 3 , 3135 ] . tlr 3 signaling is distinct from the previously described tlr 7 and 9 signaling pathways . instead of utilizing myd88 as an adaptor protein , tlr 3 uses trif . as with myd88 , trif also recruits additional proteins necessary for downstream signaling , including traf - family - member - associated nfb - activator - binding kinase 1 ( tbk1 ) , traf6 , and receptor - interacting protein 1 ( rip1 ) . trif interaction with tbk1 is necessary for the activation of irf3 , which is a transcription factor involved in the production of ifn- . tlr 3 can also activate nfb by the interaction of trif with traf6 or rip1 , which allow the transcription factor to migrate to the nucleus to induce target gene transcription [ 24 , 28 ] . the multiple proteins involved in tlr signaling yield many opportunities to inhibit this process . several endogenous inhibitors have been identified that halt , or at least impair , the signaling cascade , dampening the tlr - mediated production of inflammatory cytokines and type i ifn . the ability to modulate tlr signaling helps to maintain a homeostatic balance important for antimicrobial immunity while also preventing collateral damage to self - tissues . the inhibitory proteins important in this process target the receptors themselves , adapter molecules , and key kinases , as well as transcription factors to diminish the tlr - mediated production of inflammatory cytokines and type i ifn . triad3a acts as an e3 ubiquitin ligase promoting the degradation of tlr 4 and 9 . the degradation of these tlrs was shown to impair nfb activity upon stimulation with lps and cpg dna whereas sirna knockdown of triad3a enhanced lps and cpg dna - mediated nfb activity indicating a prominent role of triad3a in modulating tlr response . although triad3a appears to act specifically on tlr 4 and 9 , other inhibitors provide a more global impact on tlr signaling . as mentioned earlier , myd88 is an adapter protein downstream of all tlrs except tlr 3 . upon stimulation with lps or tnf , a splice - variant of myd88 , myd88s , can be induced which prevents activation of nfb . this short form of myd88 fails to recruit irak4 , impairing the ability to phosphorylate irak1 , therefore preventing the activation of nfb , although maintaining the ability to activate the transcription factor ap-1 . in addition , recently , an et al . found that the phosphatase , shp-1 , selectively impairs activation of nfb and subsequent production of proinflammatory cytokines while also inhibiting irak1 resulting in an increase in ifn- . kobayashi et al . showed that irak - m , which lacks the kinase activity of its counterparts irak1 and irak4 , suppresses the production of proinflammatory cytokines and proposed a model whereby irak - m prevents the dissociation of the irak1/irak4/myd88 complex to inhibit downstream nfb activation . likewise , the splice variant irak1c acts in a similar manner by interacting with myd88 and irak4 . like irak - m , irak1c can not be phosphorylated by irak4 and thereby inhibits the dissociation of the complex , preventing activation of nfb . two other inhibitors have been identified downstream of the irak kinases : tumor necrosis factor--induced protein 3 ( tnfaip3 , or a20 ) and irf4 . a20 is a ubiquitin - editing enzyme that deubiquitinates traf6 to inhibit the release of nfb from ib , thus inhibiting subsequent nfb - mediated gene transcription [ 3 , 44 ] . irf4 inhibits tlr signaling by competing for the same binding site on myd88 with irf5 . irf5 engages myd88 downstream of tlr stimulation in order to promote production of proinflammatory cytokines . by irf4 competing with irf5 for binding , we know that tlrs are involved in protective immunity to invading microorganisms , but what are the consequences of misregulated tlr activation meaning situations in which the tlr pathways are turned on too easily or activated by self - stimulus ? tlrs have been identified as instigators in type i diabetes , rheumatoid arthritis , and systemic lupus erythematosus . the nucleic acid binding tlrs are particularly implicated in sle , an exceedingly complex and variable disorder . sle is a systemic autoimmune disease characterized most commonly by antinuclear antibodies ( anas ) . modern , effective treatment options are lacking for sle , in that the primary treatment methods are currently corticosteroids , antimalarial , or anti - inflammatory drugs . there has not been an fda - approved lupus treatment in over 40 years . common denominator , so to speak , for all lupus patients , which relates back to the complexity of the disease . however , the relatively new idea that toll - like receptor pathways play crucial roles in lupus pathogenesis shows promise for potential drug targets . the role of nucleic acid binding tlr 7 has become quite apparent in both animal models of the disease and human patients . this receptor promotes autoantibodies and cytokines responsible for chronic inflammation [ 14 , 46 , 47 ] . one particularly important animal model at the forefront of these observations has been the bxsb mouse . this model was derived from a cross between c57bl/6 and sb / le inbred strains , which resulted in male mice expressing an accelerated , lupus - like autoimmune disease phenotype characterized by production of anas and circulating immune complexes causing severe glomerulonephritis [ 48 , 49 ] . several subsequent studies have shown that the reason for the male bias in these mice was due to an x - to - y chromosomal translocation of a gene cluster known as y autoimmune accelerator ( yaa ) and that the primary contributor to this accelerated autoimmunity is tlr 7 overexpression [ 16 , 17 ] . fcriib mice have a characteristic lupus - like phenotype with autoantibodies directed towards chromatin . however , fcriib mice that also expressed yaa exhibited a shift in autoantibody specificity from an antichromatin to an antinucleolar ana pattern , consistent with an observed shift from chromatin to rna - binding specificities . this work further indicates a role for tlrs , in particular tlr 7 , in the development of a specific autoimmune phenotype . other groups have also described nucleic acid - binding tlr involvement in the production of autoantibodies . in one case in particular , kono and colleagues ablated tlr 3 , 7 , and 9 signaling in b6-fas and bxsb mouse models and showed that these mice expressed decreases in autoantibody production . additional work has shown that defective apoptotic cell clearance is common among sle patients , and this leads to development of antinuclear antibodies [ 5254 ] . our laboratory hypothesized that inefficient clearance of apoptotic debris triggers nucleic acid - binding toll - like receptors , which confer the b - cell response and subsequent ana production . injection of syngeneic late apoptotic thymocytes into wild type b6 mice led to anti - dsdna and antihistone antibody production whereas injection into myd88 mice had no effect , suggesting that tlr stimulation is important in development of anti - dsdna antibodies in situations of late apoptotic cell excess . further studies using tlr 7- and tlr 9-deficient recipients of late apoptotic thymocytes showed that tlr 9 had no bearing on the development of anti - dsdna and antihistone antibodies in this model , but tlr 7 did . moreover , the evidence suggested that tlr 7 promoted deposition of immune complexes in the renal glomeruli of these mice , possibly by influencing antichromatin antibody isotype . these studies suggest an important role for tlr 7 in the development of autoreactive antibodies and promotion of early events leading renal pathogenesis . the dna - binding tlr 9 has also been heavily studied in connection with murine lupus . tlr 9 deficiency in some lupus models including mrl mice can variably lead to reductions or alterations in antichromatin antibodies ; however , tlr 9 deficiency paradoxically leads to disease exacerbation in multiple models [ 13 , 14 , 5658 ] . considering endogenous self - stimuli , then , murine studies indicate a central pathogenic role for tlr 7 in lupus pathogenesis and a complex overall protective role for tlr 9 . although endogenously triggered tlr 3 does not appear to drive lupus in the murine models investigated thus far , this particular tlr may play important roles in the promotion of lupus by environmental stimuli such as certain viral infections . most importantly , in addition to studies using mouse models , connections between tlrs and human lupus have also been identified . one of the most striking connections is the presence of elevated ifn- as well as a type i ifn gene signature in lupus patients [ 60 , 61 ] . ifn- is a key player in disease progression and severity and has even been shown to induce the production of autoantibodies when administered to nonautoimmune patients . another interesting finding was remission of sle in a patient , which was attributed to unresponsiveness to both tlr 7 and 9 stimulation after development of common variable immunodeficiency- ( cvid- ) like disease . several groups have identified snps in the tlr 9 gene but have discovered that there is no correlation between these polymorphisms and sle [ 6467 ] . although there was no correlation with particular snps , other groups showed that there was an upregulation of tlr 9 expression in b cells of lupus patients lending credence to the idea that tlr 9 could be involved in autoantibody production [ 6870 ] . this finding led to the notion that the signaling proteins may be at fault in disease . recent advances in genetic analysis techniques have allowed for the identification of a large number of genes ( more than 25 ) that are associated with human lupus . at least three of the lupus - associated genes are involved in tlr signaling , including irf5 , irak1 , and tnfaip3 [ 7174 ] . the implication of these genes in lupus patients further indicates a role of tlrs in the disease phenotype . whether enhanced stimulation of the tlrs or genetic factors altering the signaling cascade are at fault , the accumulation of evidence implies that tlr pathways do , in fact , play a role in the pathogenic process of sle and remain excellent candidates for therapeutic targets to alleviate disease . although most studies related to toll - like receptors in sle have focused primarily on the nucleic acid - binding tlrs , there is still a potential role for other tlrs in autoimmune disease . komatsuda et al . looked at mrna expression of several tlrs in peripheral blood mononuclear cells ( pbmcs ) from sle patients . these studies indicated a slight increase in mrna of tlr2 , 7 , and 9 in the lupus patients compared to healthy controls . additionally , others have reported an important role for tlr2 and 4 in the production of autoantibodies in the b6 autoimmune mouse model . in these studies , tlr2- and tlr4-deficient b6mice expressed lower titers of autoantibodies , excluding those directed towards nucleosome proteins . this shows that other tlrs could be candidates for targeted therapy in autoimmunity as well . significant evidence supports the involvement of tlrs in multiple disease processes , including type i diabetes , ra , and sle . the assertion that tlrs play a role in disease pathogenesis , with lupus in particular , indicates potential for therapeutic intervention by targeting these molecules or their signaling pathways . since there are several proteins involved in tlr signaling , there are a number of targets that may be utilized for potential drugs . some of the possibilities include , but are not limited to , development of tlr antagonists , inhibitors of downstream signaling events , activation of natural inhibitory molecules , or blockade of the effector molecules produced . here we will discuss reports that highlight the potential usefulness of these different types of drugs . the development of tlr antagonists for the nucleic acid - binding tlrs has proven to be a tedious process due to the similarity between eukaryotic and noneukaryotic nucleic acids . despite this difficulty , barrat and colleagues have developed immunoregulatory dna sequences ( irs ) that can bind tlr 9 but inhibit its activation and downstream effects . they have shown that these inhibitory odns can relieve inflammation in multiple scenarios . in one instance , mice injected with immunostimulatory sequences ( isss ) and d - galactosamine developed severe inflammation and died within days . however , when coinjected with the irs , inflammation was decreased and mouse survival was prolonged . in addition to these studies , the same group developed a dual tlr 7/9 inhibitor . this oligodeoxynucleotide ( odn ) was sufficient for inhibition of both tlr 7 and 9 signaling and protection against inflammation . these irs sequences were also demonstrated to inhibit the production of ifn- by human plasmacytoid dendritic cells ( pdc ) , indicating the effectiveness of these inhibitors in human cells . because of the effectiveness in inhibiting tlr function , barrat et al . also investigated the capacity of their odns to treat a lupus - prone mouse model . several studies had previously established a potential role of ifn- in the progression of autoimmunity in ( nzb nzw ) f1 mice [ 79 , 80 ] . since ifn- appears to play a central role in human sle , this strain was selected as an ideal model for the application of irs . irs injections twice weekly in ( nzb nzw ) f1 mice resulted in decreased anas , reduced glomerulonephritis at nine months , and increased rate of survival among the treated mice compared to untreated controls . in addition to the studies by barrat , two other groups used other inhibitory odns for targeting tlr signaling in autoimmunity . the odns designed by dong et al . were injected into ( nzb nzw ) f1 mice that were subsequently analyzed for kidney function and autoantibody production characteristic of lupus . their results suggested a similar capacity of these inhibitory odns to minimize glomerulonephritis and reduce autoantibodies directed towards dna . pawar et al . used irs sequences as tlr 7 and 9 inhibitors in mrl mice and showed a reduction in inflammatory cytokine levels and autoantibody titers , as well as a decrease in resulting tissue damage . these experiments using inhibitory odns in treating lupus - prone animal models suggest strong potential for treatment of human lupus and other autoimmune diseases by targeting tlrs . other potential therapeutic targets include the downstream signaling proteins involved in the tlr signaling cascade . there are a number of potential targets for this intervention including myd88 , traf6 , and irak1 and 4 . since both tlr 7 and 9 utilize this protein , it is an excellent target to decrease the signaling that takes place in lupus . two independent groups have shown that inhibiting myd88 impairs the phosphorylation / activation of downstream kinases and nfb activity . bartfai and colleagues developed a chemical mimic to the three amino acid sequence at the conserved bb - loop of the tir domain of myd88 . the results of this mimic showed an inhibition of myd88 and il-1ri interaction by ablating phosphorylation of map kinases . describe a similar mimic ; however , they employ a peptide sequence instead of a chemical mimic . this inhibitor showed similar inhibition of myd88 signaling indicated by impaired nfb activity . they also went on to show that not only do these peptides inhibit dimerization of myd88 , but also inhibit the recruitment of irak1 and irak4 . in view of the fact that myd88 is a central mediator in tlr 7 and 9 signaling , these inhibitors of myd88 dimerization and recruitment of essential kinases could prove to be an effective treatment option in lupus by abolishing the aberrant tlr signaling that induces the elevated type i ifn levels and persistent inflammation . briefly , irak4 is a kinase that interacts with myd88 and traf6 and is associated with the activation of the downstream transcription factors in tlr signaling . based on irak4-deficient animal models , this kinase has been shown to be indispensible in tlr signaling [ 89 , 90 ] . because of this idea that irak4 is necessary for production of effectors in a tlr - dependent manner , it has been subjected to targeted therapy . however , irak4 inhibitors developed to date have yet to be analyzed for efficacy in animal models [ 9194 ] . the last actively studied therapeutic approach we will discuss targets the end - product effectors of tlr signaling as opposed to the signaling cascade itself . an important target that is now accepted to be characteristic of sle is ifn-. this cytokine is found in elevated levels among lupus patients , and high levels associate with worsened measures of disease activity . due to the apparent pathogenic nature of ifn- , inhibitory monoclonal antibodies have been developed against the cytokine as a treatment for sle . one such drug is currently in phase ii clinical trials and has shown promise thus far in relieving symptoms of lupus . among the tlr and tlr - related targets for therapy in lupus , this antibody directed towards ifn- currently , active research on this topic is concerned with negatively regulating molecules that activate tlr - dependent signaling , but what about activating some of the natural inhibitory proteins , such as triad3a , myd88s , shp-1 , irak - m , irak1c , irf4 , and a20 ? these naturally occurring inhibitors of the signaling cascade could be targeted for agonistic drugs . xie et al . reported that irak - m - deficient mice injected with tumor cells develop increased cd4 t - cell differentiation and upregulation of costimulatory molecules and other activation markers on b cells , compared to wild - type controls , indicating an inhibitory role for irak - m . although irak - m deficiency appears to have a positive role in cancer models , the inverse is likely to be true for autoimmune situations . stimulation of production or activity of irak - m may provide another alternative therapy in sle . similarly , promoting the production or activity of the other endogenous tlr signaling inhibitors , triad3a , myd88s , shp-1 , irak1c , irf4 , or a20 , could provide additional options for effective treatment for systemic autoimmunity . utilizing these endogenous proteins could provide a new avenue to decrease tlr - mediated inflammation in patients with autoimmune diseases , with each of these targets allowing a different route to decreasing production of inflammatory cytokines and subsequent risk of tissue damage . although the approaches presented here are in the context of nucleic acid - binding tlrs , the proposed strategies for tlr signaling inhibition could actually act more globally . since most tlrs can signal through the adaptor protein myd88 , these potential therapeutic targets could prevent signaling through all tlrs with the exception of tlr 3 . the specific tlr 7 and tlr 9 inhibitors discussed earlier would be ideal as a treatment method to avoid a total inhibition of the first line of microbial defense ; however , with evidence revealing potential roles for other toll - like receptors , namely , tlr 2 and 4 , in sle , a more widespread inhibition may be an important aspect worth investigating . although tlrs are extraordinarily important in pathogen recognition and normal immune function , including that of both innate and adaptive arms of the immune system , they have also been ascribed causative roles in autoimmunity . various studies ranging from lupus - prone mouse models , to genome - wide association studies in human lupus patients , to in vitro studies with patient cells have indicated a significant role for nucleic acid - binding tlrs in the progression and severity of lupus and other autoimmune diseases . from these studies , we have learned about the involvement of tlrs in lupus - prone mouse models , the association of polymorphisms in irf5 , irak1 , and tnfaip3 with human disease , and the upregulation of tlrs in sle patients . due to the accumulation of these data suggesting a detrimental role of tlr signaling in lupus , efforts are currently underway to ascertain reliable treatment methods based on the targeting of tlrs and their signaling counterparts . inhibition of tlrs and their signaling pathways have proven to be efficacious in lupus - prone mouse models and successfully inhibit ifn- production by human pdc in vitro . although the direct tlr antagonists have not been studied in human patients , inhibitors of ifn- , a primary downstream effector of tlr signaling and important disease mediator in sle , have been developed and are currently undergoing clinical trials . monoclonal antibody treatments have been successfully utilized in the setting of rheumatic diseases for some time now and are likely to comprise a new armament the realm of lupus treatment . although there has not been a new drug approved for the treatment of lupus in many years , current investigation regarding the targeting of tlrs and their downstream effectors is showing some promise and warrants high expectations .

toll - like receptors ( tlrs ) are important innate immune receptors for the identification and clearance of invading pathogens . twelve tlrs that recognize various conserved components of microorganisms are currently known . among these , the endosomal tlrs 3 , 7/8 , and 9 recognize dsrna , ssrna , and cpg dna , respectively . nucleic acid - sensing tlrs , tlr 7 in particular , have been implicated in systemic lupus erythematosus ( sle ) and are thought to exacerbate disease pathology . activation of these tlrs results in the production of inflammatory cytokines and type i interferon . genome - wide association studies , single nucleotide polymorphism analyses as well as experimental mouse models have provided evidence of tlr signaling involvement in sle and other autoimmune diseases . since activation of these receptor pathways promotes autoimmune phenotypes , inhibitory drugs that target these pathways constitute important new therapeutic strategies for the treatment of systemic autoimmunity .

1. Introduction 2. TLR Function and Signaling 3. TLRs in Autoimmunity 4. TLRs as Therapeutic Targets 5. Conclusions

toll - like receptors ( tlrs ) are pattern recognition receptors of the innate immune system that recognize specific pathogen - associated molecular patterns ( pamps ) conserved among microorganisms . there are currently twelve known tlrs in mammals , which identify common constituents of invading pathogens including double - stranded and single - stranded rna , unmethylated cpg dna , bacterial lipopolysaccharide ( lps ) , lipoproteins , and flagellin . these adapters recruit other molecules to initiate signaling cascades ultimately leading to the production of proinflammatory cytokines and type i ifn . these tlr responses are important in the functioning of both the innate and adaptive arms of immunity [ 4 , 5 ] . although tlrs are important for antimicrobial immunity , they have also been implicated in the pathogenesis of autoimmune diseases . for example , tlrs 2 and 4 have been identified as factors involved in the onset of type i diabetes mellitus [ 69 ] , and tlrs 16 are expressed by rheumatoid arthritis ( ra ) synovial fibroblasts and are thought to provoke joint inflammation in ra [ 10 , 11 ] . moreover , the nucleic acid binding tlrs 7 and 9 have been connected to both human and mouse models of systemic lupus erythematosus ( sle ) [ 1217 ] . two major possibilities arise in describing how tlrs might work in autoimmunity ; either they are stimulated by exogenous antigens , like viral ssrna , which then stimulate resident immune cells , or the tlrs recognize endogenous self - antigens to initiate and propagate inflammation and autoimmunity . the accumulation of evidence pointing towards tlrs in autoimmunity has opened the door for potential therapeutic interventions directed towards the modulation of toll - like receptors and their signaling pathways . since tlrs are normally responsive to microbial pathogens , there has been some speculation as to the application of tlr agonists as vaccine adjuvants to stimulate more robust immune responses [ 18 , 19 ] . on the other side of the spectrum , for autoimmunity , disease can result from aberrant hyperactive signaling ; therefore , the application of inhibitory , or antagonistic , tlr therapeutics is of considerable interest . the focus of this paper is to summarize the present literature documenting how modulation of toll - like receptor pathways may be used as potential methods of treatment for autoimmunity , particularly sle . under normal circumstances , tlrs aid in the identification and removal of materials that may be detrimental to the host organism ; these are usually of bacterial , viral , fungal , or protozoan origin [ 1 , 23 ] . since we are more concerned with sle , we will concentrate on the signaling that takes place upon activation of nucleic acid - binding tlrs 3 , 7/8 , and 9 . ligand binding is thought to induce a conformational change resulting in interaction or close juxtaposition of the two cytosolic toll / il-1 receptor ( tir ) domains , thus providing an interface for adaptor protein binding and subsequent signal transduction . as described by several groups , myd88 is the most common of these adaptors and has been shown to be involved in signaling through all tlrs except tlr 3 [ 2427 ] . ultimately , the activation of transcription factors nfb and irfs 5 and 7 results in their translocation into the nucleus where they initiate gene transcription and production of proinflammatory cytokines and type i ifn ( figure 1 ) [ 3 , 3135 ] . tlr 3 signaling is distinct from the previously described tlr 7 and 9 signaling pathways . as with myd88 , trif also recruits additional proteins necessary for downstream signaling , including traf - family - member - associated nfb - activator - binding kinase 1 ( tbk1 ) , traf6 , and receptor - interacting protein 1 ( rip1 ) . trif interaction with tbk1 is necessary for the activation of irf3 , which is a transcription factor involved in the production of ifn- . the multiple proteins involved in tlr signaling yield many opportunities to inhibit this process . several endogenous inhibitors have been identified that halt , or at least impair , the signaling cascade , dampening the tlr - mediated production of inflammatory cytokines and type i ifn . the ability to modulate tlr signaling helps to maintain a homeostatic balance important for antimicrobial immunity while also preventing collateral damage to self - tissues . the inhibitory proteins important in this process target the receptors themselves , adapter molecules , and key kinases , as well as transcription factors to diminish the tlr - mediated production of inflammatory cytokines and type i ifn . triad3a acts as an e3 ubiquitin ligase promoting the degradation of tlr 4 and 9 . the degradation of these tlrs was shown to impair nfb activity upon stimulation with lps and cpg dna whereas sirna knockdown of triad3a enhanced lps and cpg dna - mediated nfb activity indicating a prominent role of triad3a in modulating tlr response . although triad3a appears to act specifically on tlr 4 and 9 , other inhibitors provide a more global impact on tlr signaling . upon stimulation with lps or tnf , a splice - variant of myd88 , myd88s , can be induced which prevents activation of nfb . found that the phosphatase , shp-1 , selectively impairs activation of nfb and subsequent production of proinflammatory cytokines while also inhibiting irak1 resulting in an increase in ifn- . showed that irak - m , which lacks the kinase activity of its counterparts irak1 and irak4 , suppresses the production of proinflammatory cytokines and proposed a model whereby irak - m prevents the dissociation of the irak1/irak4/myd88 complex to inhibit downstream nfb activation . two other inhibitors have been identified downstream of the irak kinases : tumor necrosis factor--induced protein 3 ( tnfaip3 , or a20 ) and irf4 . a20 is a ubiquitin - editing enzyme that deubiquitinates traf6 to inhibit the release of nfb from ib , thus inhibiting subsequent nfb - mediated gene transcription [ 3 , 44 ] . irf4 inhibits tlr signaling by competing for the same binding site on myd88 with irf5 . irf5 engages myd88 downstream of tlr stimulation in order to promote production of proinflammatory cytokines . tlrs have been identified as instigators in type i diabetes , rheumatoid arthritis , and systemic lupus erythematosus . the nucleic acid binding tlrs are particularly implicated in sle , an exceedingly complex and variable disorder . modern , effective treatment options are lacking for sle , in that the primary treatment methods are currently corticosteroids , antimalarial , or anti - inflammatory drugs . however , the relatively new idea that toll - like receptor pathways play crucial roles in lupus pathogenesis shows promise for potential drug targets . the role of nucleic acid binding tlr 7 has become quite apparent in both animal models of the disease and human patients . one particularly important animal model at the forefront of these observations has been the bxsb mouse . this model was derived from a cross between c57bl/6 and sb / le inbred strains , which resulted in male mice expressing an accelerated , lupus - like autoimmune disease phenotype characterized by production of anas and circulating immune complexes causing severe glomerulonephritis [ 48 , 49 ] . several subsequent studies have shown that the reason for the male bias in these mice was due to an x - to - y chromosomal translocation of a gene cluster known as y autoimmune accelerator ( yaa ) and that the primary contributor to this accelerated autoimmunity is tlr 7 overexpression [ 16 , 17 ] . this work further indicates a role for tlrs , in particular tlr 7 , in the development of a specific autoimmune phenotype . other groups have also described nucleic acid - binding tlr involvement in the production of autoantibodies . in one case in particular , kono and colleagues ablated tlr 3 , 7 , and 9 signaling in b6-fas and bxsb mouse models and showed that these mice expressed decreases in autoantibody production . additional work has shown that defective apoptotic cell clearance is common among sle patients , and this leads to development of antinuclear antibodies [ 5254 ] . our laboratory hypothesized that inefficient clearance of apoptotic debris triggers nucleic acid - binding toll - like receptors , which confer the b - cell response and subsequent ana production . further studies using tlr 7- and tlr 9-deficient recipients of late apoptotic thymocytes showed that tlr 9 had no bearing on the development of anti - dsdna and antihistone antibodies in this model , but tlr 7 did . moreover , the evidence suggested that tlr 7 promoted deposition of immune complexes in the renal glomeruli of these mice , possibly by influencing antichromatin antibody isotype . these studies suggest an important role for tlr 7 in the development of autoreactive antibodies and promotion of early events leading renal pathogenesis . tlr 9 deficiency in some lupus models including mrl mice can variably lead to reductions or alterations in antichromatin antibodies ; however , tlr 9 deficiency paradoxically leads to disease exacerbation in multiple models [ 13 , 14 , 5658 ] . considering endogenous self - stimuli , then , murine studies indicate a central pathogenic role for tlr 7 in lupus pathogenesis and a complex overall protective role for tlr 9 . although endogenously triggered tlr 3 does not appear to drive lupus in the murine models investigated thus far , this particular tlr may play important roles in the promotion of lupus by environmental stimuli such as certain viral infections . most importantly , in addition to studies using mouse models , connections between tlrs and human lupus have also been identified . one of the most striking connections is the presence of elevated ifn- as well as a type i ifn gene signature in lupus patients [ 60 , 61 ] . ifn- is a key player in disease progression and severity and has even been shown to induce the production of autoantibodies when administered to nonautoimmune patients . another interesting finding was remission of sle in a patient , which was attributed to unresponsiveness to both tlr 7 and 9 stimulation after development of common variable immunodeficiency- ( cvid- ) like disease . recent advances in genetic analysis techniques have allowed for the identification of a large number of genes ( more than 25 ) that are associated with human lupus . at least three of the lupus - associated genes are involved in tlr signaling , including irf5 , irak1 , and tnfaip3 [ 7174 ] . the implication of these genes in lupus patients further indicates a role of tlrs in the disease phenotype . whether enhanced stimulation of the tlrs or genetic factors altering the signaling cascade are at fault , the accumulation of evidence implies that tlr pathways do , in fact , play a role in the pathogenic process of sle and remain excellent candidates for therapeutic targets to alleviate disease . although most studies related to toll - like receptors in sle have focused primarily on the nucleic acid - binding tlrs , there is still a potential role for other tlrs in autoimmune disease . these studies indicated a slight increase in mrna of tlr2 , 7 , and 9 in the lupus patients compared to healthy controls . additionally , others have reported an important role for tlr2 and 4 in the production of autoantibodies in the b6 autoimmune mouse model . in these studies , tlr2- and tlr4-deficient b6mice expressed lower titers of autoantibodies , excluding those directed towards nucleosome proteins . significant evidence supports the involvement of tlrs in multiple disease processes , including type i diabetes , ra , and sle . the assertion that tlrs play a role in disease pathogenesis , with lupus in particular , indicates potential for therapeutic intervention by targeting these molecules or their signaling pathways . since there are several proteins involved in tlr signaling , there are a number of targets that may be utilized for potential drugs . some of the possibilities include , but are not limited to , development of tlr antagonists , inhibitors of downstream signaling events , activation of natural inhibitory molecules , or blockade of the effector molecules produced . the development of tlr antagonists for the nucleic acid - binding tlrs has proven to be a tedious process due to the similarity between eukaryotic and noneukaryotic nucleic acids . in one instance , mice injected with immunostimulatory sequences ( isss ) and d - galactosamine developed severe inflammation and died within days . in addition to these studies , the same group developed a dual tlr 7/9 inhibitor . this oligodeoxynucleotide ( odn ) was sufficient for inhibition of both tlr 7 and 9 signaling and protection against inflammation . these irs sequences were also demonstrated to inhibit the production of ifn- by human plasmacytoid dendritic cells ( pdc ) , indicating the effectiveness of these inhibitors in human cells . several studies had previously established a potential role of ifn- in the progression of autoimmunity in ( nzb nzw ) f1 mice [ 79 , 80 ] . since ifn- appears to play a central role in human sle , this strain was selected as an ideal model for the application of irs . irs injections twice weekly in ( nzb nzw ) f1 mice resulted in decreased anas , reduced glomerulonephritis at nine months , and increased rate of survival among the treated mice compared to untreated controls . in addition to the studies by barrat , two other groups used other inhibitory odns for targeting tlr signaling in autoimmunity . their results suggested a similar capacity of these inhibitory odns to minimize glomerulonephritis and reduce autoantibodies directed towards dna . used irs sequences as tlr 7 and 9 inhibitors in mrl mice and showed a reduction in inflammatory cytokine levels and autoantibody titers , as well as a decrease in resulting tissue damage . these experiments using inhibitory odns in treating lupus - prone animal models suggest strong potential for treatment of human lupus and other autoimmune diseases by targeting tlrs . other potential therapeutic targets include the downstream signaling proteins involved in the tlr signaling cascade . there are a number of potential targets for this intervention including myd88 , traf6 , and irak1 and 4 . since both tlr 7 and 9 utilize this protein , it is an excellent target to decrease the signaling that takes place in lupus . in view of the fact that myd88 is a central mediator in tlr 7 and 9 signaling , these inhibitors of myd88 dimerization and recruitment of essential kinases could prove to be an effective treatment option in lupus by abolishing the aberrant tlr signaling that induces the elevated type i ifn levels and persistent inflammation . briefly , irak4 is a kinase that interacts with myd88 and traf6 and is associated with the activation of the downstream transcription factors in tlr signaling . based on irak4-deficient animal models , this kinase has been shown to be indispensible in tlr signaling [ 89 , 90 ] . because of this idea that irak4 is necessary for production of effectors in a tlr - dependent manner , it has been subjected to targeted therapy . the last actively studied therapeutic approach we will discuss targets the end - product effectors of tlr signaling as opposed to the signaling cascade itself . this cytokine is found in elevated levels among lupus patients , and high levels associate with worsened measures of disease activity . due to the apparent pathogenic nature of ifn- , inhibitory monoclonal antibodies have been developed against the cytokine as a treatment for sle . among the tlr and tlr - related targets for therapy in lupus , this antibody directed towards ifn- currently , active research on this topic is concerned with negatively regulating molecules that activate tlr - dependent signaling , but what about activating some of the natural inhibitory proteins , such as triad3a , myd88s , shp-1 , irak - m , irak1c , irf4 , and a20 ? stimulation of production or activity of irak - m may provide another alternative therapy in sle . similarly , promoting the production or activity of the other endogenous tlr signaling inhibitors , triad3a , myd88s , shp-1 , irak1c , irf4 , or a20 , could provide additional options for effective treatment for systemic autoimmunity . utilizing these endogenous proteins could provide a new avenue to decrease tlr - mediated inflammation in patients with autoimmune diseases , with each of these targets allowing a different route to decreasing production of inflammatory cytokines and subsequent risk of tissue damage . although the approaches presented here are in the context of nucleic acid - binding tlrs , the proposed strategies for tlr signaling inhibition could actually act more globally . since most tlrs can signal through the adaptor protein myd88 , these potential therapeutic targets could prevent signaling through all tlrs with the exception of tlr 3 . the specific tlr 7 and tlr 9 inhibitors discussed earlier would be ideal as a treatment method to avoid a total inhibition of the first line of microbial defense ; however , with evidence revealing potential roles for other toll - like receptors , namely , tlr 2 and 4 , in sle , a more widespread inhibition may be an important aspect worth investigating . various studies ranging from lupus - prone mouse models , to genome - wide association studies in human lupus patients , to in vitro studies with patient cells have indicated a significant role for nucleic acid - binding tlrs in the progression and severity of lupus and other autoimmune diseases . from these studies , we have learned about the involvement of tlrs in lupus - prone mouse models , the association of polymorphisms in irf5 , irak1 , and tnfaip3 with human disease , and the upregulation of tlrs in sle patients . due to the accumulation of these data suggesting a detrimental role of tlr signaling in lupus , efforts are currently underway to ascertain reliable treatment methods based on the targeting of tlrs and their signaling counterparts . although the direct tlr antagonists have not been studied in human patients , inhibitors of ifn- , a primary downstream effector of tlr signaling and important disease mediator in sle , have been developed and are currently undergoing clinical trials . monoclonal antibody treatments have been successfully utilized in the setting of rheumatic diseases for some time now and are likely to comprise a new armament the realm of lupus treatment . although there has not been a new drug approved for the treatment of lupus in many years , current investigation regarding the targeting of tlrs and their downstream effectors is showing some promise and warrants high expectations .

their main distinction from other types of polymers lies in their densely crosslinked molecular structure . this crosslinking leads to a number of favorable thermal and mechanical properties including high strength and modulus , high creep resistance , high glass transition temperature , low shrinkage , and better chemical resistance . these properties in conjunction with ease of processing have made epoxy resins an attractive choice for use in many engineering components and structures . they have found huge applications in aerospace , automotive , packaging , coating , and microelectric industries . in recent years , researchers have developed and investigated polymer nanocomposites based on a wide variety of micro-/nanoscale fillers including clay particles [ 16 ] , aluminum particles , tio2 particles , graphenes [ 9 , 10 ] , carbon nanotubes [ 1113 ] , and halloysites [ 4 , 14 ] to improve the mechanical properties of epoxy polymers . among the various reinforcements , large aspect ratio layered silicates or clays are especially attractive for enhancing the barrier properties and , hence , can be used to improve the resistance to moisture degradation . epoxy polymers are characteristically hydrophilic , which means that they have strong affinity towards water . this nature of epoxy resins makes them susceptible to high moisture absorption ; in general , depending on the nature of the epoxy resin , the equilibrium moisture uptake can be in the range of 17% . absorbed moisture usually degrades the functional , structural , and mechanical properties of the polymer matrix [ 4 , 1619 ] . it has been reported that mechanical and thermal properties of epoxy - based systems are severely affected by moisture absorption in comparison to other matrix materials , such as bismaleimide ( bmi ) , polyetheretherketone ( peek ) , and cyanate ester . water absorption into a polymer matrix leads to change in both chemical and physical characteristics and affects the mechanical properties through different mechanisms such as plasticization , crazing , hydrolysis , and swelling . plasticization is the most important physical change that occurs through the interaction of the water molecules with polar groups in the matrix , which can severely depress the glass transition temperature [ 12 , 18 , 2124 ] . for example , high moisture absorption capability of tgddm / dds epoxy resin , which is about 7 wt% , reduces the system tg from 260c to 130c [ 2527 ] . in general , for most epoxy systems , tg is reduced by 20c/1 wt% of moisture intake . the decrease in modulus of epoxy has also been reported to be due to plasticization according to several studies [ 2932 ] . other studies showed that the decrease in modulus resulted from crazing [ 3335 ] , where the absorbed water acted as a crazing agent continuously decreasing the mechanical strength of epoxies with exposure time in water . this was supported by sem micrographs of epoxies , which have shown cavities and fractured fibrils that could be explained by a moisture induced crazing mechanism . the aforementioned chemical changes mainly include chain scission and hydrolysis [ 22 , 36 ] . plasticization is considered reversible upon drying , while other effects of moisture absorption are irreversible . in recent years , effect of moisture absorption on the mechanical properties of neat epoxy and clay - epoxy nanocomposite has been frequently studied . zhao and li reported that tensile strength and modulus decreased for both neat epoxy and nanocomposites upon moisture absorption , while the tensile strain increased significantly for moisture absorbed samples . although failure occurred in brittle manner , effect of plasticization was found in sem images , which showed shear yielding for both neat epoxy and nanocomposite samples after being exposed to moisture . alamri and low reported lower flexural strength and modulus for halloysite , nanoclay , and nanosilicon carbide nanocomposites as a result of moisture absorption . al - qadhi et al . studied the effect of moisture absorption on the tensile properties of clay - epoxy nanocomposites and found that tensile strength and modulus decrease as a result of water uptake . wang et al . investigated the effect of hydrothermal degradation on mechanical properties such as tensile strength , modulus , and fracture toughness with immersion duration . for dgeba epoxy systems , fracture toughness and modulus were not influenced much with immersion time , while tensile strength decreased for nanocomposites . tensile and flexural properties of nanoclay reinforced composite laminates and cnt reinforced nanocomposites have also been found to be affected adversely as a result of moisture absorption [ 12 , 40 ] . according to the study conducted by buck et al . , at elevated temperature , combination of moisture and sustained load significantly reduced ultimate tensile strength of e - glass / vinyl - ester composite materials . a study on elastic modulus of epoxy polymer after absorption - desorption cycle showed recovery of property from wet condition , although modulus remained at a lower value than the as - prepared samples for lower filler volume . for higher volume fraction of reinforcement , elastic modulus improved to a value which was more than the elastic modulus of the as - prepared samples . phua et al . also reported recovery of tensile properties after refrying omtt - pbs nanocomposites . a similar study conducted by ferguson and qu showed recovery of elastic properties from moisture saturated state after a desorption cycle . however , de'ne`ve and shanahan did not observe any recovery of elastic modulus after absorption - desorption cycle at elevated temperature . it is evident from the published works that moisture absorption can severely alter mechanical properties of epoxies by decreasing the elastic modulus [ 12 , 29 , 33 ] , tensile strength [ 3 , 12 ] , shear modulus [ 30 , 31 ] , flexural strength and flexural modulus [ 40 , 43 ] , yield stress , and ultimate stress as water uptake increases . most of the research on polymer - clay nanocomposites has been mainly focused on investigating the effect of moisture absorption on mechanical properties such as elastic modulus and tensile strength . although fracture toughness is a very important property for these nanocomposites as these are used in various structural applications , the effects of moisture absorption on fracture toughness of polymer - clay nanocomposites have not been studied extensively . durability of polymer / clay nanocomposites is still needed to be studied in depth , particularly for hygrothermal aging in which the degradation of the mechanical properties and loss of integrity of these nanocomposites occur from the simultaneous action of moisture and temperature . this study on clay - epoxy nanocomposites was designed to investigate the effect of hygrothermal aging on mechanical properties of these nanocomposites . two different clay particles were used to investigate the effect of clay structure on the permanent property changes due to hygrothermal aging . a drying cycle was employed to quantify the recovery of the properties after hygrothermal aging . this was helpful to understand the extent of permanent degradation that occurred by the combined application of elevated temperature and moisture . mechanical properties in terms of fracture toughness , flexural strength , and flexural modulus are the properties that were studied . scanning electron microscopy and fourier transform spectroscopy were conducted to further elucidate the underlying fracture mechanisms of these preconditioned specimens . the epoxy resin used for this study is epon 862 , which is a diglycidyl ether of bisphenol f. the curing agent used for this resin system was a moderately reactive , low viscosity aliphatic amine curing agent , epikure 3274 . both of these chemicals were supplied by miller - stephenson chemical company , inc . , , arlington heights , il ) modified with a quaternary amine ( trimethyl stearyl ) . somasif mae ( co - op chemical co. , japan ) , which was the other clay particle used for this study , is a synthetic mica modified with dimethyl dihydrogenated tallow ammonium chloride . epoxy was preheated to 65c before desired amount of clay was introduced and mixed using mechanical mixer for 12 hours . to reduce the viscosity of the mixture and to facilitate mixing , temperature was maintained at 65c using a hot plate for the entire duration of mixing . the mixture was then degassed for around 30 minutes to remove any entrapped air bubbles . bubble - free mixture of clay and epoxy was then shear - mixed using a high - speed shear disperser ( t-25 ultra turrax , ika works inc . , north carolina , usa ) for 30 minutes . during this process curing agent was added to the mixture at 100 : 40 weight ratio and carefully hand - mixed to avoid introduction of any air bubble . after it was properly mixed , the final slurry containing epoxy and clay was poured into an aluminum mold and cured at room temperature for 24 hours followed by postcuring at 121c for 6 hours . the final sample had a nominal dimension of 177.80 mm 152.50 mm 12.70/6.35 mm . to study the effect of loading percentage , the weight fraction of the clay in the nanocomposite was varied from 0.5 to 2.0% . after specimens were cut into the final required dimension according to the astm standards d5045 and d790 , they were subjected to degradation . specimens from each nanocomposite were taken and submerged in purified deionized boiling water for 24 hours . water saturated specimens were dried in an oven at 110c for 6 hours to remove moisture from the samples leaving only permanent degradation in the form of bonded water . critical stress intensity factor , kic , was determined by single edge notch bend ( senb ) test as per the astm d5045 on universal testing machine ( instron 5567 , norwood , ma ) in a displacement - controlled mode with fixed crosshead speed of 10 mm / min . nominal dimension for the senb test samples was 67.30 mm 15.20 mm 6.35 mm . a notch was created using precision diamond saw ( mk-370 , mk diamond products inc . , torrance , ca , usa ) and , afterwards , a sharp precrack with ratio of 0.45 < a / w < 0.55 was created by tapping a fresh razor blade into the notch . fracture toughness for the specimens was calculated in terms of critical stress intensity factor , kic . the crack length , a , was measured using an optical microscope ( nikon l150 ) which has a traveling plate with graduations : ( 1)kic = pbwf(aw ) , where p = maximum applied force ( n ) , b = thickness of the specimen ( mm ) , w = width of the specimen ( mm ) , and f(a / w ) = geometry factor , and it is given by the following equation : ( 2)f(aw)=3(s / w)a / w2(1 + 2(a / w))(1a / w)3/2 [(2.153.93(aw)+2.7(aw)2)1.99(aw)(1aw)iiiiiii(2.153.93(aw)+2.7(aw)2 ) ] , where s = support span ( mm ) and a = length of the precrack ( mm ) . flexural strength and flexural modulus were determined using three - point bend ( 3pb ) test according to astm d790 on universal testing machine ( instron 5567 , norwood , ma ) . the nominal dimension for the flexural test specimens was 55.90 mm 12.70 mm 6.35 mm . consider ( 3)r = zl26d , where r = rate of crosshead motion ( mm / min ) , l = support span ( mm ) , d = depth of beam ( mm ) , and z = rate of straining of the outer fiber ( mm / mm / min ) = 0.01 . the flexural strength and flexural modulus were calculated using the following equations , respectively : ( 4)f , max=3pmaxl2bd2,(5)eb = l3m4bd3 , where f , max = flexural strength ( mpa ) , eb = flexural modulus ( mpa ) , pmax = maximum load on the load - deflection curve ( n ) , l = support span ( mm ) , b = width of beam tested ( mm ) , d = depth of beam tested ( mm ) , and m = slope of the tangent to the initial straight - line portion of the load - deflection curve ( n / mm of deflection ) . surface morphology of the fractured specimens from senb tests was observed using scanning electron microscopy ( hitachi s-4800 fesem , dallas , tx ) . as polymer materials are nonconductive to electrons , all fracture surfaces were sputtered with gold - palladium alloy before sem imaging . ftir spectroscopy measurements were performed using atr - ftir spectrometer ( nicolet is10 , waltham , ma ) using 64 scans at a resolution of 2.0 cm . table 2 shows the relative weight changes that occurred in the specimens after 24 hours of boiling water absorption and 6 hours of high temperature desorption cycle . for the studied material systems , percentage weight gain after absorption cycle showed no change as a function of clay loading percentage . this observation was different from the findings reported by glaskova and aniskevich for clay - epoxy nanocomposites . according to their study , moisture absorption was found to have increased slightly with the increase of clay weight percentage . the reason why a different clay - epoxy nanocomposite system behaves differently in moisture absorption test is still not clear and further investigation is required to understand it . in this study , although both clays are structurally different , the observed percentage weight gain for both nanocomposite systems was found approximately to be the same . these two observations led to the conclusion that moisture diffusion process primarily depended on the polymer system under investigation . moisture desorption data showed that most of the absorbed water is free water , which can be driven out of the system by drying . for neat epoxy , this is possibly due to the fact that presence of clay hindered the moisture diffusion process in and out of the epoxy polymers . an increasing trend in the amount of retained moisture for higher clay loading nanocomposites also supported the aforementioned statement . amount of water retained after the desorption cycle has been found to be almost similar for both clay - epoxy nanocomposite systems . the critical stress intensity factors as a function of clay loading percentage for nanomer i.28e and somasif mae clay - epoxy nanocomposites are shown in figure 1 . critical stress intensity factor , kic , increased 28% for the as - prepared 0.5 wt% nanomer i.28e clay - epoxy nanocomposite compared to neat epoxy . the reason behind this observation can be attributed to the layered structure of the clay . clay in a polymer material physically blocks , bifurcates , and deflects the crack path , compelling the crack to travel longer path , which in turn results in higher toughness in a clay - polymer nanocomposite . the toughening effect of clay on epoxy polymer started to decrease with any additional clay reinforcement . this is a common behavior for several types of epoxy - clay nanocomposites and has been reported in previous studies conducted on clay - epoxy nanocomposites [ 3 , 6 , 45 , 46 ] . depending on the processing technique and epoxy - clay interaction , there is an optimum weight percentage for which the property enhancement can be maximized . any further addition of clay negates the positive effect by forming agglomerates due to improper exfoliation of the clay platelets and thus results in stress concentration forcing the material to fail at lower loads . for moisture saturated epoxy - clay nanocomposites , kic was found to be lower compared to the as - prepared nanocomposite samples for all the nanomer i.28e nanocomposites . as water molecules diffuse into the nanofiller - epoxy interface , debonding and weakening of the interface occur , resulting in poor stress transfer between the filler and the epoxy matrix [ 43 , 47 , 48 ] . redried neat epon 862 , free of void - filling water , showed 29% reduction in fracture toughness compared to the as - prepared neat epon 862 samples . addition of 0.5 wt% of clay resulted in 16% reduction in fracture toughness when compared to the as - prepared samples , which was significantly less compared to 29% reduction of neat epoxy . for 2.0 wt% nanomer i.28e clay - epoxy nanocomposite , however , the standard deviation of the as - prepared sample was much higher , which could possibly mean that the dried and the as - prepared samples have no difference in toughness . for the as - prepared and moisture saturated samples , somasif mae nanocomposites showed comparable trend in fracture toughness data ; clay reinforcement successfully improved the baseline epoxy properties and moisture absorption degraded the mechanical properties for all clay percentages . however , the permanent degradation after absorption - desorption cycle was found to be more prominent in the case of somasif mae clay nanocomposites compared to nanomer i.28e clay nanocomposites . the recovery of property after 6 hours of drying was negligible for somasif mae clay nanocomposites , whereas nanomer i.28e nanocomposites showed significant recovery of property after drying . the difference in property recovery of these two clay - epoxy nanocomposites can be attributed to the structural differences of the two clay particles and has been further investigated through sem and ftir technique . flexural modulus for the neat epoxy and clay - epoxy nanocomposites was determined from 3pb test and has been plotted against clay loading percentage in figure 2 for nanomer i.28e and somasif mae clay . flexural modulus increased almost linearly for both clays as a function of clay loading percentage . according to previous studies on epoxy polymers , incorporation of hard substance such as clay in polymer matrix results in higher modulus . when a load is applied on epoxy , the polymer chains slide past each other and deform . once layered silicates such as clay particles are introduced in a polymer system , it restricts the motion of the polymer chain sliding and makes the matrix less pliable . as the clay content increases , it is more difficult for the polymer chains to untangle and move . this increase in restriction of polymer chains is responsible for the increase in modulus as the clay percentage increases . for the hygrothermally conditioned specimens , this behavior observed is mostly due to the presence of water inside the epoxy system , which increases the ductility of the epoxy system . water acts as an effective plasticizer and can diffuse into the nanofiller - polymer interface and weaken the bonding between them [ 40 , 43 ] . presence of water in epoxy system also results in an increase in free volume through rupture of hydrogen bonding between polymer chains , which increases the chain mobility and eases the segmental motion when a load is applied to the composite . these physical changes can be attributed to the observed lower modulus for water absorbed specimens . other mechanisms affecting the polymer such as hydrolysis and chain scission may also be responsible for lowering the modulus . once hydrolysis and chain scission take place , less bonding between the polymers makes it more deformable resulting in lower modulus for aged samples [ 22 , 36 , 51 ] . the effect of hygrothermal aging was more severe in neat epoxy than in the nanocomposites . for neat epon 862 , flexural modulus decreased 20% after hygrothermal aging , whereas it was only 13% and 11% for 0.5 wt% of nanomer i.28e and somasif mae clay - epoxy nanocomposites , respectively . the high aspect ratio of the clay platelets provides resistance against polymer chain mobility in a water absorbed ductile polymer leading to the observed lower degradation of flexural modulus values in comparison to neat polymer . for nanomer i.28e clay - epoxy samples conditioned at 110c for 6 h , recovery of flexural modulus once redried , free water residing in the microvoids was evaporated , and the effect of plasticization was not prominent anymore . as a result , the ductility of the polymer reduced and the recovery of mechanical properties from moisture absorbed state occurred . nevertheless , in case of somasif mae clay - epoxy samples , modulus recovery was negligible after the desorption cycle . due to the structural difference between the two clay particles , it is possible that the interface of somasif mae clay - epoxy is being more affected by the hygrothermal degradation than the nanomer i.28e clay - epoxy interface . flexural strengths for the epoxy and clay - epoxy nanocomposites were determined using three - point bend ( 3pb ) test and are plotted against clay loading percentage in figure 3 for nanomer i.28e and somasif mae clay . figure 3 shows that the addition of nanomer i.28e clay provided negligible improvement in flexural strength compared to neat epoxy . the maximum improvement in flexural strength was found to be less than 10% for both clay - epoxy systems from the base flexural strength of neat epoxy . similar observation has been reported in the literature , where addition of nanoclay particles did not significantly improve the flexural strength of the system . furthermore , when 2.0 wt% of clay is added to the nanocomposite , flexural strength value dropped to a lower value than the neat epoxy . increasing the amount of nanoparticles more than a certain amount has been found to reduce the flexural strength in earlier studies . it can also be observed that moisture absorbed nanocomposites showed significant reduction in flexural strength . for 1.0 wt% of i.28e clay - epoxy nanocomposites , reduction in flexural strength of nanocomposites after moisture absorption has been previously reported in the literature [ 4 , 43 , 5456 ] and has been attributed to the degradation of interface region , which in turn reduces stress transfer between the nanofiller and the matrix . for redried samples , as most of the moisture is driven out of the system and plasticization effect was minimal , flexural strength for these samples recovered almost fully . for instance , flexural strength recovers to 95% of its original value for 1.0 wt% of nanomer i.28e clay - epoxy nanocomposite . almost similar trend was observed for somasif mae clay - epoxy nanocomposites , where addition of clay did not change the property significantly , and after 24 h of hygrothermal aging property decreased to a lower value compared to the as - prepared samples . however , the severity of degradation was much less in both clay - epoxy systems compared to neat epoxy system . well dispersed high aspect ratio clay platelets have the capability of crack deflection and crack arresting , which can lead to the observed higher flexural strength in wet clay - epoxy samples in comparison to neat epoxy samples [ 48 , 57 ] . for redried nanocomposites , similar trend was observed for both material systems and it was found that flexural strength recovers almost fully . in this study , flexural strength has not been largely affected by the addition of clay into the epoxy . this may as well mean that flexural strength has been primarily governed by the flexural strength of the epoxy . as the strength of neat epoxy was marginally affected by the absorption - desorption cycle , so did the strength of clay - epoxy nanocomposites . the scanning electron micrographs of the fracture surface of tested neat epoxy and clay - epoxy nanocomposites are shown in figures 48 . sem micrograph of the fracture surface of the as - prepared neat polymer ( figure 4(a ) ) showed characteristic brittle failure with a smooth fracture surface . this mirror - like fracture surface is an indication of poor fracture toughness of epoxy and has been reported in previous studies conducted on epoxy polymers . for the redried neat epoxy specimen ( figure 4(b ) ) , a network of microcracks throughout the fracture surface is found . according to their study , absorbed moisture enhances craze initiation and propagation in polymer which can result in the formation of microcracks or fibrils in the polymer system . in this study , lower fracture toughness for dried neat polymer compared to unaged neat polymer can be attributed to the formation of these microcracks . the sem micrographs of clay incorporated epoxy systems showed significantly rougher fracture surface compared to the neat polymer . clay , if present in a system , physically blocks and slows down the crack propagation and the resulting fracture surface of the nanocomposite shows river - markings instead of smooth fracture surface found in the neat polymer . these river - markings provide clear indication of the enhanced toughening mechanism in polymeric materials due to clay incorporation and support the observed higher fracture toughness for clay - epoxy nanocomposites compared to the neat epoxy polymer . fracture surfaces of nanofiller reinforced polymer nanocomposites showing higher surface roughness have been reported in prior studies [ 4 , 6 , 59 ] . comparing the fracture surfaces of the as - prepared nanomer i.28e and somasif mae nanocomposite systems ( figures 5(a ) , 6(a ) , 7(a ) , and 8(a ) ) , it was observed that the as - prepared somasif mae clay nanocomposite has considerably less amount of river - markings , which is indicative of poor toughening in somasif mae clay nanocomposites . this observation supported the difference of critical stress intensity factor measured for these two nanocomposite systems . poor adhesion between somasif mae clay and epoxy resulted in less energy requirement during new surface formation , which can be attributed as the reason of these nanocomposites showing less fracture toughness than nanomer i.28e clay nanocomposites for the same clay loading percentage . fracture surface of moisture absorbed nanomer i.28e clay nanocomposites ( figures 5(b ) and 7(b ) ) showed less number of river - markings ( i.e. , lower critical stress intensity factor ) than the as - prepared nanocomposites . sem micrograph of somasif clay - epoxy nanocomposites ( figures 6(b ) and 8(b ) ) showed the presence of shear leaps . as shear yielding requires less energy to form new surface , moisture absorbed specimens had lower fracture toughness than the as - prepared specimens . although shear yielding was found to be the principle mechanism of failure in these specimens , some form of crack bifurcation and crack pinning was also present in these fracture surfaces . fracture surface micrographs of redried nanomer i.28e nanocomposites ( figures 5(c ) and 7(c ) ) showed more roughness than the moisture absorbed specimens indicating higher fracture energy absorbance for these specimens . micrographs of redried somasif mae clay - epoxy samples ( figures 6(c ) and 8(c ) ) showed significantly less rough fracture surface than the redried nanomer i.28e samples . it is important to note that these redried somasif mae clay - epoxy fracture surfaces showed very little resistance against the crack propagation even after most of the water was driven out of the system . this observation led to the speculation that absorbed moisture could have weakened the interface of the somasif mae clay particles and the epoxy matrix . the replacement of oh ( hydroxyl ) groups from the octahedral layer of the clay by the f ( fluorine ) groups makes the somasif mae clay particles much more hydrophobic compared to the nanomer i.28e clay particles . the higher hydrophobicity of the somasif mae clay particles could have exerted an additional force on the moisture absorbed interface and weakened the bond between the clay particles and the epoxy chains . this might have in turn resulted in the poor adhesion / less fracture energy absorption in the redried somasif mae clay - epoxy nanocomposites . ftir spectra of the as - prepared and redried neat epoxy are shown in figure 9 . from the ftir spectra , it was evident that as a result of the absorption - desorption cycle the epoxy underwent some permanent chemical changes . the drying cycle used in this study removed most of the water that was absorbed by the polymer system , and it was believed that the remaining water ( only about 7% of the total moisture uptake ) is chemically bound to the polymer system . ftir spectra ( figure 7 ) showed band at 32003400 cm , which is characteristic oh stretching of the hydroxyl group . figure 10 shows the ftir spectra for nanomer i.28e and somasif mae clay powder before and after the absorption - desorption cycle . nanomer i.28e clay showed another peak at 915 cm region , which is characteristic al oh peak . in somasif mae , the oh groups present in the corner of the octahedral layer of nanomer i.28e were substituted with f , which explains the absence of the peak at 915 cm . both clays in as - is condition showed 32003400 cm band for hydroxyl groups . however , the band was weaker in case of somasif mae compared to nanomer i.28e clay because of the hydrophobic nature of the somasif mae clay . it was interesting to note that the overall changes the clay powders underwent as a result of absorption - desorption cycle are fairly small and can be considered as insignificant . property deterioration due to moisture absorption has been one of the most important areas of interest in polymer research for the last few years . to be used as structural materials , it is of utmost importance to understand the reversible and irreversible changes occurring in polymeric materials as a result of moisture absorption . this study was conducted to elucidate the effect of moisture absorption on the mechanical properties of clay reinforced epoxy polymers . fracture toughness , flexural strength , and flexural modulus were determined for two different clay - epoxy nanocomposites following the astm standards . the effects of hygrothermal aging and subsequent redrying on the mechanical properties of these polymer nanocomposites were investigated . after removing the free water by drying , the irreversible effect or the permanent damage due to hygrothermal aging on the clay - epoxy nanocomposite systems was determined . irrespective of the clay reinforcement type , all the studied properties were degraded due to hygrothermal aging . several physical and chemical changes , such as interface weakening , hydrolysis , and chain scission , are responsible for the observed effect . the permanent damage or degradation was severe in case of fracture toughness and flexural modulus . permanent damage was found to be the highest for somasif mae clay reinforced specimens between two clay - epoxy nanocomposite systems . after studying the sem micrographs of the fracture surfaces , it was speculated that moisture absorption had higher negative impact on the interface of somasif mae clay and epoxy matrix compared to the other clay - epoxy system . the hydrophobic nature of the somasif mae clay due to the presence of f ( fluorine ) in the structure may have created additional tension between the polymer crosslinks in presence of moisture . ftir spectra of the clay particles treated with the same absorption - desorption cycle provided proof that both nanoparticles undergo minimal chemical change and retain their respective original chemistry . although incorporation of clay in epoxy matrix did not fully stop the degradation , it had positive effects to some extent . it was observed that the studied properties in general were less severely degraded for clay - epoxy nanocomposites compared to neat epoxy samples . therefore , clay particles could be successfully used to reinforce polymer materials to reduce the severity of property deterioration caused by the moisture absorption . however , the chemistry between the clay particles and polymer matrix and more specifically the chemical structure of the clay particles should be carefully considered to attain the best possible resistance against the property deterioration .

hydrophilic nature of epoxy polymers can lead to both reversible and irreversible / permanent changes in epoxy upon moisture absorption . the permanent changes leading to the degradation of mechanical properties due to combined effect of moisture and elevated temperature on epon 862 , nanomer i.28e , and somasif mae clay - epoxy nanocomposites are investigated in this study . the extent of permanent degradation on fracture and flexural properties due to the hygrothermal aging is determined by drying the epoxy and their clay - epoxy nanocomposites after moisture absorption . significant permanent damage is observed for fracture toughness and flexural modulus , while the extent of permanent damage is less significant for flexural strength . it is also observed that permanent degradation in somasif mae clay - epoxy nanocomposites is higher compared to nanomer i.28e clay - epoxy nanocomposites . fourier transform infrared ( ftir ) spectroscopy revealed that both clays retained their original chemical structure after the absorption - desorption cycle without undergoing significant changes . scanning electron microscopy ( sem ) images of the fracture surfaces provide evidence that somasif mae clay particles offered very little resistance to crack propagation in case of redried specimens when compared to nanomer i.28e counterpart . the reason for the observed higher extent of permanent degradation in somasif mae clay - epoxy system has been attributed to the weakening of the filler - matrix interface .

1. Introduction 2. Materials and Characterization 3. Results and Discussions 4. Conclusion

this crosslinking leads to a number of favorable thermal and mechanical properties including high strength and modulus , high creep resistance , high glass transition temperature , low shrinkage , and better chemical resistance . in recent years , researchers have developed and investigated polymer nanocomposites based on a wide variety of micro-/nanoscale fillers including clay particles [ 16 ] , aluminum particles , tio2 particles , graphenes [ 9 , 10 ] , carbon nanotubes [ 1113 ] , and halloysites [ 4 , 14 ] to improve the mechanical properties of epoxy polymers . this nature of epoxy resins makes them susceptible to high moisture absorption ; in general , depending on the nature of the epoxy resin , the equilibrium moisture uptake can be in the range of 17% . absorbed moisture usually degrades the functional , structural , and mechanical properties of the polymer matrix [ 4 , 1619 ] . it has been reported that mechanical and thermal properties of epoxy - based systems are severely affected by moisture absorption in comparison to other matrix materials , such as bismaleimide ( bmi ) , polyetheretherketone ( peek ) , and cyanate ester . plasticization is considered reversible upon drying , while other effects of moisture absorption are irreversible . in recent years , effect of moisture absorption on the mechanical properties of neat epoxy and clay - epoxy nanocomposite has been frequently studied . zhao and li reported that tensile strength and modulus decreased for both neat epoxy and nanocomposites upon moisture absorption , while the tensile strain increased significantly for moisture absorbed samples . alamri and low reported lower flexural strength and modulus for halloysite , nanoclay , and nanosilicon carbide nanocomposites as a result of moisture absorption . studied the effect of moisture absorption on the tensile properties of clay - epoxy nanocomposites and found that tensile strength and modulus decrease as a result of water uptake . investigated the effect of hydrothermal degradation on mechanical properties such as tensile strength , modulus , and fracture toughness with immersion duration . tensile and flexural properties of nanoclay reinforced composite laminates and cnt reinforced nanocomposites have also been found to be affected adversely as a result of moisture absorption [ 12 , 40 ] . a study on elastic modulus of epoxy polymer after absorption - desorption cycle showed recovery of property from wet condition , although modulus remained at a lower value than the as - prepared samples for lower filler volume . however , de'ne`ve and shanahan did not observe any recovery of elastic modulus after absorption - desorption cycle at elevated temperature . it is evident from the published works that moisture absorption can severely alter mechanical properties of epoxies by decreasing the elastic modulus [ 12 , 29 , 33 ] , tensile strength [ 3 , 12 ] , shear modulus [ 30 , 31 ] , flexural strength and flexural modulus [ 40 , 43 ] , yield stress , and ultimate stress as water uptake increases . most of the research on polymer - clay nanocomposites has been mainly focused on investigating the effect of moisture absorption on mechanical properties such as elastic modulus and tensile strength . although fracture toughness is a very important property for these nanocomposites as these are used in various structural applications , the effects of moisture absorption on fracture toughness of polymer - clay nanocomposites have not been studied extensively . durability of polymer / clay nanocomposites is still needed to be studied in depth , particularly for hygrothermal aging in which the degradation of the mechanical properties and loss of integrity of these nanocomposites occur from the simultaneous action of moisture and temperature . this study on clay - epoxy nanocomposites was designed to investigate the effect of hygrothermal aging on mechanical properties of these nanocomposites . two different clay particles were used to investigate the effect of clay structure on the permanent property changes due to hygrothermal aging . this was helpful to understand the extent of permanent degradation that occurred by the combined application of elevated temperature and moisture . mechanical properties in terms of fracture toughness , flexural strength , and flexural modulus are the properties that were studied . scanning electron microscopy and fourier transform spectroscopy were conducted to further elucidate the underlying fracture mechanisms of these preconditioned specimens . the epoxy resin used for this study is epon 862 , which is a diglycidyl ether of bisphenol f. the curing agent used for this resin system was a moderately reactive , low viscosity aliphatic amine curing agent , epikure 3274 . the crack length , a , was measured using an optical microscope ( nikon l150 ) which has a traveling plate with graduations : ( 1)kic = pbwf(aw ) , where p = maximum applied force ( n ) , b = thickness of the specimen ( mm ) , w = width of the specimen ( mm ) , and f(a / w ) = geometry factor , and it is given by the following equation : ( 2)f(aw)=3(s / w)a / w2(1 + 2(a / w))(1a / w)3/2 [(2.153.93(aw)+2.7(aw)2)1.99(aw)(1aw)iiiiiii(2.153.93(aw)+2.7(aw)2 ) ] , where s = support span ( mm ) and a = length of the precrack ( mm ) . the flexural strength and flexural modulus were calculated using the following equations , respectively : ( 4)f , max=3pmaxl2bd2,(5)eb = l3m4bd3 , where f , max = flexural strength ( mpa ) , eb = flexural modulus ( mpa ) , pmax = maximum load on the load - deflection curve ( n ) , l = support span ( mm ) , b = width of beam tested ( mm ) , d = depth of beam tested ( mm ) , and m = slope of the tangent to the initial straight - line portion of the load - deflection curve ( n / mm of deflection ) . surface morphology of the fractured specimens from senb tests was observed using scanning electron microscopy ( hitachi s-4800 fesem , dallas , tx ) . this observation was different from the findings reported by glaskova and aniskevich for clay - epoxy nanocomposites . the reason why a different clay - epoxy nanocomposite system behaves differently in moisture absorption test is still not clear and further investigation is required to understand it . in this study , although both clays are structurally different , the observed percentage weight gain for both nanocomposite systems was found approximately to be the same . for neat epoxy , this is possibly due to the fact that presence of clay hindered the moisture diffusion process in and out of the epoxy polymers . amount of water retained after the desorption cycle has been found to be almost similar for both clay - epoxy nanocomposite systems . the critical stress intensity factors as a function of clay loading percentage for nanomer i.28e and somasif mae clay - epoxy nanocomposites are shown in figure 1 . critical stress intensity factor , kic , increased 28% for the as - prepared 0.5 wt% nanomer i.28e clay - epoxy nanocomposite compared to neat epoxy . the reason behind this observation can be attributed to the layered structure of the clay . this is a common behavior for several types of epoxy - clay nanocomposites and has been reported in previous studies conducted on clay - epoxy nanocomposites [ 3 , 6 , 45 , 46 ] . for moisture saturated epoxy - clay nanocomposites , kic was found to be lower compared to the as - prepared nanocomposite samples for all the nanomer i.28e nanocomposites . as water molecules diffuse into the nanofiller - epoxy interface , debonding and weakening of the interface occur , resulting in poor stress transfer between the filler and the epoxy matrix [ 43 , 47 , 48 ] . redried neat epon 862 , free of void - filling water , showed 29% reduction in fracture toughness compared to the as - prepared neat epon 862 samples . addition of 0.5 wt% of clay resulted in 16% reduction in fracture toughness when compared to the as - prepared samples , which was significantly less compared to 29% reduction of neat epoxy . for 2.0 wt% nanomer i.28e clay - epoxy nanocomposite , however , the standard deviation of the as - prepared sample was much higher , which could possibly mean that the dried and the as - prepared samples have no difference in toughness . for the as - prepared and moisture saturated samples , somasif mae nanocomposites showed comparable trend in fracture toughness data ; clay reinforcement successfully improved the baseline epoxy properties and moisture absorption degraded the mechanical properties for all clay percentages . however , the permanent degradation after absorption - desorption cycle was found to be more prominent in the case of somasif mae clay nanocomposites compared to nanomer i.28e clay nanocomposites . the recovery of property after 6 hours of drying was negligible for somasif mae clay nanocomposites , whereas nanomer i.28e nanocomposites showed significant recovery of property after drying . the difference in property recovery of these two clay - epoxy nanocomposites can be attributed to the structural differences of the two clay particles and has been further investigated through sem and ftir technique . flexural modulus for the neat epoxy and clay - epoxy nanocomposites was determined from 3pb test and has been plotted against clay loading percentage in figure 2 for nanomer i.28e and somasif mae clay . for the hygrothermally conditioned specimens , this behavior observed is mostly due to the presence of water inside the epoxy system , which increases the ductility of the epoxy system . for neat epon 862 , flexural modulus decreased 20% after hygrothermal aging , whereas it was only 13% and 11% for 0.5 wt% of nanomer i.28e and somasif mae clay - epoxy nanocomposites , respectively . the high aspect ratio of the clay platelets provides resistance against polymer chain mobility in a water absorbed ductile polymer leading to the observed lower degradation of flexural modulus values in comparison to neat polymer . for nanomer i.28e clay - epoxy samples conditioned at 110c for 6 h , recovery of flexural modulus once redried , free water residing in the microvoids was evaporated , and the effect of plasticization was not prominent anymore . nevertheless , in case of somasif mae clay - epoxy samples , modulus recovery was negligible after the desorption cycle . due to the structural difference between the two clay particles , it is possible that the interface of somasif mae clay - epoxy is being more affected by the hygrothermal degradation than the nanomer i.28e clay - epoxy interface . flexural strengths for the epoxy and clay - epoxy nanocomposites were determined using three - point bend ( 3pb ) test and are plotted against clay loading percentage in figure 3 for nanomer i.28e and somasif mae clay . figure 3 shows that the addition of nanomer i.28e clay provided negligible improvement in flexural strength compared to neat epoxy . similar observation has been reported in the literature , where addition of nanoclay particles did not significantly improve the flexural strength of the system . for 1.0 wt% of i.28e clay - epoxy nanocomposites , reduction in flexural strength of nanocomposites after moisture absorption has been previously reported in the literature [ 4 , 43 , 5456 ] and has been attributed to the degradation of interface region , which in turn reduces stress transfer between the nanofiller and the matrix . for instance , flexural strength recovers to 95% of its original value for 1.0 wt% of nanomer i.28e clay - epoxy nanocomposite . almost similar trend was observed for somasif mae clay - epoxy nanocomposites , where addition of clay did not change the property significantly , and after 24 h of hygrothermal aging property decreased to a lower value compared to the as - prepared samples . however , the severity of degradation was much less in both clay - epoxy systems compared to neat epoxy system . well dispersed high aspect ratio clay platelets have the capability of crack deflection and crack arresting , which can lead to the observed higher flexural strength in wet clay - epoxy samples in comparison to neat epoxy samples [ 48 , 57 ] . in this study , flexural strength has not been largely affected by the addition of clay into the epoxy . this may as well mean that flexural strength has been primarily governed by the flexural strength of the epoxy . as the strength of neat epoxy was marginally affected by the absorption - desorption cycle , so did the strength of clay - epoxy nanocomposites . the scanning electron micrographs of the fracture surface of tested neat epoxy and clay - epoxy nanocomposites are shown in figures 48 . this mirror - like fracture surface is an indication of poor fracture toughness of epoxy and has been reported in previous studies conducted on epoxy polymers . in this study , lower fracture toughness for dried neat polymer compared to unaged neat polymer can be attributed to the formation of these microcracks . these river - markings provide clear indication of the enhanced toughening mechanism in polymeric materials due to clay incorporation and support the observed higher fracture toughness for clay - epoxy nanocomposites compared to the neat epoxy polymer . comparing the fracture surfaces of the as - prepared nanomer i.28e and somasif mae nanocomposite systems ( figures 5(a ) , 6(a ) , 7(a ) , and 8(a ) ) , it was observed that the as - prepared somasif mae clay nanocomposite has considerably less amount of river - markings , which is indicative of poor toughening in somasif mae clay nanocomposites . poor adhesion between somasif mae clay and epoxy resulted in less energy requirement during new surface formation , which can be attributed as the reason of these nanocomposites showing less fracture toughness than nanomer i.28e clay nanocomposites for the same clay loading percentage . fracture surface of moisture absorbed nanomer i.28e clay nanocomposites ( figures 5(b ) and 7(b ) ) showed less number of river - markings ( i.e. sem micrograph of somasif clay - epoxy nanocomposites ( figures 6(b ) and 8(b ) ) showed the presence of shear leaps . fracture surface micrographs of redried nanomer i.28e nanocomposites ( figures 5(c ) and 7(c ) ) showed more roughness than the moisture absorbed specimens indicating higher fracture energy absorbance for these specimens . micrographs of redried somasif mae clay - epoxy samples ( figures 6(c ) and 8(c ) ) showed significantly less rough fracture surface than the redried nanomer i.28e samples . it is important to note that these redried somasif mae clay - epoxy fracture surfaces showed very little resistance against the crack propagation even after most of the water was driven out of the system . this observation led to the speculation that absorbed moisture could have weakened the interface of the somasif mae clay particles and the epoxy matrix . the replacement of oh ( hydroxyl ) groups from the octahedral layer of the clay by the f ( fluorine ) groups makes the somasif mae clay particles much more hydrophobic compared to the nanomer i.28e clay particles . the higher hydrophobicity of the somasif mae clay particles could have exerted an additional force on the moisture absorbed interface and weakened the bond between the clay particles and the epoxy chains . this might have in turn resulted in the poor adhesion / less fracture energy absorption in the redried somasif mae clay - epoxy nanocomposites . from the ftir spectra , it was evident that as a result of the absorption - desorption cycle the epoxy underwent some permanent chemical changes . the drying cycle used in this study removed most of the water that was absorbed by the polymer system , and it was believed that the remaining water ( only about 7% of the total moisture uptake ) is chemically bound to the polymer system . figure 10 shows the ftir spectra for nanomer i.28e and somasif mae clay powder before and after the absorption - desorption cycle . nanomer i.28e clay showed another peak at 915 cm region , which is characteristic al oh peak . in somasif mae , the oh groups present in the corner of the octahedral layer of nanomer i.28e were substituted with f , which explains the absence of the peak at 915 cm . however , the band was weaker in case of somasif mae compared to nanomer i.28e clay because of the hydrophobic nature of the somasif mae clay . it was interesting to note that the overall changes the clay powders underwent as a result of absorption - desorption cycle are fairly small and can be considered as insignificant . property deterioration due to moisture absorption has been one of the most important areas of interest in polymer research for the last few years . to be used as structural materials , it is of utmost importance to understand the reversible and irreversible changes occurring in polymeric materials as a result of moisture absorption . this study was conducted to elucidate the effect of moisture absorption on the mechanical properties of clay reinforced epoxy polymers . fracture toughness , flexural strength , and flexural modulus were determined for two different clay - epoxy nanocomposites following the astm standards . after removing the free water by drying , the irreversible effect or the permanent damage due to hygrothermal aging on the clay - epoxy nanocomposite systems was determined . irrespective of the clay reinforcement type , all the studied properties were degraded due to hygrothermal aging . the permanent damage or degradation was severe in case of fracture toughness and flexural modulus . permanent damage was found to be the highest for somasif mae clay reinforced specimens between two clay - epoxy nanocomposite systems . after studying the sem micrographs of the fracture surfaces , it was speculated that moisture absorption had higher negative impact on the interface of somasif mae clay and epoxy matrix compared to the other clay - epoxy system . the hydrophobic nature of the somasif mae clay due to the presence of f ( fluorine ) in the structure may have created additional tension between the polymer crosslinks in presence of moisture . ftir spectra of the clay particles treated with the same absorption - desorption cycle provided proof that both nanoparticles undergo minimal chemical change and retain their respective original chemistry . it was observed that the studied properties in general were less severely degraded for clay - epoxy nanocomposites compared to neat epoxy samples .

breakthroughs in research have allowed the characterization of malignancies according to their unique gene expression , which has allowed the pragmatic targeting of many cancer types based on their specific gene expression patterns . for example , trastuzumab improves the overall and progression - free survival in human epidermal receptor 2- ( her2- ) positive breast cancer [ 13 ] . the receptor - specific monoclonal antibodies bevacizumab [ 4 , 5 ] and cetuximab have shown remarkable outcome in vascular growth factor receptor- ( vegf- ) positive and epidermal growth factor receptor ( egfr)-positive cancer , respectively . individualized profiling and targeting systems provide novel tools to improve both prognostic accuracy and individualized treatment for patients . in addition , each of the classical pillars of cancer therapy(1 ) surgical resection , ( 2 ) chemotherapy , and ( 3 ) radiation therapy has made significant technological strides , and numerous clinical studies have improved our ability to effectively apply and combine these modalities . vaccines that prevent the spread of the human papilloma virus ( hpv ) promise to dramatically reduce cervical cancer ; conversely , viruses are being developed that directly attack carcinogenic cells . advancements such as functional imaging , magnetic resonance imaging , immunohistochemistry , and flow cytometry have recently refined our ability to tease out , subdivide , prognosticate , and define the myriad of permutations in this uniquely complex and malignant disease . a growing arsenal of prevention strategies and screening technologies has allowed physicians to diagnose and treat cancer earlier in its progression than ever before . all of these evolving modalities and strategies to manage cancer have helped result in a pattern of continuously dwindling cancer - related morbidity and mortality in the united states . however , despite this progress , cancer remains the 2nd most common cause of death in the united states . our continuing inability to cure cancer is largely attributed to the ability of cancer cells to spread and repopulate after initial therapies have eliminated all detectable signs of disease . new interventions that reduce this capacity could have a far - reaching impact on our ability to prevent recurrences , extend survival , and cure many types of cancer . thus , better understanding the mechanisms of cancer progression for the development of antirepopulation therapies is likely to offer significant clinical benefit . the cancer stem cell ( csc ) hypothesis has emerged within this line of investigation . this hypothesis has helped explain how cancer might recur and metastasize despite effective initial treatment and thus represents a promising new front in the war on cancer . a paradigm shift in our understanding of cancer tumorigenesis emerged in 1994 when john dick and colleagues demonstrated that human acute myeloid leukemia ( aml ) has a hierarchical organization that originates from a primitive hematopoietic cell . this popularized a concept first proposed over a century ago : that cancer growth within a given neoplastic process may be dependent upon only a small fraction of progenitor cells [ 15 , 16 ] . these cancer cells that retain their normal stem cell properties of self - renewal and pluripotency are often referred to as cscs . within the framework of the csc hypothesis , normally dormant stem cells may inadvertently acquire tumorigenic dna mutations and become cscs which inappropriately begin dividing and direct the neoplastic process . multiple studies have recently provided compelling support of the csc hypothesis [ 1719 ] . despite controversies surrounding the csc hypothesis [ 20 , 21 ] , substantial evidence has emerged that supports its role in cancer including aml , brain , breast , colon , head and neck , lung , liver , melanoma , pancreatic , prostate , and squamous cell cancer ( figure 1 ) . however , the csc hypothesis has not been fully established and will likely evolve as unknown molecular targets capable of promoting tumorigenesis continue to be discovered . moreover , the translation from the theoretical benefit of csc eradication into its actual clinical benefit has to be experimentally demonstrated . another poorly understood nuance is that certain cancer types may be relatively independent of fractional csc populations , operating more consistently with classical stochastic or clonal evolution models . although there are limitations to the csc hypothesis , it is evident that cancer often possesses functionally defined cscs , and is likely to be at least partially dependent on cscs for growth and survival . in cancer types where neoplastic growth and differentiation depend on cscs , complete eradication of this population may be curative . furthermore , agents that force cscs to rapidly differentiate en masse within such cancer types may limit disease progression . alternatively , suppressing residual cscs after initial tumor debulking may sustain remissions and extend the progression - free survival of patients receiving csc suppressive therapy . considering these distinct therapeutic potentials of targeting cscs , it appears that csc - targeted therapies could be an effective complement to traditional treatment approaches such as surgery , chemotherapy , and radiation therapy . indeed , it is possible that these traditional strategies leave behind residual cscs which are capable of spreading and regenerating tumors , leading to cancer recurrence and metastasis . moreover , these recurring tumors often acquire resistance to chemotherapy and radiation [ 36 , 37 ] . multiple investigators have demonstrated the ability of cscs to develop resistance traits after induction chemo- and radiation therapy . . this heterogeneity may be responsible for the evolution of resistance to first - line therapies in recurrent cancer since treatment - resistant cells within a heterogeneous tumor population may be selected for during induction therapy . in addition , csc heterogeneity may make the pharmacological eradication of the entire csc population difficult since these cells may exhibit variable expression of drug - targeted genetic markers . this task is complicated by the possibility that cancer may exhibit fluctuating phenotypes , frequencies , and biological properties within an individual patient . furthermore , existing microenvironmental signaling pathways may recruit or promote csc functions , perhaps through neoplastic clonal dedifferentiation processes [ 40 , 41 ] . until these challenges are overcome , csc - targeting therapies will not reach their full potential . regardless , research surrounding the csc hypothesis has already helped generate numerous potential pharmacological interventions , and combinations of these csc - specific therapeutic approaches with traditional cancer treatment strategies may show synergistic benefits since their mechanisms of action are distinct and complementary . as previously mentioned , cancer recurrence may be partly due to the fact that conventional therapies such as chemo- and radiation therapy fail to specifically target cscs . instead , these therapies likely enrich csc populations by preferentially killing differentiated cancer cells that had little potential to sustain cancer growth . numerous studies indicate that cscs are resistant to chemo- and radiotherapy and are therefore preferentially preserved when cancer cells are targeted by these approaches [ 15 , 4245 ] . interestingly , during differentiation therapy for the treatment of acute promyelocytic leukemia ( apml ) , all - transretinoic acid and arsenic trioxide are used to induce the differentiation of cscs down their hematopoietic lineage . the dramatic anticancer effects of combined modality differentiation therapy in leukemia also demonstrate how synergy between independent therapeutic approaches can achieve remarkable outcomes in cancer therapy . thus , differentiation treatment of apml serves to illustrate ( 1 ) the relative impotence of differentiated cells in cancer , ( 2 ) the potential therapeutic benefit of specifically targeting cscs , and ( 3 ) the potential synergy between csc - specific therapies and existing modalities . in recent years , an effort has been made to successfully identify stem cells in multiple human malignancies , including hematological , breast , colorectal , brain , pancreatic , and maxillofacial cancer [ 22 , 25 , 32 , 34 , 36 , 4850 ] . much attention has been directed to specific cell - surface proteins . among these , cd133/prominin-1 is a cell - surface molecule thought to be a stem cell marker for multiple cancer types , including cns , colon , hepatocellular , pancreatic , prostate , and renal cancer . demonstrated that freshly excised small cell and nonsmall cell lung cancers tissues contain a small subset of cd133-positive cells capable of generating long - term lung tumor spheres in vitro and differentiating into tumors in vivo . matsumoto et al . elucidated a mechanistic relationship between cd133 and the hypoxia - inducible factor-1 ( hif-1 ) , a downstream molecule in the mammalian target of rapamycin ( mtor ) cell signaling pathway , suggesting a role for mtor in the regulation of cd133 expression . in addition to cell - surface markers , many investigators have focused on the selective overexpression of certain genes normally present in progenitor cells . leukemia cells , which have been transformed from the normally present partially committed cells responsible for physiological cellular maintenance , undergo mutations that result in self - perpetuated renewal capabilities . these cells can be identified by selective gene overexpression [ 24 , 5357 ] . in an attempt to link cellular pathways to gene expression patterns in lung cscs , stevenson et al . compiled and tested a model of 100 signature genes to determine embryonic stemness . cells with a high embryonic stemness score were found to affect multiple cellular processes , including ras , myc , chromosomal instability , and cellular invasiveness . seo and colleagues demonstrated increased expression of 13 genes in side - population ( sp ) a549 nonsmall cell lung cancer cells , as compared to non - sp cells . stemness of cancer cells by quantifying gene expression signatures , and he has shown that this model may predict therapeutic outcomes . bmi1 pathway algorithm is based on a collective signature of 9 individual gene characteristics : tez , eed pathway , suz12/polii , suz12 , nanog / sox2/oct4 , pcg - tf , bcd - tf , esc pattern , and bmi1 pathway . this multifactorial model allowed the stratification of patients into high - risk and low - risk groups in a retrospective analysis of large cohorts of breast , prostate , lung , and ovarian cancer patients . it remains to be confirmed whether an individual cellular marker can accurately identify normal stem cells or cscs or whether a multifactorial phenotypic model is required . cscs have been found to exhibit a number of genetic and cellular adaptations that confer resistance to classical therapeutic approaches . these include relative dormancy / slow cell cycle kinetics , efficient dna repair , high expression of multidrug - resistance - type membrane transporters , and resistance to apoptosis ( figure 2 ) . radiotherapy and most types of chemotherapy exert their antineoplastic function by disrupting cancer cell dna integrity ; therefore , it is possible that the oncogenic resistance of cscs results from increased expression of dna integrity - maintenance systems . in addition , increased expression of drug efflux pumps may promote oncogenic resistance against cytotoxic chemotherapeutic agents [ 62 , 63 ] . normal , noncancerous stem cells exhibit well - fortified dna mutation defense systems that typically serve to prevent mutation into carcinogenic cscs . unfortunately , when mutations that create cscs do occur , the inherent defense systems of stem cells serve to protect them from dna - targeting chemo- and radiation therapy . the chemo- and radioresistance of cscs has now been demonstrated in numerous experiments , although the mechanisms underlying this resistance are not fully understood . in one experiment , radiation was shown to cause equal levels of damage to all cancer cells , but cscs were able to repair this damage more rapidly . one potential modulator of csc resistance to dna - targeting agents is the family of checkpoint kinases 1/2 ( chk1/2 kinases ) , which become activated after genotoxic stress and arrest the cell cycle to allow dna repair . these kinases have higher basal and inducible activities in cscs than in nonstem cells . supporting the role of chk1/2 kinases in cscs , chk1/2 inhibitors partially reverse the resistance of glioblastoma cscs to radiation - induced cell death [ 65 , 66 ] . in addition to augmented dna repair systems , cscs may also exhibit changes in telomerase function , which allows resistance to chromosomal degradation in these rapidly dividing cells . telomerase is a complex ribonucleoprotein enzyme that synthesizes and maintains telomeric repeats at the ends of chromosomal strands . sustained telomerase function is critical in conferring cellular immortality , as telomeres are otherwise shortened with each cell division , eventually triggering cellular senescence . telomerase function was recently shown to be downregulated in brain cscs , and several drugs that interfere with telomerase function are already in clinical trials , including arsenic trioxide , grn163l , and vaccines [ 6872 ] . an important component of the dna integrity defense systems in normal stem cells is the relatively high expression of efflux transporters from the atp - binding cassette ( abc ) gene family . these pumps allow normal stem cells to preserve their genome more effectively against chemical mutagens in an attempt to prevent carcinogenesis . similar to the way that cscs may derive resistance to dna damage from the preexisting dna repair systems in normal stem cells , cscs may also derive resistance to chemical mutagens ( e.g. , chemotherapy ) through the expression of drug efflux pumps in normal stem cells from which they were derived . moreover , the relatively high expression of these transporters may be used to identify cscs within a neoplasm . drugs that block the function of efflux transporters or that down - regulate their expression have the potential to overcome csc chemoresistance . although multidrug transporters are not likely to significantly influence the direct cytotoxicity of radiation - based therapies , chemotherapy or chemoradiation therapy may benefit from blockade of multidrug efflux pumps in cscs . resistance to therapy might also be conferred to cscs through the activation of the akt pathway [ 75 , 76 ] and the overamplification of apoptosis inhibitor proteins . this was first demonstrated in chemoresistant hepatocellular carcinoma cscs , which were found to preferentially activate akt / pkb and bcl-2 cell survival pathways . this suggests that characterization of akt and bcl-2 expression in cscs may have significant clinical utility . fms - like tyrosine kinase 3 ( flt3 ) receptor signaling is an important hematopoietic growth pathway upstream of akt . flt3 receptors are often mutated in aml and are associated with a high relapse rate and poor prognosis . inhibition of flt3 signaling with cep701 reduces the tumorigenicity of xenografts , and cep 701 has reached phase 2 clinical trials . the mitochondrial pathway of apoptosis is triggered by cytochrome c release and second mitochondria - derived activator of caspase ( smac ) activation . smac , in association with direct inhibitor of apoptosis binding protein with low pi ( smac / diablo ) , promotes apoptosis via neutralization of inhibitor of apoptosis ( iap ) proteins . most human cancers have high levels of iaps , including the x - linked inhibitor of apoptosis protein ( xiap ) isoform , which are associated with poor treatment responses . based on these observations , vellanki et al . found that the inherent radioresistance of glioblastoma cscs could be alleviated by promoting apoptosis with an xiap inhibitor . importantly , this treatment had no undesirable radiosensitizing effects on normal rat neurons or glial cells . another promising molecular target to promote apoptosis in cscs is nuclear factor b ( nfb ) . nfb is a transcription factor believed to be intricately involved in the development and progression of certain cancer types . nuclear factor nb ( nfnb ) , a cousin of nfb , is an antiapoptotic transcription factor that is activated in leukemias [ 86 , 87 ] , pancreatic adenocarcinoma , and melanoma . although these nuclear factors are not as well studied as akt , they may offer promising drug targets . nf-b inhibitors include npi-0052 ( salinosporamides a ) , which is in phase i clinical trials , and tdzd-8 ( parthenolide ) , which is still in preclinical testing . oxygen is a well - known radiosensitizing agent due to its ability to form radiation - induced reactive oxygen species that can damage dna . accordingly , radioresistance in breast cscs may be mediated by increased production of free - radical scavengers . considering the dependence of radiotherapy on oxygen free radicals , it has long been postulated that areas of low oxygen tension within tumors create microenvironments that are relatively protected from radiation - induced damage . unexpectedly , it was discovered that cscs reside along perivascular areas and are thus likely to be well oxygenated . this may help explain the efficacy of antiangiogenic therapies such as bevacizumab in that such therapies may be csc - specific . theoretically , csc compartment hypoxia may be induced by antiangiogenic therapies , conferring radioresistance to the cscs , although this has yet to be demonstrated in vivo , and the clinical significance of this remains unknown . still , we speculate that radiation might be more effective in treating cancer if it is administered before any antiangiogenic chemotherapies are applied . vermeulen and colleagues recently discovered another interesting role of the microenvironment , specifically in the promotion of cancer cell stemness . importantly , wnt signaling in these cells depended on costimulation by c - met signaling . activated myofibroblasts in the tumor microenvironment were responsible for c - met activation through production of hepatocyte growth factor ( hgf ) . thus , inhibition of stromal - produced hgf or the subsequent activation of c - met signaling via c - met inhibitors may represent additional approaches to target cscs . an important result of the well - documented csc resistance to radiation and chemotherapy is that these therapies often serve to enrich the resistant csc subpopulation , perhaps even selecting for more resistant clones within a heterogeneous csc population . evidence of radiation - induced enrichment has been shown in both brain [ 15 , 45 ] and breast cscs . furthermore , radiation has little effect on the ability of remaining cscs to regrow tumors . thus , csc enrichment may be the basis for the relative inability of most single modality cancer treatment strategies to control long - term cancer growth . csc - specific pharmaceutical interventions are being developed that may eliminate both primary and acquired csc chemoresistance . this may dramatically improve the treatment of cancer by abrogating the potential for csc - induced tumor regrowth and systemic disease spread after initial treatment . showing that pancreatic cscs could survive and expand after serial exposures to gemcitabine , this chemoresistance was overcome by the use of cd44 or abc transporter inhibitors . it is now well established that combination therapy helps prevent the development of cancer resistance , except in a select group of cancer types where a single pharmaceutically correctable mutation exists . for example , many clinical trials have shown improvements in cancer survival with the use of concurrent chemo- and radiation therapy . this likely reflects the broadly held belief that the best chance for curing cancer is during the first round of therapy before the selection pressure promotes the evolution of resistant cscs . in later rounds of therapy , not only has the cancer had time to grow and spread further , but it has also evolved resistance to previously encountered therapies . unfortunately , coadministration of chemotherapy and radiation therapy is not effective against all types of cancer , and it is not always feasible due to its potential significant toxicity . thus , it will be important to design preclinical studies and clinical trials that evaluate potential synergistic benefits of adding csc - targeted therapies to traditional cancer regimens . as new data supports a role for the csc hypothesis in solid tumors in addition to hematologic malignancies , outcomes following the surgical resection of solid tumors may significantly improve . if induction approaches can be effectively augmented with anti - csc therapies , then followup surgical resection may show improved curative outcomes . theoretically , csc - specific induction chemotherapies should offer an immediate reduction in csc metastatic potential and should reduce any hematogenous and lymphatic csc micrometastases that would otherwise diminish the efficacy of surgical resection . considering its powerful therapeutic potential , csc - targeted therapies may be particularly valuable in surgically challenging malignancies such as pancreatic and brain cancer . in order to more effectively target cscs , molecular proliferation and survival mechanisms of cscs many institutions have developed large banks of malignant tissues with coordinated clinical data , and this resource is being actively mined . techniques for concentrating , isolating , and enriching cscs from resected tumors are also rapidly evolving , and cell culture and xenograft models that allow us to transplant and sustain cscs are maturing . finally , these advances have been translated into the development of several therapeutic opportunities . here , we will review some of the prominent classes of drugs that will potentially yield clinical benefits in the near future . an update of clinical trials assessing these targets is illustrated in table 1 ( data from http://clinicaltrials.gov/ ) . developmental pathways that direct the differentiation of normal stem cells represent attractive targets for drug discovery . in particular , the roles of notch and wnt/-catenin signaling have been examined , and both have been implicated in the development and progression of several types of leukemia [ 100 , 101 ] . for instance , wnt signaling serves an important role in promoting the proliferation of immature thymocytes . the nonsteroidal anti - inflammatory drug ( nsaid ) etodolac inhibits wnt signaling and may be of benefit in the treatment of chronic lymphocytic leukemia . in fact , all nsaids may have anti - wnt properties and thus potentially have anticancer properties . the wnt/-catenin pathway promotes genomic instability and dna damage tolerance that may be enhanced by dna damage in cscs . the pathway has been shown to promote genomic instability in colon cancer and possibly promotes conversion of normal stem cells to cscs in gliomas . it has been postulated that the wnt/-catenin pathway may promote genomic instability after irradiation , thus allowing tumor cells to both survive after irradiation and to develop additional adaptive mutations . wnt inhibitors have been designed to therapeutically prevent this possibility and include icg-001 , fungal derivatives pkf115 - 854 and cgp049090 , as well as monoclonal antibodies against wnt-1 and wnt-2 ( figure 3 ) . the notch/-secretase / jagged signaling pathway is an important regulator of differentiation and helps control cell fate . the notch ligands , jagged 1 & 2 and delta1 ( d1 ) to delta3 ( d3 ) , induce the release of the notch intracellular ( notch - ic ) domain via enzymatic proteolytic cleavage by - and -secretases . notch - ic translocates to the nucleus where it induces transcription of notch responsive genes [ 106 , 107 ] . notch signaling pathways are activated in both breast cscs and in endothelial cells in response to radiation . inhibition of notch signaling via -secretase inhibitors can potentially block csc self - renewal and decrease medulloblastoma growth , and significant efforts to downregulate notch signaling are underway . currently available notch signaling inhibitors include mk-0752 , a -secretase inhibitor that is in clinical development for the treatment of leukemia ( figure 4 ) . in addition to the classical notch pathway , other routes may be used to modulate the carcinogenic potential of elevated notch signaling in cscs . in particular , the delta / notch - like epidermal growth factor - related receptor ( dner ) can be induced by histone deacetylase inhibition to inhibit the growth of and induce the differentiation of glioblastoma neurospheres and xenografts . this provides a basis for the manipulation of noncanonical signaling pathways for therapeutic intervention against cscs . the hedgehog signaling pathway may represent an important modulator of csc carcinogenesis with significant therapeutic implications [ 113117 ] . similar to notch signaling , hedgehog signaling may also benefit from expanded drug discovery efforts within noncanonical pathways . already , hedgehog inhibitors have been shown to inhibit medulloblastoma growth in mice , and at least 3 different hedgehog inhibitors have reached phase i clinical trials ( figure 5 ) . as mentioned previously , the cell - surface molecule cd133 is believed to be a stem cell marker for multiple cancer types . its tumor - initiating function has been demonstrated in cns cancer , where only cd133 cells from brain tumor biopsy samples were able to reform tumors in in vivo mouse models . a recent study by wang et al . demonstrated the potential therapeutic use of targeting cd133 to direct therapy specifically towards cscs . they conjugated single - walled carbon nanotubules ( swnts ) , which allow localized hyperthermia treatment , to anti - cd133 monoclonal antibodies , and cultured these products with both cd133 and cd133 glioblastoma ( gbm ) cells . a mixture of cd133 and cd133 cells were then exposed to near - infrared laser light , and the cd133 gbm cells were selectively destroyed . with the advent of multidisciplinary approaches to cancer therapy , significant strides have been made in the treatment of cancer . now with new discoveries relating to cscs , we have yet another mechanism of therapeutic arsenal that may prove beneficial in combination with current therapeutic modalities . the basic foundations for csc - targeted therapy are actively being discovered , and there are already several pharmacologic agents available that are capable of specifically modulating csc intracellular signaling . still , much remains unknown about the basic signaling mechanisms of cscs that confer resistance to treatment , and better methods for the disruption of csc signaling must be developed to fully integrate the csc hypothesis into our treatment paradigms . interestingly , cscs may not necessarily need to be eradicated to prevent cancer progression if they can be forced to differentiate down their lineage en masse as they do in the treatment of apml . it is important for future studies to focus on the discovery of new molecular targets for the development of better pharmaceutical agents to eliminate or differentiate cscs and that these agents be studied in tandem with traditional cancer therapies .

despite advances in treatment , cancer remains the 2nd most common cause of death in the united states . poor cure rates may result from the ability of cancer to recur and spread after initial therapies have seemingly eliminated detectable signs of disease . a growing body of evidence supports a role for cancer stem cells ( cscs ) in tumor regrowth and spread after initial treatment . thus , targeting cscs in combination with traditional induction therapies may improve treatment outcomes and survival rates . unfortunately , cscs tend to be resistant to chemo- and radiation therapy , and a better understanding of the mechanisms underlying csc resistance to treatment is necessary . this paper provides an update on evidence that supports a fundamental role for cscs in cancer progression , summarizes potential mechanisms of csc resistance to treatment , and discusses classes of drugs currently in preclinical or clinical testing that show promise at targeting cscs .

1. Introduction 2. The CSC Hypothesis 3. Frequent Cancer Recurrence May Be due to the Preferential Killing of Differentiated Cells While Leaving CSCs behind 4. The Detection and Identification of CSCs 5. Mechanisms of CSC Resistance to Chemotherapy and Radiation 6. Induction Therapy May Enrich CSCs 7. Concluding Remarks

breakthroughs in research have allowed the characterization of malignancies according to their unique gene expression , which has allowed the pragmatic targeting of many cancer types based on their specific gene expression patterns . in addition , each of the classical pillars of cancer therapy(1 ) surgical resection , ( 2 ) chemotherapy , and ( 3 ) radiation therapy has made significant technological strides , and numerous clinical studies have improved our ability to effectively apply and combine these modalities . vaccines that prevent the spread of the human papilloma virus ( hpv ) promise to dramatically reduce cervical cancer ; conversely , viruses are being developed that directly attack carcinogenic cells . advancements such as functional imaging , magnetic resonance imaging , immunohistochemistry , and flow cytometry have recently refined our ability to tease out , subdivide , prognosticate , and define the myriad of permutations in this uniquely complex and malignant disease . a growing arsenal of prevention strategies and screening technologies has allowed physicians to diagnose and treat cancer earlier in its progression than ever before . all of these evolving modalities and strategies to manage cancer have helped result in a pattern of continuously dwindling cancer - related morbidity and mortality in the united states . however , despite this progress , cancer remains the 2nd most common cause of death in the united states . our continuing inability to cure cancer is largely attributed to the ability of cancer cells to spread and repopulate after initial therapies have eliminated all detectable signs of disease . new interventions that reduce this capacity could have a far - reaching impact on our ability to prevent recurrences , extend survival , and cure many types of cancer . thus , better understanding the mechanisms of cancer progression for the development of antirepopulation therapies is likely to offer significant clinical benefit . the cancer stem cell ( csc ) hypothesis has emerged within this line of investigation . this hypothesis has helped explain how cancer might recur and metastasize despite effective initial treatment and thus represents a promising new front in the war on cancer . a paradigm shift in our understanding of cancer tumorigenesis emerged in 1994 when john dick and colleagues demonstrated that human acute myeloid leukemia ( aml ) has a hierarchical organization that originates from a primitive hematopoietic cell . these cancer cells that retain their normal stem cell properties of self - renewal and pluripotency are often referred to as cscs . within the framework of the csc hypothesis , normally dormant stem cells may inadvertently acquire tumorigenic dna mutations and become cscs which inappropriately begin dividing and direct the neoplastic process . multiple studies have recently provided compelling support of the csc hypothesis [ 1719 ] . despite controversies surrounding the csc hypothesis [ 20 , 21 ] , substantial evidence has emerged that supports its role in cancer including aml , brain , breast , colon , head and neck , lung , liver , melanoma , pancreatic , prostate , and squamous cell cancer ( figure 1 ) . however , the csc hypothesis has not been fully established and will likely evolve as unknown molecular targets capable of promoting tumorigenesis continue to be discovered . moreover , the translation from the theoretical benefit of csc eradication into its actual clinical benefit has to be experimentally demonstrated . another poorly understood nuance is that certain cancer types may be relatively independent of fractional csc populations , operating more consistently with classical stochastic or clonal evolution models . although there are limitations to the csc hypothesis , it is evident that cancer often possesses functionally defined cscs , and is likely to be at least partially dependent on cscs for growth and survival . in cancer types where neoplastic growth and differentiation depend on cscs , complete eradication of this population may be curative . alternatively , suppressing residual cscs after initial tumor debulking may sustain remissions and extend the progression - free survival of patients receiving csc suppressive therapy . considering these distinct therapeutic potentials of targeting cscs , it appears that csc - targeted therapies could be an effective complement to traditional treatment approaches such as surgery , chemotherapy , and radiation therapy . moreover , these recurring tumors often acquire resistance to chemotherapy and radiation [ 36 , 37 ] . multiple investigators have demonstrated the ability of cscs to develop resistance traits after induction chemo- and radiation therapy . this heterogeneity may be responsible for the evolution of resistance to first - line therapies in recurrent cancer since treatment - resistant cells within a heterogeneous tumor population may be selected for during induction therapy . in addition , csc heterogeneity may make the pharmacological eradication of the entire csc population difficult since these cells may exhibit variable expression of drug - targeted genetic markers . this task is complicated by the possibility that cancer may exhibit fluctuating phenotypes , frequencies , and biological properties within an individual patient . furthermore , existing microenvironmental signaling pathways may recruit or promote csc functions , perhaps through neoplastic clonal dedifferentiation processes [ 40 , 41 ] . until these challenges are overcome , csc - targeting therapies will not reach their full potential . regardless , research surrounding the csc hypothesis has already helped generate numerous potential pharmacological interventions , and combinations of these csc - specific therapeutic approaches with traditional cancer treatment strategies may show synergistic benefits since their mechanisms of action are distinct and complementary . as previously mentioned , cancer recurrence may be partly due to the fact that conventional therapies such as chemo- and radiation therapy fail to specifically target cscs . instead , these therapies likely enrich csc populations by preferentially killing differentiated cancer cells that had little potential to sustain cancer growth . numerous studies indicate that cscs are resistant to chemo- and radiotherapy and are therefore preferentially preserved when cancer cells are targeted by these approaches [ 15 , 4245 ] . interestingly , during differentiation therapy for the treatment of acute promyelocytic leukemia ( apml ) , all - transretinoic acid and arsenic trioxide are used to induce the differentiation of cscs down their hematopoietic lineage . the dramatic anticancer effects of combined modality differentiation therapy in leukemia also demonstrate how synergy between independent therapeutic approaches can achieve remarkable outcomes in cancer therapy . thus , differentiation treatment of apml serves to illustrate ( 1 ) the relative impotence of differentiated cells in cancer , ( 2 ) the potential therapeutic benefit of specifically targeting cscs , and ( 3 ) the potential synergy between csc - specific therapies and existing modalities . in recent years , an effort has been made to successfully identify stem cells in multiple human malignancies , including hematological , breast , colorectal , brain , pancreatic , and maxillofacial cancer [ 22 , 25 , 32 , 34 , 36 , 4850 ] . much attention has been directed to specific cell - surface proteins . among these , cd133/prominin-1 is a cell - surface molecule thought to be a stem cell marker for multiple cancer types , including cns , colon , hepatocellular , pancreatic , prostate , and renal cancer . demonstrated that freshly excised small cell and nonsmall cell lung cancers tissues contain a small subset of cd133-positive cells capable of generating long - term lung tumor spheres in vitro and differentiating into tumors in vivo . elucidated a mechanistic relationship between cd133 and the hypoxia - inducible factor-1 ( hif-1 ) , a downstream molecule in the mammalian target of rapamycin ( mtor ) cell signaling pathway , suggesting a role for mtor in the regulation of cd133 expression . leukemia cells , which have been transformed from the normally present partially committed cells responsible for physiological cellular maintenance , undergo mutations that result in self - perpetuated renewal capabilities . these cells can be identified by selective gene overexpression [ 24 , 5357 ] . compiled and tested a model of 100 signature genes to determine embryonic stemness . cells with a high embryonic stemness score were found to affect multiple cellular processes , including ras , myc , chromosomal instability , and cellular invasiveness . stemness of cancer cells by quantifying gene expression signatures , and he has shown that this model may predict therapeutic outcomes . bmi1 pathway algorithm is based on a collective signature of 9 individual gene characteristics : tez , eed pathway , suz12/polii , suz12 , nanog / sox2/oct4 , pcg - tf , bcd - tf , esc pattern , and bmi1 pathway . this multifactorial model allowed the stratification of patients into high - risk and low - risk groups in a retrospective analysis of large cohorts of breast , prostate , lung , and ovarian cancer patients . it remains to be confirmed whether an individual cellular marker can accurately identify normal stem cells or cscs or whether a multifactorial phenotypic model is required . cscs have been found to exhibit a number of genetic and cellular adaptations that confer resistance to classical therapeutic approaches . these include relative dormancy / slow cell cycle kinetics , efficient dna repair , high expression of multidrug - resistance - type membrane transporters , and resistance to apoptosis ( figure 2 ) . normal , noncancerous stem cells exhibit well - fortified dna mutation defense systems that typically serve to prevent mutation into carcinogenic cscs . unfortunately , when mutations that create cscs do occur , the inherent defense systems of stem cells serve to protect them from dna - targeting chemo- and radiation therapy . the chemo- and radioresistance of cscs has now been demonstrated in numerous experiments , although the mechanisms underlying this resistance are not fully understood . one potential modulator of csc resistance to dna - targeting agents is the family of checkpoint kinases 1/2 ( chk1/2 kinases ) , which become activated after genotoxic stress and arrest the cell cycle to allow dna repair . in addition to augmented dna repair systems , cscs may also exhibit changes in telomerase function , which allows resistance to chromosomal degradation in these rapidly dividing cells . telomerase function was recently shown to be downregulated in brain cscs , and several drugs that interfere with telomerase function are already in clinical trials , including arsenic trioxide , grn163l , and vaccines [ 6872 ] . an important component of the dna integrity defense systems in normal stem cells is the relatively high expression of efflux transporters from the atp - binding cassette ( abc ) gene family . these pumps allow normal stem cells to preserve their genome more effectively against chemical mutagens in an attempt to prevent carcinogenesis . similar to the way that cscs may derive resistance to dna damage from the preexisting dna repair systems in normal stem cells , cscs may also derive resistance to chemical mutagens ( e.g. , chemotherapy ) through the expression of drug efflux pumps in normal stem cells from which they were derived . resistance to therapy might also be conferred to cscs through the activation of the akt pathway [ 75 , 76 ] and the overamplification of apoptosis inhibitor proteins . this suggests that characterization of akt and bcl-2 expression in cscs may have significant clinical utility . flt3 receptors are often mutated in aml and are associated with a high relapse rate and poor prognosis . inhibition of flt3 signaling with cep701 reduces the tumorigenicity of xenografts , and cep 701 has reached phase 2 clinical trials . smac , in association with direct inhibitor of apoptosis binding protein with low pi ( smac / diablo ) , promotes apoptosis via neutralization of inhibitor of apoptosis ( iap ) proteins . based on these observations , vellanki et al . another promising molecular target to promote apoptosis in cscs is nuclear factor b ( nfb ) . nfb is a transcription factor believed to be intricately involved in the development and progression of certain cancer types . nuclear factor nb ( nfnb ) , a cousin of nfb , is an antiapoptotic transcription factor that is activated in leukemias [ 86 , 87 ] , pancreatic adenocarcinoma , and melanoma . although these nuclear factors are not as well studied as akt , they may offer promising drug targets . nf-b inhibitors include npi-0052 ( salinosporamides a ) , which is in phase i clinical trials , and tdzd-8 ( parthenolide ) , which is still in preclinical testing . unexpectedly , it was discovered that cscs reside along perivascular areas and are thus likely to be well oxygenated . this may help explain the efficacy of antiangiogenic therapies such as bevacizumab in that such therapies may be csc - specific . theoretically , csc compartment hypoxia may be induced by antiangiogenic therapies , conferring radioresistance to the cscs , although this has yet to be demonstrated in vivo , and the clinical significance of this remains unknown . vermeulen and colleagues recently discovered another interesting role of the microenvironment , specifically in the promotion of cancer cell stemness . activated myofibroblasts in the tumor microenvironment were responsible for c - met activation through production of hepatocyte growth factor ( hgf ) . thus , inhibition of stromal - produced hgf or the subsequent activation of c - met signaling via c - met inhibitors may represent additional approaches to target cscs . an important result of the well - documented csc resistance to radiation and chemotherapy is that these therapies often serve to enrich the resistant csc subpopulation , perhaps even selecting for more resistant clones within a heterogeneous csc population . furthermore , radiation has little effect on the ability of remaining cscs to regrow tumors . thus , csc enrichment may be the basis for the relative inability of most single modality cancer treatment strategies to control long - term cancer growth . this may dramatically improve the treatment of cancer by abrogating the potential for csc - induced tumor regrowth and systemic disease spread after initial treatment . it is now well established that combination therapy helps prevent the development of cancer resistance , except in a select group of cancer types where a single pharmaceutically correctable mutation exists . for example , many clinical trials have shown improvements in cancer survival with the use of concurrent chemo- and radiation therapy . in later rounds of therapy , not only has the cancer had time to grow and spread further , but it has also evolved resistance to previously encountered therapies . unfortunately , coadministration of chemotherapy and radiation therapy is not effective against all types of cancer , and it is not always feasible due to its potential significant toxicity . thus , it will be important to design preclinical studies and clinical trials that evaluate potential synergistic benefits of adding csc - targeted therapies to traditional cancer regimens . as new data supports a role for the csc hypothesis in solid tumors in addition to hematologic malignancies , outcomes following the surgical resection of solid tumors may significantly improve . if induction approaches can be effectively augmented with anti - csc therapies , then followup surgical resection may show improved curative outcomes . considering its powerful therapeutic potential , csc - targeted therapies may be particularly valuable in surgically challenging malignancies such as pancreatic and brain cancer . in order to more effectively target cscs , molecular proliferation and survival mechanisms of cscs many institutions have developed large banks of malignant tissues with coordinated clinical data , and this resource is being actively mined . techniques for concentrating , isolating , and enriching cscs from resected tumors are also rapidly evolving , and cell culture and xenograft models that allow us to transplant and sustain cscs are maturing . here , we will review some of the prominent classes of drugs that will potentially yield clinical benefits in the near future . an update of clinical trials assessing these targets is illustrated in table 1 ( data from http://clinicaltrials.gov/ ) . developmental pathways that direct the differentiation of normal stem cells represent attractive targets for drug discovery . in particular , the roles of notch and wnt/-catenin signaling have been examined , and both have been implicated in the development and progression of several types of leukemia [ 100 , 101 ] . the nonsteroidal anti - inflammatory drug ( nsaid ) etodolac inhibits wnt signaling and may be of benefit in the treatment of chronic lymphocytic leukemia . the wnt/-catenin pathway promotes genomic instability and dna damage tolerance that may be enhanced by dna damage in cscs . the pathway has been shown to promote genomic instability in colon cancer and possibly promotes conversion of normal stem cells to cscs in gliomas . wnt inhibitors have been designed to therapeutically prevent this possibility and include icg-001 , fungal derivatives pkf115 - 854 and cgp049090 , as well as monoclonal antibodies against wnt-1 and wnt-2 ( figure 3 ) . the notch ligands , jagged 1 & 2 and delta1 ( d1 ) to delta3 ( d3 ) , induce the release of the notch intracellular ( notch - ic ) domain via enzymatic proteolytic cleavage by - and -secretases . notch - ic translocates to the nucleus where it induces transcription of notch responsive genes [ 106 , 107 ] . inhibition of notch signaling via -secretase inhibitors can potentially block csc self - renewal and decrease medulloblastoma growth , and significant efforts to downregulate notch signaling are underway . in particular , the delta / notch - like epidermal growth factor - related receptor ( dner ) can be induced by histone deacetylase inhibition to inhibit the growth of and induce the differentiation of glioblastoma neurospheres and xenografts . this provides a basis for the manipulation of noncanonical signaling pathways for therapeutic intervention against cscs . the hedgehog signaling pathway may represent an important modulator of csc carcinogenesis with significant therapeutic implications [ 113117 ] . similar to notch signaling , hedgehog signaling may also benefit from expanded drug discovery efforts within noncanonical pathways . already , hedgehog inhibitors have been shown to inhibit medulloblastoma growth in mice , and at least 3 different hedgehog inhibitors have reached phase i clinical trials ( figure 5 ) . as mentioned previously , the cell - surface molecule cd133 is believed to be a stem cell marker for multiple cancer types . a recent study by wang et al . they conjugated single - walled carbon nanotubules ( swnts ) , which allow localized hyperthermia treatment , to anti - cd133 monoclonal antibodies , and cultured these products with both cd133 and cd133 glioblastoma ( gbm ) cells . a mixture of cd133 and cd133 cells were then exposed to near - infrared laser light , and the cd133 gbm cells were selectively destroyed . with the advent of multidisciplinary approaches to cancer therapy , significant strides have been made in the treatment of cancer . now with new discoveries relating to cscs , we have yet another mechanism of therapeutic arsenal that may prove beneficial in combination with current therapeutic modalities . the basic foundations for csc - targeted therapy are actively being discovered , and there are already several pharmacologic agents available that are capable of specifically modulating csc intracellular signaling . still , much remains unknown about the basic signaling mechanisms of cscs that confer resistance to treatment , and better methods for the disruption of csc signaling must be developed to fully integrate the csc hypothesis into our treatment paradigms . interestingly , cscs may not necessarily need to be eradicated to prevent cancer progression if they can be forced to differentiate down their lineage en masse as they do in the treatment of apml . it is important for future studies to focus on the discovery of new molecular targets for the development of better pharmaceutical agents to eliminate or differentiate cscs and that these agents be studied in tandem with traditional cancer therapies .

vascular endothelial dysfunction is present in healthy subjects with risk factors for atherosclerosis years before the appearance of atheromatous plaques and can be assessed non - invasively through the endothelium - dependent method of flow mediated dilation ( fmd ) ( 6 ) . a fmd test results in arterial dilation due to increased nitric oxide secretion by the endothelium in response to increased shear stress ( 21 ) . in addition , other factors have been suggested to contribute to fmd , including a balance between vasodilators ( i.e. , bradykinin , adenosine , vascular endothelial growth factor , and prostacyclin ) and vasoconstrictors ( i.e. , endothelin , prostanoids , and angiotensin ii ) ( 11 ) . the consumption of a high fat meal ( hfm ) has been shown to induce transient vascular dysfunction in healthy , young men ( 4 , 29 , 46 ) and is thought to occur due to the oxidation of postprandial triglyceride - rich lipoproteins ( 38 , 46 ) , hyperglycemia ( 47 ) , or hyperinsulinemia ( 2 ) . conversely , endurance training improves vascular function in diseased populations ( 20 , 28 ) as well as in healthy , young men ( 7 , 18 , 22 ) by increasing nitric oxide availability ( 16 ) . a cross - sectional study demonstrated that young , endurance - trained men had higher fmd responses of the brachial artery than sedentary young men ( 22 ) . the acute effects of a single hfm on vascular function in individuals who perform different amounts of physical activity is unknown . the purpose of this study was to determine if different levels of aerobic capacity evoke a differential effect on vascular function as measured by fmd prior to and following a single hfm in apparently , healthy young men . the primary aim was to determine if individuals who were highly trained had a protective effect against the negative impact of a hfm on vascular function . it was hypothesized that fmd would be similar between groups at baseline , as demonstrated previously ( 15 ) , and would be less impaired postprandially in highly trained individuals compared to moderately active individuals . a secondary aim was to determine if blood lipids , insulin , or glucose were correlated to fmd at baseline or postprandial a hfm . apparently healthy , highly trained ( n = 10 ) and moderately active ( n = 10 ) young men between the ages of 19 and 26 years were recruited to participate in the study . no subject had a history of disease , smoked or was presently taking any medications or dietary supplements that may have an impact on vascular function . subjects with a body mass index ( bmi ) 28 kgm were excluded to eliminate obesity having an impact on endothelium - dependent vasodilation ( 44 ) . highly - trained subjects were members of a division i university cross country team and had participated in high - intensity endurance training for at least two years . the moderately active subjects reported occasional endurance / recreational exercise ( i.e. , jogging , basketball , cycling , etc . ) but were not consistently exercising ( > 3 daysweek , 20 minday ) over the past six months . self - reported physical activity levels were obtained from each subject prior to participation in the study to determine training status . all subjects signed a written informed consent approved by the review board of the university of louisville . aerobic capacity , defined by the maximal oxygen consumption ( vo2max ) , of each subject was measured using a treadmill ramp protocol and indirect open circuit spirometry ( parvomedics , sandy , ut ) . subjects reported to the laboratory for vascular testing within two - weeks of the vo2max test . they were instructed to abstain from exercise for 24-hours to avoid any confounding influences of exercise on lipid metabolism ( 35 ) , to fast for 12-hours ( 38 ) , and to avoid caffeine and alcohol consumption for 12-hours prior to vascular testing . body composition was determined using bioelectrical impedance ( rjl systems , clinton township , mi ) . total body resistance and reactance to an alternating electrical current were used to calculate fat mass ( fm ) . lean body mass ( lbm ) was calculated using a validated multiple regression equation : lbm = 8.98751 + 0.36273 [ height ( cm)/resistance ) + 0.21411 ( height ) + 0.13290 ( weight ( kg ) ] ( 41 ) . endothelium - dependent fmd of the brachial artery was determined non - invasively using high - resolution ultrasound with an upper arm cuff occlusion to induce reactive hyperemia as previously described ( 17 , 33 , 42 , 45 ) . briefly , subjects were placed supine , in a quiet , dark , temperature controlled room for 10-min . vascular function was measured in the right brachial artery using quantitative doppler ultrasound ( philips hdi 5000 , seattle , wa ) by a single investigator . the brachial artery was imaged longitudinally , 2 cm above the antecubital fossa by b - mode ultrasound , using a 125 mhz linear array transducer . reactive hyperemia was induced by inflation of a pneumatic cuff placed on the upper arm ( 34 cm above the transducer ) proximal to the transducer using a rapid cuff inflator ( hokanson e20 , bellevue , wa ) at 6080 mmhg above the systolic pressure for five - minutes . a five - minute period was measured both before and after cuff occlusion to determine baseline brachial artery diameter and fmd after cuff occlusion , respectively . peak fmd for each individual was determined as the greatest diameter following cuff occlusion release and expressed as a percent change from baseline . a video file was collected throughout the entire test ( ulead video studio 7 , taipei , taiwan ) to allow data analysis after the test . a custom - made software program using labview version 7.1 ( national instruments , austin , tx ) measured the changes in the brachial diameter beat by beat during diastole throughout the testing period and thereby allowed second by second data collection . blood pressure was measured throughout the entire test to verify that changes in blood flow were not dependent on changes in blood pressure . ten ml of blood was collected from an antecubital vein to obtain fasting triglycerides ( tg ) , total cholesterol ( tc ) , high density lipoproteins ( hdl - c ) , low density lipoproteins ( ldl - c ) , insulin , and glucose . samples were centrifuged and the serum was stored at -80 c for less than six months until analyzed . tg , tc , and hdl - c were measured enzymatically by reflectance spectrophotometry and ldl - c was calculated by the friedewald formula ( 40 ) . insulin was measured by an electrochemiluminescent double monoclonal immunometric assay ( roche diagnostics , indianapolis , in ) . glucose was measured via an enzymatic method ( ortho clinical diagnostics , raritan , nj ) using glucose oxidase coupled to peroxidase . following the baseline blood draw , subjects consumed a meal consisting of an enormous omelet sandwich [ 740 kcals , 46 g fat , 16 g saturated fat , 330 mg cholesterol ( 56% fat , 24% carbohydrate , 20% protein ) ] and a medium order of hash browns [ ( 310 kcals , 20 g fat , 5.5 g saturated fat , 0 mg cholesterol ( 58% fat , 40% carbohydrate , 2% protein ) ] ( burger king corporation , miami , fl ) with water . the meal fat content used in this study was consistent with other literature which has shown adverse effects of an acute meal on endothelial function ( 8 , 29 ) . between testing sessions subjects rested quietly in the laboratory while reading and were not allowed to consume any other food or drink other than water . an independent samples t - test was used to determine any mean differences between group characteristics . a two - way repeated measures anova was performed to assess differences within subjects for vascular and blood variables prior to and following the hfm . correlational analyses were used to determine any relationship between fmd and blood variables at baseline , two- , and four - hour postprandial . apparently healthy , highly trained ( n = 10 ) and moderately active ( n = 10 ) young men between the ages of 19 and 26 years were recruited to participate in the study . no subject had a history of disease , smoked or was presently taking any medications or dietary supplements that may have an impact on vascular function . subjects with a body mass index ( bmi ) 28 kgm were excluded to eliminate obesity having an impact on endothelium - dependent vasodilation ( 44 ) . highly - trained subjects were members of a division i university cross country team and had participated in high - intensity endurance training for at least two years . the moderately active subjects reported occasional endurance / recreational exercise ( i.e. , jogging , basketball , cycling , etc . ) but were not consistently exercising ( > 3 daysweek , 20 minday ) over the past six months . self - reported physical activity levels were obtained from each subject prior to participation in the study to determine training status . all subjects signed a written informed consent approved by the review board of the university of louisville . aerobic capacity , defined by the maximal oxygen consumption ( vo2max ) , of each subject was measured using a treadmill ramp protocol and indirect open circuit spirometry ( parvomedics , sandy , ut ) . subjects reported to the laboratory for vascular testing within two - weeks of the vo2max test . they were instructed to abstain from exercise for 24-hours to avoid any confounding influences of exercise on lipid metabolism ( 35 ) , to fast for 12-hours ( 38 ) , and to avoid caffeine and alcohol consumption for 12-hours prior to vascular testing . body composition was determined using bioelectrical impedance ( rjl systems , clinton township , mi ) . total body resistance and reactance to an alternating electrical current lean body mass ( lbm ) was calculated using a validated multiple regression equation : lbm = 8.98751 + 0.36273 [ height ( cm)/resistance ) + 0.21411 ( height ) + 0.13290 ( weight ( kg ) ] ( 41 ) . endothelium - dependent fmd of the brachial artery was determined non - invasively using high - resolution ultrasound with an upper arm cuff occlusion to induce reactive hyperemia as previously described ( 17 , 33 , 42 , 45 ) . briefly , subjects were placed supine , in a quiet , dark , temperature controlled room for 10-min . vascular function was measured in the right brachial artery using quantitative doppler ultrasound ( philips hdi 5000 , seattle , wa ) by a single investigator . the brachial artery was imaged longitudinally , 2 cm above the antecubital fossa by b - mode ultrasound , using a 125 mhz linear array transducer . reactive hyperemia was induced by inflation of a pneumatic cuff placed on the upper arm ( 34 cm above the transducer ) proximal to the transducer using a rapid cuff inflator ( hokanson e20 , bellevue , wa ) at 6080 mmhg above the systolic pressure for five - minutes . a five - minute period was measured both before and after cuff occlusion to determine baseline brachial artery diameter and fmd after cuff occlusion , respectively . peak fmd for each individual was determined as the greatest diameter following cuff occlusion release and expressed as a percent change from baseline . a video file was collected throughout the entire test ( ulead video studio 7 , taipei , taiwan ) to allow data analysis after the test . a custom - made software program using labview version 7.1 ( national instruments , austin , tx ) measured the changes in the brachial diameter beat by beat during diastole throughout the testing period and thereby allowed second by second data collection . blood pressure was measured throughout the entire test to verify that changes in blood flow were not dependent on changes in blood pressure . ten ml of blood was collected from an antecubital vein to obtain fasting triglycerides ( tg ) , total cholesterol ( tc ) , high density lipoproteins ( hdl - c ) , low density lipoproteins ( ldl - c ) , insulin , and glucose . samples were centrifuged and the serum was stored at -80 c for less than six months until analyzed . tg , tc , and hdl - c were measured enzymatically by reflectance spectrophotometry and ldl - c was calculated by the friedewald formula ( 40 ) . insulin was measured by an electrochemiluminescent double monoclonal immunometric assay ( roche diagnostics , indianapolis , in ) . glucose was measured via an enzymatic method ( ortho clinical diagnostics , raritan , nj ) using glucose oxidase coupled to peroxidase . following the baseline blood draw , subjects consumed a meal consisting of an enormous omelet sandwich [ 740 kcals , 46 g fat , 16 g saturated fat , 330 mg cholesterol ( 56% fat , 24% carbohydrate , 20% protein ) ] and a medium order of hash browns [ ( 310 kcals , 20 g fat , 5.5 g saturated fat , 0 mg cholesterol ( 58% fat , 40% carbohydrate , 2% protein ) ] ( burger king corporation , miami , fl ) with water . the meal fat content used in this study was consistent with other literature which has shown adverse effects of an acute meal on endothelial function ( 8 , 29 ) . between testing sessions subjects rested quietly in the laboratory while reading and were not allowed to consume any other food or drink other than water . an independent samples t - test was used to determine any mean differences between group characteristics . a two - way repeated measures anova was performed to assess differences within subjects for vascular and blood variables prior to and following the hfm . correlational analyses were used to determine any relationship between fmd and blood variables at baseline , two- , and four - hour postprandial . the highly trained and moderately active groups were not different in age , height , weight or lbm ( table 1 ) . as expected , differences were found between groups for vo2max ( p < 0.001 ) . body mass index ( p = 0.014 ) and fm ( p = 0.029 ) were found to be higher in the moderately active compared to the highly trained group . resting brachial artery diameter was not different between groups ( 0.48 0.04 vs. 0.46 0.05 cm , highly trained vs. moderately active , respectively ) ( p = 0.398 ) at any time point , suggesting that any differences found in fmd across time were not due to changes in baseline diameter . flow mediated dilation was not different between groups ( p = 0.108 ) across time points ( figure 1 ) . however , a time effect was found after the hfm ( p < 0.001 ) as fmd was reduced ( 3740% ) from baseline to two - hour postprandial ( p < 0.001 ) in both groups . fmd from baseline to four - hour postprandial was decreased by 20% in the highly trained group while the moderately active group remained at a 37% reduction ( p < 0.001 ) . when fmd was normalized to baseline diameter to control for diameter differences between subjects , the results were the same , with two- and four - hour fmd reduced from baseline in both groups ( p < 0.001 ) and no difference between groups ( p = 0.257 ) . the average time to peak dilation was not different between groups ( p = 0.518 ) nor did it differ over time ( 88.2 15.0 and 97.5 18.3 s , highly trained vs. moderately active , respectively ) ( p = 0.167 ) . blood pressure was not different between groups ( p = 0.890 ) , did not change throughout the test ( p = 0.689 ) , and did not differ across time point ( p = 0.783 ) suggesting that blood pressure did not affect the blood velocity ( i.e. shear stress stimulus ) on the vessel . no between group differences were found in tg levels ( p = 0.093 ) across all time points ( table 2 ) . however , a time effect for both groups was found ( p < 0.001 ) . compared to baseline , tg increased by approximately 43% and 52% ( p < 0.001 ) at two- and four - hour postprandial , respectively , with no difference between two- and four - hour postprandial ( p = 0.323 ) . total cholesterol was not different between groups ( p = 0.310 ) or across time ( p = 0.138 ) . an interaction effect was found between time and group ( p = 0.049 ) for ldl - c . levels of ldl - c were found to be different across time ( p = 0.002 ) . the highly trained group had a reduction in postprandial ldl - c from baseline by 6.0% ( p = 0.05 ) at four - hour postprandial but not at two - hour ( p = 0.110 ) . while in the moderately active group ldl - c was reduced from baseline by 19% ( p = 0.010 ) and 11% ( p < 0.001 ) at two- and four - hour postprandial , respectively . however , no between group differences in ldl - c were found at any time period ( p = 0.457 ) . no differences in hdl - c were found between groups at any time point ( p = 0.634 ) . the hdl - c two - hour postprandial period for both groups was reduced compared to baseline ( p = 0.024 ) and four - hour postprandial ( p = 0.014 ) . no differences were determined between baseline and four - hour postprandial ( p = 0.572 ) . glucose concentrations were found to be greater at two - hour postprandial compared to baseline in both groups ( p = 0.003 ) ( table 3 ) . at four - hour postprandial , glucose levels decreased and were lower than the two - hour measurement ( p = 0.05 ) but were not different from baseline . no between group differences were found for serum glucose concentrations at any time point ( p = 0.895 ) . serum insulin concentration was greater at two- ( p < 0.001 ) and four - hour ( p = 0.003 ) postprandial a hfm compared to baseline for both groups ( table 3 ) . furthermore , insulin was lower at four - hour compared to two - hour ( p < 0.001 ) . in addition , the moderately active individuals demonstrated greater insulin concentrations compared to the highly trained group at all time periods ( p = 0.056 ) . baseline fmd was correlated to baseline tg and hdl - c levels ( table 4 ) . no significant correlations between fmd and any blood markers were found at two - hour postprandial . at four - hours the primary finding of this study was that flow mediated dilation of the brachial artery was impaired following the consumption of a single high - fat meal , independent of aerobic capacity . no differences in fmd were detected between groups at any time point suggesting that a high aerobic capacity does not augment or protect vascular function prior to or following a hfm . these findings demonstrate that vascular function is not improved , and can be impaired by a single hfm , to the same extent whether the subject is highly trained or moderately active . in addition , our finding that resting brachial artery diameter was unaffected by the test meal is in agreement with previous studies ( 29 , 36 , 45 ) and is important as an increased diameter prior to occlusion results in a decreased fmd response after reactive hyperemia ( 17 ) . when we normalized fmd to resting diameter in order to control for diameter differences between subjects , we found similar changes to those of fmd alone . our findings of reduced endothelial function after a hfm confirms the results of several studies using healthy , young men ( 4 , 8 , 29 , 46 ) . on the other hand , several others have found conflicting results ( 13 , 39 ) . we found an approximate 40% reduction in fmd at two- and four - hour postprandial which is consistent with the results of tsai et al . discrepancies in studies which have found no changes may be explained by age of the subject , the dietary lipid composition of the meals ( 10 ) , the timing of the vascular measurements , and/or the gender of the subjects studied ( 32 ) . a potential mechanism for the reduction in fmd postprandially may be increased serum insulin concentrations . our results indicated that insulin levels at two- and four - hour postprandial compared to baseline were elevated . acute hyperinsulinemia has been shown to have a negative impact on the fmd in healthy subjects ( 2 ) . while other work has found that the degree of hyperinsulinemia independently predicted decreases in fmd in healthy volunteers ( 3 ) . the mechanism for the reduction in fmd with hyperinsulinemia is unknown but it may be due to oxidative stress ( 2 ) . another potential mechanism to explain our findings may be elevated blood lipid concentrations as research has indicated that impairments in fmd after a hfm are due to increased oxidation of elevated tg levels ( 4 , 38 , 46 ) . elevations in tg results in a rise in superoxide anion production ( 4 ) which impairs vascular function through direct inactivation of nitric oxide and increases in lipid oxidation ( 27 ) ; thereby , limiting nitric oxide availability necessary for arterial dilation . despite our observation of differing levels of ldl - c during the postprandial period , we do not believe that this mechanism contributed to our results as brachial artery fmd has not correlated with several traditional cardiovascular risk factors , including ldl - c in healthy men ( 48 ) . our findings support this as no significant correlations were found between fmd and ldl - c in this study . interestingly , our correlational analyses did not suggest that any one variable was strongly related to postprandial fmd . thus , other factors such as increased oxidative stress , free radicals , apolipoprotein b , reduced endogenous antioxidants , inflammatory cytokines , and/or neutrophils may be responsible for the decrease in fmd at two- and four - hour postprandial . future studies should examine these factors to determine if they exert detrimental effects on vascular function following a hfm . our results indicated that possessing a high aerobic capacity did not protect individuals from the acute negative effects of a hfm on vascular function to a greater extent than those who are moderately active . we did not find a difference in fmd at baseline between the highly trained and moderately active men ( 11.1 vs. 9.6% , respectively ) . results are mixed in regards to the effect of exercise training improving ( 7 , 22 ) or having no effect ( 12 , 15 ) on vascular function in young , healthy individuals . goto et al . ( 18 ) found an augmentation in endothelium - dependent dilation in young ( 25 2.5 years ) , healthy men following 12-weeks of moderate - intensity aerobic exercise , but not following the same period of mild or high - intensity exercise . it was postulated by these authors that the elevated oxidative stress levels seen in individuals performing high - intensity exercise may have impaired endothelium - dependent dilation through a reduction in nitric oxide bioavailability . increasing exercise intensity has been shown to progressively increase the production of nitric oxide ( 30 ) . it is possible that the high - intensity exercise performed by our highly trained group increased nitric oxide production while at the same time resulted in increased oxidative stress thus counteracting the beneficial effects of increased nitric oxide production . we did not measure nitric oxide production , however , so this can only be postulated based on the current research . future research warrants looking at this mechanism as well as other factors modulating endothelial function ( i.e. bradykinin , prostacyclin , endothelin ) ( 11 ) . the young age of our subjects may be another reason why we did not find a difference in fmd between our subjects . it has been shown that age is an important contributor to vascular function and has an interactive effect with disease and lifestyle on cardiovascular health ( 25 ) . indeed , the fmd response is preserved in older athletes compared to their age - matched , sedentary counterparts and is similar to the responses seen in both younger athletic and sedentary individuals ( 15 ) . thus , we may not have seen a difference in vascular function either prior to or after the hfm because our subjects were young enough that they did not exhibit the arterial stiffening that occurs due to aging . in addition , the health status of our subjects may explain our findings as all of our subjects were considered healthy ( i.e. no history of disease or smoking ) via self - reported medical history . research has demonstrated that exercise training improves endothelium - dependent dilation in diseased individuals ( 20 , 28 ) as well as in aged individuals ( 15 ) , with inconsistent results observed in young , healthy individuals ( 7 , 12 , 24 , 32 ) . vascular dysfunction induced by a high - fat meal may occur due to enhanced oxidative stress ( 4 , 46 ) . in contrast to our hypothesis , our study demonstrated that high aerobic capacity did not offer increased protection against the acute , negative effects of a high - fat meal on vascular function . several mechanisms have been suggested by which exercise may protect endothelial function following a high - fat meal ( i.e. increased shear stress , diminished oxidative stress and inflammation ( 46 ) , increased antioxidant enzyme activity ( 31 ) , and the release of anti - inflammatory cytokines ( 43 ) ) . the scarcity of studies that have investigated the effects of exercise training on postprandial endothelial function further demonstrates the need for future research to expand our understanding of the role of diet , exercise , age , and health status on cardiovascular health . it is possible that we may not have seen differences in fmd between our groups due to methodology ( i.e. upper arm versus forearm occlusion ) or the population ( i.e. diseased ( 20 , 28 ) or aged ( 12 , 15 , 34 ) that was used . in the present study we induced fmd by utilizing the proximal cuff position method . in young , healthy subjects brachial artery fmd has been found to be significantly higher after upper arm occlusion compared to forearm occlusion and thus proposed as a better means of evaluating vascular function in this population ( 1 , 5 ) . occlusion of the forearm has been suggested to provide a more accurate assessment of endothelial dysfunction induced by smoking ( 19 ) . the partial attenuation of fmd with the no synthase inhibitor n - monomethyl - l - arginine ( l - nmma ) following upper arm occlusion demonstrates that dilation induced by the proximal occlusion method is not entirely mediated by no ( 14 ) . the regional ischemia produced by occlusion of the upper arm is associated with the release of vasodilators ( i.e. potassium , adenosine , atp ) and changes in local ph that could explain the greater dilation observed with proximal occlusion ( 14 ) . however , we chose to use the upper arm occlusion method due to our population studied and because previous studies examining the effect of a high fat meal on fmd have also utilized this method ( 4 , 29 , 36 , 37 ) . exercise training improves endothelium - dependent dilation in asymptomatic older individuals ( 15 ) , as well as patients with chronic heart failure ( 20 ) and type 2 diabetes ( 28 ) . however , the beneficial effects of exercise training on vascular function in young individuals exhibiting normal arterial function have not been consistently observed ( 7 , 12 , 24 , 32 ) . future studies warrant investigation into the effects of both cuff position and aging on postprandial vascular function . another possibility is that the difference in aerobic capacity between subjects may not have been large enough to elicit difference in fmd at baseline . however , this does not seem likely as a difference in fmd with a population that had similar differences in vo2max has been found previously ( 22 ) . alternatively , differences in postprandial fmd between the highly trained and moderately active group may have become apparent if we had extended the study ( i.e. six - hour postprandial hfm ) as tsai et al . ( 46 ) found a reduction in fmd at six - hour postprandial in healthy , young men . the increased calories or the type of fat used in our meal could have contributed to the impairment of postprandial vascular function seen in this study rather than the fat intake . however , we do not think this is likely as an isocaloric low - fat meal has no effect on fmd when compared to a hfm ( 23 , 38 ) . furthermore , a meal high in saturated fat , which was comparable to our study , resulted in reduced fmd while meals high in carbohydrates , monounsaturated fat , or polyunsaturated fat had no effect on fmd ( 23 ) . a limitation to the study is the lack of chronic dietary information for our subjects as chronic high fat ( 26 ) or low fat ( 9 ) diets may alter fmd . although the chronic dietary patterns between our subjects may have been different , we compared the fmd response for each individual to baseline ; thus , the changes in fmd are indicative of the acute response to a hfm . another limitation to this study is the lack of a true sedentary group as a control . despite being classified as moderately - active , this group still displayed a vo2max of 47.3 7.1 mlkgmin and thus can be considered as possessing a high level of fitness . the inclusion of a sedentary control group with a lower aerobic capacity may have allowed us to detect differences between groups not seen in the present study design and to further elucidate the role of fitness level on postprandial fmd . future studies should include individuals with lower aerobic capacities and different age populations to allow for further investigation into the relationship between fitness , aging and postprandial endothelial function . this study demonstrated that fmd was similar with differing aerobic capacities and was reduced in both highly trained and moderately active young men for up to four - hours after consuming a hfm . our results suggested that a high aerobic capacity did not augment fmd and did not offer increased protection against the acute , negative effects of a hfm on vascular function compared to individuals possessing a moderate aerobic capacity . moderate - intensity exercisers demonstrated similar fmd responses compared to elite runners , suggesting that exercise performed at a moderate - intensity is sufficient to elicit positive responses in overall cardiovascular risk and vascular health . future studies are warranted with a larger sample size to determine the mechanism for the decrease in fmd after a hfm and to determine if differences may exist between groups if followed for a longer period of time ( i.e. six- or eight - hours postprandial ) . these findings are significant as it demonstrates that moderately active individuals have similar acute responses to a hfm compared to highly trained individuals , suggesting that increased aerobic capacity and/or high intensity aerobic exercise is not necessary to maintain endothelial function .

the consumption of a high - fat meal induces transient vascular dysfunction . aerobic exercise enhances vascular function in healthy individuals . our purpose was to determine if different levels of aerobic capacity impact vascular function , as measured by flow mediated dilation , following a high - fat meal . flow mediated dilation of the brachial artery was determined before , two- and four - hours postprandial a high - fat meal in young males classified as highly trained ( n = 10 ; vo2max = 74.6 5.2 mlkgmin1 ) or moderately active ( n = 10 ; vo2max = 47.3 7.1 mlkgmin1 ) . flow mediated dilation was reduced at two- ( p < 0.001 ) and four - hours ( p < 0.001 ) compared to baseline for both groups but was not different between groups at any time point ( p = 0.108 ) . triglycerides and insulin increased at two- ( p < 0.001 ) and four - hours ( p < 0.05 ) in both groups . ldl - c was reduced at four - hours ( p = 0.05 ) in highly trained subjects , and two- and four - hours ( p 0.01 ) in moderately active subjects . hdl - c decreased at two- ( p = 0.024 ) and four - hours ( p = 0.014 ) in both groups . glucose increased at two - hours postprandial for both groups ( p = 0.003 ) . our results indicate that a high - fat meal results in reduced endothelium - dependent vasodilation in highly trained and moderately active individuals with no difference between groups . thus , high aerobic capacity does not protect against transient reductions in vascular function after the ingestion of a single high - fat meal compared to individuals who are moderately active .

INTRODUCTION METHOD Participants Protocol Statistical Analysis RESULTS DISCUSSION

vascular endothelial dysfunction is present in healthy subjects with risk factors for atherosclerosis years before the appearance of atheromatous plaques and can be assessed non - invasively through the endothelium - dependent method of flow mediated dilation ( fmd ) ( 6 ) . , bradykinin , adenosine , vascular endothelial growth factor , and prostacyclin ) and vasoconstrictors ( i.e. the consumption of a high fat meal ( hfm ) has been shown to induce transient vascular dysfunction in healthy , young men ( 4 , 29 , 46 ) and is thought to occur due to the oxidation of postprandial triglyceride - rich lipoproteins ( 38 , 46 ) , hyperglycemia ( 47 ) , or hyperinsulinemia ( 2 ) . conversely , endurance training improves vascular function in diseased populations ( 20 , 28 ) as well as in healthy , young men ( 7 , 18 , 22 ) by increasing nitric oxide availability ( 16 ) . a cross - sectional study demonstrated that young , endurance - trained men had higher fmd responses of the brachial artery than sedentary young men ( 22 ) . the acute effects of a single hfm on vascular function in individuals who perform different amounts of physical activity is unknown . the purpose of this study was to determine if different levels of aerobic capacity evoke a differential effect on vascular function as measured by fmd prior to and following a single hfm in apparently , healthy young men . the primary aim was to determine if individuals who were highly trained had a protective effect against the negative impact of a hfm on vascular function . it was hypothesized that fmd would be similar between groups at baseline , as demonstrated previously ( 15 ) , and would be less impaired postprandially in highly trained individuals compared to moderately active individuals . a secondary aim was to determine if blood lipids , insulin , or glucose were correlated to fmd at baseline or postprandial a hfm . apparently healthy , highly trained ( n = 10 ) and moderately active ( n = 10 ) young men between the ages of 19 and 26 years were recruited to participate in the study . subjects with a body mass index ( bmi ) 28 kgm were excluded to eliminate obesity having an impact on endothelium - dependent vasodilation ( 44 ) . highly - trained subjects were members of a division i university cross country team and had participated in high - intensity endurance training for at least two years . the moderately active subjects reported occasional endurance / recreational exercise ( i.e. subjects reported to the laboratory for vascular testing within two - weeks of the vo2max test . endothelium - dependent fmd of the brachial artery was determined non - invasively using high - resolution ultrasound with an upper arm cuff occlusion to induce reactive hyperemia as previously described ( 17 , 33 , 42 , 45 ) . vascular function was measured in the right brachial artery using quantitative doppler ultrasound ( philips hdi 5000 , seattle , wa ) by a single investigator . the brachial artery was imaged longitudinally , 2 cm above the antecubital fossa by b - mode ultrasound , using a 125 mhz linear array transducer . a five - minute period was measured both before and after cuff occlusion to determine baseline brachial artery diameter and fmd after cuff occlusion , respectively . peak fmd for each individual was determined as the greatest diameter following cuff occlusion release and expressed as a percent change from baseline . a custom - made software program using labview version 7.1 ( national instruments , austin , tx ) measured the changes in the brachial diameter beat by beat during diastole throughout the testing period and thereby allowed second by second data collection . ten ml of blood was collected from an antecubital vein to obtain fasting triglycerides ( tg ) , total cholesterol ( tc ) , high density lipoproteins ( hdl - c ) , low density lipoproteins ( ldl - c ) , insulin , and glucose . tg , tc , and hdl - c were measured enzymatically by reflectance spectrophotometry and ldl - c was calculated by the friedewald formula ( 40 ) . correlational analyses were used to determine any relationship between fmd and blood variables at baseline , two- , and four - hour postprandial . apparently healthy , highly trained ( n = 10 ) and moderately active ( n = 10 ) young men between the ages of 19 and 26 years were recruited to participate in the study . subjects with a body mass index ( bmi ) 28 kgm were excluded to eliminate obesity having an impact on endothelium - dependent vasodilation ( 44 ) . highly - trained subjects were members of a division i university cross country team and had participated in high - intensity endurance training for at least two years . the moderately active subjects reported occasional endurance / recreational exercise ( i.e. subjects reported to the laboratory for vascular testing within two - weeks of the vo2max test . endothelium - dependent fmd of the brachial artery was determined non - invasively using high - resolution ultrasound with an upper arm cuff occlusion to induce reactive hyperemia as previously described ( 17 , 33 , 42 , 45 ) . vascular function was measured in the right brachial artery using quantitative doppler ultrasound ( philips hdi 5000 , seattle , wa ) by a single investigator . the brachial artery was imaged longitudinally , 2 cm above the antecubital fossa by b - mode ultrasound , using a 125 mhz linear array transducer . a five - minute period was measured both before and after cuff occlusion to determine baseline brachial artery diameter and fmd after cuff occlusion , respectively . a custom - made software program using labview version 7.1 ( national instruments , austin , tx ) measured the changes in the brachial diameter beat by beat during diastole throughout the testing period and thereby allowed second by second data collection . ten ml of blood was collected from an antecubital vein to obtain fasting triglycerides ( tg ) , total cholesterol ( tc ) , high density lipoproteins ( hdl - c ) , low density lipoproteins ( ldl - c ) , insulin , and glucose . tg , tc , and hdl - c were measured enzymatically by reflectance spectrophotometry and ldl - c was calculated by the friedewald formula ( 40 ) . insulin was measured by an electrochemiluminescent double monoclonal immunometric assay ( roche diagnostics , indianapolis , in ) . correlational analyses were used to determine any relationship between fmd and blood variables at baseline , two- , and four - hour postprandial . the highly trained and moderately active groups were not different in age , height , weight or lbm ( table 1 ) . as expected , differences were found between groups for vo2max ( p < 0.001 ) . body mass index ( p = 0.014 ) and fm ( p = 0.029 ) were found to be higher in the moderately active compared to the highly trained group . resting brachial artery diameter was not different between groups ( 0.48 0.04 vs. 0.46 0.05 cm , highly trained vs. moderately active , respectively ) ( p = 0.398 ) at any time point , suggesting that any differences found in fmd across time were not due to changes in baseline diameter . flow mediated dilation was not different between groups ( p = 0.108 ) across time points ( figure 1 ) . however , a time effect was found after the hfm ( p < 0.001 ) as fmd was reduced ( 3740% ) from baseline to two - hour postprandial ( p < 0.001 ) in both groups . fmd from baseline to four - hour postprandial was decreased by 20% in the highly trained group while the moderately active group remained at a 37% reduction ( p < 0.001 ) . when fmd was normalized to baseline diameter to control for diameter differences between subjects , the results were the same , with two- and four - hour fmd reduced from baseline in both groups ( p < 0.001 ) and no difference between groups ( p = 0.257 ) . the average time to peak dilation was not different between groups ( p = 0.518 ) nor did it differ over time ( 88.2 15.0 and 97.5 18.3 s , highly trained vs. moderately active , respectively ) ( p = 0.167 ) . blood pressure was not different between groups ( p = 0.890 ) , did not change throughout the test ( p = 0.689 ) , and did not differ across time point ( p = 0.783 ) suggesting that blood pressure did not affect the blood velocity ( i.e. no between group differences were found in tg levels ( p = 0.093 ) across all time points ( table 2 ) . however , a time effect for both groups was found ( p < 0.001 ) . compared to baseline , tg increased by approximately 43% and 52% ( p < 0.001 ) at two- and four - hour postprandial , respectively , with no difference between two- and four - hour postprandial ( p = 0.323 ) . total cholesterol was not different between groups ( p = 0.310 ) or across time ( p = 0.138 ) . an interaction effect was found between time and group ( p = 0.049 ) for ldl - c . levels of ldl - c were found to be different across time ( p = 0.002 ) . the highly trained group had a reduction in postprandial ldl - c from baseline by 6.0% ( p = 0.05 ) at four - hour postprandial but not at two - hour ( p = 0.110 ) . while in the moderately active group ldl - c was reduced from baseline by 19% ( p = 0.010 ) and 11% ( p < 0.001 ) at two- and four - hour postprandial , respectively . however , no between group differences in ldl - c were found at any time period ( p = 0.457 ) . no differences in hdl - c were found between groups at any time point ( p = 0.634 ) . the hdl - c two - hour postprandial period for both groups was reduced compared to baseline ( p = 0.024 ) and four - hour postprandial ( p = 0.014 ) . no differences were determined between baseline and four - hour postprandial ( p = 0.572 ) . glucose concentrations were found to be greater at two - hour postprandial compared to baseline in both groups ( p = 0.003 ) ( table 3 ) . at four - hour postprandial , glucose levels decreased and were lower than the two - hour measurement ( p = 0.05 ) but were not different from baseline . no between group differences were found for serum glucose concentrations at any time point ( p = 0.895 ) . serum insulin concentration was greater at two- ( p < 0.001 ) and four - hour ( p = 0.003 ) postprandial a hfm compared to baseline for both groups ( table 3 ) . furthermore , insulin was lower at four - hour compared to two - hour ( p < 0.001 ) . in addition , the moderately active individuals demonstrated greater insulin concentrations compared to the highly trained group at all time periods ( p = 0.056 ) . baseline fmd was correlated to baseline tg and hdl - c levels ( table 4 ) . no significant correlations between fmd and any blood markers were found at two - hour postprandial . at four - hours the primary finding of this study was that flow mediated dilation of the brachial artery was impaired following the consumption of a single high - fat meal , independent of aerobic capacity . no differences in fmd were detected between groups at any time point suggesting that a high aerobic capacity does not augment or protect vascular function prior to or following a hfm . these findings demonstrate that vascular function is not improved , and can be impaired by a single hfm , to the same extent whether the subject is highly trained or moderately active . in addition , our finding that resting brachial artery diameter was unaffected by the test meal is in agreement with previous studies ( 29 , 36 , 45 ) and is important as an increased diameter prior to occlusion results in a decreased fmd response after reactive hyperemia ( 17 ) . we found an approximate 40% reduction in fmd at two- and four - hour postprandial which is consistent with the results of tsai et al . our results indicated that insulin levels at two- and four - hour postprandial compared to baseline were elevated . elevations in tg results in a rise in superoxide anion production ( 4 ) which impairs vascular function through direct inactivation of nitric oxide and increases in lipid oxidation ( 27 ) ; thereby , limiting nitric oxide availability necessary for arterial dilation . despite our observation of differing levels of ldl - c during the postprandial period , we do not believe that this mechanism contributed to our results as brachial artery fmd has not correlated with several traditional cardiovascular risk factors , including ldl - c in healthy men ( 48 ) . our findings support this as no significant correlations were found between fmd and ldl - c in this study . thus , other factors such as increased oxidative stress , free radicals , apolipoprotein b , reduced endogenous antioxidants , inflammatory cytokines , and/or neutrophils may be responsible for the decrease in fmd at two- and four - hour postprandial . future studies should examine these factors to determine if they exert detrimental effects on vascular function following a hfm . our results indicated that possessing a high aerobic capacity did not protect individuals from the acute negative effects of a hfm on vascular function to a greater extent than those who are moderately active . we did not find a difference in fmd at baseline between the highly trained and moderately active men ( 11.1 vs. 9.6% , respectively ) . results are mixed in regards to the effect of exercise training improving ( 7 , 22 ) or having no effect ( 12 , 15 ) on vascular function in young , healthy individuals . ( 18 ) found an augmentation in endothelium - dependent dilation in young ( 25 2.5 years ) , healthy men following 12-weeks of moderate - intensity aerobic exercise , but not following the same period of mild or high - intensity exercise . it was postulated by these authors that the elevated oxidative stress levels seen in individuals performing high - intensity exercise may have impaired endothelium - dependent dilation through a reduction in nitric oxide bioavailability . it is possible that the high - intensity exercise performed by our highly trained group increased nitric oxide production while at the same time resulted in increased oxidative stress thus counteracting the beneficial effects of increased nitric oxide production . indeed , the fmd response is preserved in older athletes compared to their age - matched , sedentary counterparts and is similar to the responses seen in both younger athletic and sedentary individuals ( 15 ) . thus , we may not have seen a difference in vascular function either prior to or after the hfm because our subjects were young enough that they did not exhibit the arterial stiffening that occurs due to aging . research has demonstrated that exercise training improves endothelium - dependent dilation in diseased individuals ( 20 , 28 ) as well as in aged individuals ( 15 ) , with inconsistent results observed in young , healthy individuals ( 7 , 12 , 24 , 32 ) . vascular dysfunction induced by a high - fat meal may occur due to enhanced oxidative stress ( 4 , 46 ) . in contrast to our hypothesis , our study demonstrated that high aerobic capacity did not offer increased protection against the acute , negative effects of a high - fat meal on vascular function . several mechanisms have been suggested by which exercise may protect endothelial function following a high - fat meal ( i.e. the scarcity of studies that have investigated the effects of exercise training on postprandial endothelial function further demonstrates the need for future research to expand our understanding of the role of diet , exercise , age , and health status on cardiovascular health . in young , healthy subjects brachial artery fmd has been found to be significantly higher after upper arm occlusion compared to forearm occlusion and thus proposed as a better means of evaluating vascular function in this population ( 1 , 5 ) . the regional ischemia produced by occlusion of the upper arm is associated with the release of vasodilators ( i.e. however , we chose to use the upper arm occlusion method due to our population studied and because previous studies examining the effect of a high fat meal on fmd have also utilized this method ( 4 , 29 , 36 , 37 ) . exercise training improves endothelium - dependent dilation in asymptomatic older individuals ( 15 ) , as well as patients with chronic heart failure ( 20 ) and type 2 diabetes ( 28 ) . however , the beneficial effects of exercise training on vascular function in young individuals exhibiting normal arterial function have not been consistently observed ( 7 , 12 , 24 , 32 ) . alternatively , differences in postprandial fmd between the highly trained and moderately active group may have become apparent if we had extended the study ( i.e. however , we do not think this is likely as an isocaloric low - fat meal has no effect on fmd when compared to a hfm ( 23 , 38 ) . furthermore , a meal high in saturated fat , which was comparable to our study , resulted in reduced fmd while meals high in carbohydrates , monounsaturated fat , or polyunsaturated fat had no effect on fmd ( 23 ) . a limitation to the study is the lack of chronic dietary information for our subjects as chronic high fat ( 26 ) or low fat ( 9 ) diets may alter fmd . although the chronic dietary patterns between our subjects may have been different , we compared the fmd response for each individual to baseline ; thus , the changes in fmd are indicative of the acute response to a hfm . despite being classified as moderately - active , this group still displayed a vo2max of 47.3 7.1 mlkgmin and thus can be considered as possessing a high level of fitness . the inclusion of a sedentary control group with a lower aerobic capacity may have allowed us to detect differences between groups not seen in the present study design and to further elucidate the role of fitness level on postprandial fmd . this study demonstrated that fmd was similar with differing aerobic capacities and was reduced in both highly trained and moderately active young men for up to four - hours after consuming a hfm . our results suggested that a high aerobic capacity did not augment fmd and did not offer increased protection against the acute , negative effects of a hfm on vascular function compared to individuals possessing a moderate aerobic capacity . future studies are warranted with a larger sample size to determine the mechanism for the decrease in fmd after a hfm and to determine if differences may exist between groups if followed for a longer period of time ( i.e. six- or eight - hours postprandial ) . these findings are significant as it demonstrates that moderately active individuals have similar acute responses to a hfm compared to highly trained individuals , suggesting that increased aerobic capacity and/or high intensity aerobic exercise is not necessary to maintain endothelial function .

osteonecrosis of the femoral head ( onfh ) , of which avascular necrosis comprises the principal part , is a progressive and disabling condition in young and middle - aged patients . typically , patients are asymptomatic in early stages while eventually requiring a total hip replacement ( thr ) . morbid vascularity ( i.e. , thrombophilia ) in cancellous bones is etiological determinants of this disease . vascular endothelial growth factor ( vegf ) is a critical mediator of vasculogenesis and angiogenesis . it has been reported that erythropoietin protects the femoral head against glucocorticoids ( gcs)-induced osteonecrosis in part by stimulating the expression of vegf . in support of these findings , being the most potent and effective therapy for immune - related diseases , gcs have been a mainstay of therapeutic recourse for most inflammatory disorders . among patients younger than 40 years of age , administration of gcs is the most prominent causative factor ( about 60% ) for onfh . besides thrombophilia and other genetic propensities , gcs have been proven to be crucial in early stages of steroid - induced necrosis of femoral head ( sinfh ) . because their treatment results in reduced blood flow and hypercoagulability , gcs damage the bone microvascular endothelial cells ( bmecs ) of the femoral head . among several pathogenic extrapolations for sinfh , the vascular hypothesis - in that local microvascular dysfunction leads to decreased blood flow in the femoral head has become the most widely supported . based on the specific microstructures of the femoral head those effects that gcs particularly have on bmecs would destroy the prop of osseous microcirculation , hereby leading to sinfh . in the current study , we assumed that gcs significantly impact the transcriptome of bmecs during the early stage of sinfh . we simulated sinfh of early stage by gcs - treatment in cultured bmecs and investigated their transcriptomes by microarray analyses , expecting to provide clues for remedying sinfh . the protocol described herein were approved by the ethics reviewing council of china - japan friendship hospital , which abides by the declaration of helsinki on ethical principles for medical research involving human subjects ( institutional clinical trials register number is 2014 - 34 , beijing 100029 , china ) . written informed consent was obtained from all participants . all patients were enrolled at china - japan friendship hospital between october 1 , 2013 and february 28 , 2014 , with diagnosis classified as traumatic transcervical fracture . any traumatic fracture complicated with other etiological mechanisms ( e.g. , osteonecrosis , arthritis or osteoarthritis ) or secondary fracture related to precipitating factors , such as osteoporosis , the current study was performed in beijing key lab for immune - mediated inflammatory diseases , which is affiliated to china - japan friendship hospital , china . to harvest bmecs for microarray analysis , cancellous bone of femoral head the crunched cancellous bones were promptly shipped to the cell culture facility , where they were digested overnight with 0.2% of type one collagenase and subsequent trypsinized of 20 min . after centrifugation , bmecs were collected and plated in 100 mm gelatin - precoated culture dishes , cultured at 37c . complete culture medium was m199 medium ( thermo fisher scientific , waltham , ma , usa ) supplemented with 2 mmol / l l - glutamine , 100 g / ml streptomycin and 100 iu / ml penicillin ( sigma - aldrich ) , 20% fetal bovine serum , and recombinant human vegf 165 ( rhvegf 165 , sino biological inc . , the medium was changed after 24 h to remove nonadherent cells . once grown to 80% confluency , cells were detached by trypsinization and passaged to two duplicates of cultures with totally 10 cells per culture . once the bmecs in each culture had grown to 80% confluence , one of the duplicate cultures was treated with hydrocortisone ( tianjin kingyork group co. ltd . , tianjin , china ) to final concentration of 0.1 mg / ml for 24 h ( i.e. , gcs - treated experimental sample ) . the paired - control culture , which was from the same patient , was left untreated . in total , eight pairs of duplicate cultures from eight different patients were investigated in this study . for analysis of cell surface markers , the cells were grown on sterile coverslips in a basal culture medium for 48 h , then washed and fixed in 4% paraformaldehyde following permeabilization with 0.1% triton x-100 . after blocking with 5% bovine serum albumin , cells were incubated overnight at 4c with specific primary antibody , which was one of cd31 ( pecam-1 ) , cd54 ( icam-1 ) , cd106 ( vcam-1 ) and cd144 ( ve - cadherin ) . after washing off the primary antibody , the fluorescein isothiocyanate conjugated secondary antibody and hoechst 33342 were added and incubated for 1 h at room temperature . the slides were mounted using anti - fading mounting medium and digital slides were acquired using a fluorescence microscope ix71 ( olympus , tokyo , japan ) . for negative controls , total rna was extracted from cells using the trizol reagent and purified with mirvana mirna isolation kit ( ambion , austin , tx , usa ) . the purity and concentration of rna were determined from od260/280 readings using spectrophotometer ( nanodrop nd-1000 ) . rna integrity was determined by capillary electrophoresis using the rna 6000 nano lab - on - a - chip kit and the bioanalyzer 2100 ( agilent technologies , santa clara , ca , usa ) . only rna extracts with rna integrity number values higher yields of cdna were labeled with a fluorescent dye ( cy5 and cy3-dctp ) using capitalbio crna amplification and labeling kit ( capitalbio , beijing , china ) . the labeled crnas from lncrnas and mrnas were purified and hybridized to agilent human lncrna + mrna array v3.0 ( agilent ) . the labeled and purified micrornas were hybridized with agilent human microrna microarray release 19.0 according to the manufacturer 's instructions . images were scanned with the agilent microarray scanner , gridded , and analyzed using agilent feature extraction software version 10.10 ( agilent ) . the raw data were summarized and normalized using the genespring software v12.0 ( agilent ) . to identify differentially expressed genes , we used threshold values of 2 absolute fold change and a benjamini - hochberg corrected p 0.05 . the data were log2 transformed and median centered by genes using the adjust data function of cluster 3.0 software ( open source ) . these genes were classified according to the go analysis provided by molecular annotation system 3.0 ( http://bioinfo.capitalbio.com/mas3/ ) . signaling pathway analysis of these genes the lncrnas were classified according to whether the corresponding transcript was mapped to the opposite strand of an exon , to an intron , or within an intergenic region of a protein - coding gene . the cis - acting lncrna prediction was carried out based on their tight correlation ( pearson 's correlation coefficient minimum of 0.99 ) to a group of differentially expressed protein - coding genes . the trans - prediction was conducted using blat tools ( standalone blat v. 35 1 fast sequence search command line tool ) to compare the full sequence of the lncrna with the 3-untranslated region ( 3 utr ) of its co - expression mrnas . the target prediction were performed by their tight correlation ( pearson 's correlation coefficient minimum of 0.7 ) to a group of differentially expressed protein - coding genes . the correlated genes were intersected with an assembly of target genes predicted by anyone of 9 online programs , including miranda ( http://www.microrna.org/microrna/home.do ) , mirdb ( http://mirdb.org/mirdb/ ) , dianamt , etc . to validate the microarray results , quantitative real - time polymerase chain reaction ( qrt - pcr ) was performed using the lightcycler - faststart dna master sybr green i pcr kit ( roche , basel , switzerland ) according to the manufacturer 's specifications . the comparative threshold cycle ( ct ) method was used for the calculation of amplification fold . to verify the microrna expression profile , stem - loop microrna qrt - pcr was performed using the small rna u6 for an internal control . the relative quantity of each microrna in each sample , normalized to u6 rna and relative to the expression in control samples , was calculated using the comparative ct ( ct ) method , with the equation of rq = 2 , where ct = ( ctmicrorna ctu6 ) exp ( ctmicrorna ctu6 ) control the protocol described herein were approved by the ethics reviewing council of china - japan friendship hospital , which abides by the declaration of helsinki on ethical principles for medical research involving human subjects ( institutional clinical trials register number is 2014 - 34 , beijing 100029 , china ) . written informed consent was obtained from all participants . all patients were enrolled at china - japan friendship hospital between october 1 , 2013 and february 28 , 2014 , with diagnosis classified as traumatic transcervical fracture . any traumatic fracture complicated with other etiological mechanisms ( e.g. , osteonecrosis , arthritis or osteoarthritis ) or secondary fracture related to precipitating factors , such as osteoporosis , the current study was performed in beijing key lab for immune - mediated inflammatory diseases , which is affiliated to china - japan friendship hospital , china . to harvest bmecs for microarray analysis , cancellous bone of femoral head the crunched cancellous bones were promptly shipped to the cell culture facility , where they were digested overnight with 0.2% of type one collagenase and subsequent trypsinized of 20 min . after centrifugation , bmecs were collected and plated in 100 mm gelatin - precoated culture dishes , cultured at 37c . complete culture medium was m199 medium ( thermo fisher scientific , waltham , ma , usa ) supplemented with 2 mmol / l l - glutamine , 100 g / ml streptomycin and 100 iu / ml penicillin ( sigma - aldrich ) , 20% fetal bovine serum , and recombinant human vegf 165 ( rhvegf 165 , sino biological inc . , the medium was changed after 24 h to remove nonadherent cells . once grown to 80% confluency , cells were detached by trypsinization and passaged to two duplicates of cultures with totally 10 cells per culture . once the bmecs in each culture had grown to 80% confluence , one of the duplicate cultures was treated with hydrocortisone ( tianjin kingyork group co. ltd . , tianjin , china ) to final concentration of 0.1 mg / ml for 24 h ( i.e. , gcs - treated experimental sample ) . the paired - control culture , which was from the same patient , was left untreated . in total , eight pairs of duplicate cultures from eight different patients were investigated in this study . for analysis of cell surface markers , the cells were grown on sterile coverslips in a basal culture medium for 48 h , then washed and fixed in 4% paraformaldehyde following permeabilization with 0.1% triton x-100 . after blocking with 5% bovine serum albumin , cells were incubated overnight at 4c with specific primary antibody , which was one of cd31 ( pecam-1 ) , cd54 ( icam-1 ) , cd106 ( vcam-1 ) and cd144 ( ve - cadherin ) . after washing off the primary antibody , the fluorescein isothiocyanate conjugated secondary antibody and hoechst 33342 were added and incubated for 1 h at room temperature . the slides were mounted using anti - fading mounting medium and digital slides were acquired using a fluorescence microscope ix71 ( olympus , tokyo , japan ) . for negative controls , total rna was extracted from cells using the trizol reagent and purified with mirvana mirna isolation kit ( ambion , austin , tx , usa ) . the purity and concentration of rna were determined from od260/280 readings using spectrophotometer ( nanodrop nd-1000 ) . rna integrity was determined by capillary electrophoresis using the rna 6000 nano lab - on - a - chip kit and the bioanalyzer 2100 ( agilent technologies , santa clara , ca , usa ) . only rna extracts with rna integrity number values higher yields of cdna were labeled with a fluorescent dye ( cy5 and cy3-dctp ) using capitalbio crna amplification and labeling kit ( capitalbio , beijing , china ) . the labeled crnas from lncrnas and mrnas were purified and hybridized to agilent human lncrna + mrna array v3.0 ( agilent ) . the labeled and purified micrornas were hybridized with agilent human microrna microarray release 19.0 according to the manufacturer 's instructions . images were scanned with the agilent microarray scanner , gridded , and analyzed using agilent feature extraction software version 10.10 ( agilent ) . the raw data were summarized and normalized using the genespring software v12.0 ( agilent ) . to identify differentially expressed genes , we used threshold values of 2 absolute fold change and a benjamini - hochberg corrected p 0.05 . the data were log2 transformed and median centered by genes using the adjust data function of cluster 3.0 software ( open source ) . these genes were classified according to the go analysis provided by molecular annotation system 3.0 ( http://bioinfo.capitalbio.com/mas3/ ) . signaling pathway analysis of these genes was performed with kegg pathway database ( http://www.kegg.jp/kegg/pathway.html ) . the lncrnas were classified according to whether the corresponding transcript was mapped to the opposite strand of an exon , to an intron , or within an intergenic region of a protein - coding gene . the cis - acting lncrna prediction was carried out based on their tight correlation ( pearson 's correlation coefficient minimum of 0.99 ) to a group of differentially expressed protein - coding genes . the trans - prediction was conducted using blat tools ( standalone blat v. 35 1 fast sequence search command line tool ) to compare the full sequence of the lncrna with the 3-untranslated region ( 3 utr ) of its co - expression mrnas . the target prediction were performed by their tight correlation ( pearson 's correlation coefficient minimum of 0.7 ) to a group of differentially expressed protein - coding genes . the correlated genes were intersected with an assembly of target genes predicted by anyone of 9 online programs , including miranda ( http://www.microrna.org/microrna/home.do ) , mirdb ( http://mirdb.org/mirdb/ ) , dianamt , etc . to validate the microarray results , quantitative real - time polymerase chain reaction ( qrt - pcr ) was performed using the lightcycler - faststart dna master sybr green i pcr kit ( roche , basel , switzerland ) according to the manufacturer 's specifications . the comparative threshold cycle ( ct ) method was used for the calculation of amplification fold . to verify the microrna expression profile , stem - loop microrna qrt - pcr was performed using the small rna u6 for an internal control . the relative quantity of each microrna in each sample , normalized to u6 rna and relative to the expression in control samples , was calculated using the comparative ct ( ct ) method , with the equation of rq = 2 , where ct = ( ctmicrorna ctu6 ) exp ( ctmicrorna ctu6 ) control consistent with previous observations , a greater percentage of lncrnas was detected above background compared to encoding ones . we found that 78% of lncrnas interrogated on the microarray exhibited expression above background and that 0.9% of these were significantly differentially expressed between gcs - treated and untreated bmecs . by contrast , 63% of protein - coding mrna transcripts on the microarray were expressed above background , and 2.2% of these were significantly differentially expressed [ table 1 ] . in addition , statistical analysis showed that lncrnas expressed above background were scattered on all chromosomes . the ratio ( expressed probes / total probes ) expressed from nuclear chromosome was lower than the mitochondrial genome , although few of them were differentially expressed [ figure 1a ] . when we evaluated the expression levels of lncrnas in paired samples ( gcs - treated to untreated ratio ) by log fold - change , 166 lncrnas were found to be significantly down - regulated , and 73 lncrnas were significantly up - regulated in the gcs - treated bmecs . there were more than twice as many lncrnas down - regulated as there were lncrnas up - regulated [ figure 1b ] . conversely , differentially up - regulated mrnas ( 336 out of 518 ) were more common than significantly down - regulated mrnas ( 182 out of 518 ) . in line with our expectations , some annotated lncrnas were differentially expressed in gcs - treated bmecs , such as nav2-it1 , nav2-as5 , nav2-as1 , ntm - it2 , and arhgef19-as1 . we analyzed the genomic location which partly reflects regulatory mechanisms - of the differentially expressed lncrnas . we performed this analysis based on the lncrna orientation to local protein - coding genes by categorizing their associated protein - coding genes as small nucleolar , divergent , intronic , antisense and intergenic according to our modified definition [ figure 1c ] . all the records have been submitted to gene expression omnibus ( geo ) of national center for biotechnology information , have been approved , and assigned geo accession numbers referred as gse60333 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60333 ) . this superseries record provides access to two linked subseries that are gse60332 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60332 ) and gse60093 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60093 ) , along with platform gpl19072 for agilent-052909 cbc_lncrnamrna_v3 ( probe name version ) , which is initially submitted by us ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gpl19072 ) . summary of microarray analysis results * significant differential expression was defined as probes with p0.05 and absolute fold - change 2 . overall profiles of the glucocorticoid versus control transcriptomes of bone microvascular endothelial cells ( bmecs ) . ( a ) relative distribution of expressed lncrnas ( expressed probes / total probes ) derived from each chromosome ; ( b ) relative chromosomal distribution of differentially regulated ( up- or down - regulated probe number / total probes number ) lncrnas ; ( c ) annotation of genomic context of differentially expressed lncrnas ; ( d ) validation of microarray results by quantitative real - time polymerase chain reaction . to validate the microarray data , we investigated seven randomly selected rnas ( 2 micrornas , 2 lncrnas and 3 mrnas ) which were identified to be differentially expressed after gcs administration using quantitative real - time pcr ( qrt - pcr ) . u6 served as an internal control for microrna and gapdh served as an internal control for lncrna and mrna detection , respectively . the qrt - pcr results showed that expression levels of ensg00000259007.1 ( probe name , p4493 ) , xloc_011117 ( probe name , p19376 ) , peroxisome proliferator - activated receptor gamma , bmp binding endothelial regulator and smad family member 3 were concordant with microarray data with a strong correlation [ figure 1d ] . as for micrornas , the percentages for above - background expression and the differential expression were 18% and 1.4% , respectively [ table 1 ] . hierarchical clustering analysis for the 16 sets of paired data ( two data each patient ) was summarized in a dendrogram [ figure 2a ] . eight sets of data from each group were combined for significance analysis of microarrays . after filtering out micrornas that were undetectable , of them , 5 micrornas were up - regulated by more than 2.5-fold , while 11 micrornas were down - regulated by more than 2.5-fold after 24-h treatment . only 5 micrornas among them were consistently up / down - regulated at least 2.5-fold at all 8 samples after treatment with corrected p 0.05 [ figure 2b ] . except for hsa - mir-339 - 5p that was up - regulated , ( a ) hierarchical clustered heat maps showing the log2 transformed expression values for differentially expressed micrornas between glucocorticoids ( gcs)-treated and untreated bone microvascular endothelial cells of eight patients ; ( b ) scatter and volcano plots of micrornas with significant differential expression induced by gcs - treatment . summary of micrornas with significantly differentially expressed genes in gcs - treated versus control bmec cells * * significant differential expression was defined as p0.05 and absolute fold - change 2.5 . bmec : bone microvascular endothelial cell ; gcs : glucocorticoids ; fc : fold - change . the microrna target prediction was performed for all 5 micrornas that were significantly influenced by gcs - treatment . we found a total of 55 target genes were predicted to be targeted by these micrornas . we further expanded our analysis to identify protein - coding genes whose expression inversely correlated to these micrornas . some lncrnas were also implicated in specific diseases by significantly correlating with genes participating in graft - versus - host disease , type 1 diabetes , arthritis , asthma , periodontitis , systemic lupus erythematosus , graves disease , etc . , based on this dataset , we performed the enrichment analysis for go terms , functionally annotating the genes by a conditional hypergeometric test . gene functional annotations were classified into three categories : biological process , molecular function ( mf ) and cellular component . because micrornas and lncrnas were prone to exert influence on associated protein - coding genes in transcription - related processes , we submitted a list of protein - coding genes which were significantly correlated with differentially expressed noncoding rnas that is , 172 mrnas correlated with lncrnas along with a list of 214 mrnas correlated with differentially expressed micrornas , and 518 statistically differentially expressed mrnas to mas 3.0 ( http://bioinfo.capitalbio.com/mas3/ ) for go term enrichment analysis . some pivotal roles involving both mrnas and mrnas were significantly correlated with noncoding rnas . differentially expressed mrnas and their counterparts statistically correlated with noncoding rnas that were composed of micrornas and lncrnas were classified according to their go annotation terms , respectively [ figure 3a and b ] . there were similar portfolios of the highest enriched go terms that were presented by both mrnas and mrnas that significantly correlated with noncoding rnas . the gene ontology ( go ) enrichment analysis provides a controlled vocabulary to describe differentially expressed transcriptomes . the most significantly enriched terms for mrnas ( a ) and the most enriched go terms for the genes significantly correlated to differentially expressed noncoding rnas ( b ) are displayed here . for further analyses of protein - coding genes correlated with micrornas , we constructed a gene interaction network [ figure 4a ] . for example , enst00000553603.1 ( probed by p4493 ) not only correlated with many protein - coding genes , but also had positive interrelated relationships to tcons_00024496 ( probed by p20100 ) and enst00000585190.1 ( probed by p6957 ) , suggesting the complex regulatory network which p4493 could be implicated [ figure 4b ] . notwithstanding the intricate interrelationship among some lncrnas , no differentially expressed lncrnas overlapped with any annotated micrornas as far as our study is concerned . through significantly correlating key genes , noncoding rnas in addition , some signaling pathways were conspicuously modulated by noncoding rnas . among these pathways , compensatory increased foxo signaling pathway should be involved in the development of sinfh [ figure 4c ] . ( a ) co - expression network composed with differentially expressed micrornas and their correlated genes ( pearson 's correlation coefficient minimum of 0.99 ) . ( b ) co - expression network composed with differentially expressed lncrnas and their correlated genes ( pearson 's correlation coefficient minimum of 0.99 ) . ( c ) kegg pathway analysis is a functional analysis that maps genes to pathways . the foxo signaling pathway shows modulation in apoptotic signatures and may associate with onset of avascular osteonecrosis . consistent with previous observations , a greater percentage of lncrnas was detected above background compared to encoding ones . we found that 78% of lncrnas interrogated on the microarray exhibited expression above background and that 0.9% of these were significantly differentially expressed between gcs - treated and untreated bmecs . by contrast , 63% of protein - coding mrna transcripts on the microarray were expressed above background , and 2.2% of these were significantly differentially expressed [ table 1 ] . in addition , statistical analysis showed that lncrnas expressed above background were scattered on all chromosomes . the ratio ( expressed probes / total probes ) expressed from nuclear chromosome was lower than the mitochondrial genome , although few of them were differentially expressed [ figure 1a ] . when we evaluated the expression levels of lncrnas in paired samples ( gcs - treated to untreated ratio ) by log fold - change , 166 lncrnas were found to be significantly down - regulated , and 73 lncrnas were significantly up - regulated in the gcs - treated bmecs . there were more than twice as many lncrnas down - regulated as there were lncrnas up - regulated [ figure 1b ] . conversely , differentially up - regulated mrnas ( 336 out of 518 ) were more common than significantly down - regulated mrnas ( 182 out of 518 ) . in line with our expectations , some annotated lncrnas were differentially expressed in gcs - treated bmecs , such as nav2-it1 , nav2-as5 , nav2-as1 , ntm - it2 , and arhgef19-as1 . we analyzed the genomic location which partly reflects regulatory mechanisms - of the differentially expressed lncrnas . we performed this analysis based on the lncrna orientation to local protein - coding genes by categorizing their associated protein - coding genes as small nucleolar , divergent , intronic , antisense and intergenic according to our modified definition [ figure 1c ] . all the records have been submitted to gene expression omnibus ( geo ) of national center for biotechnology information , have been approved , and assigned geo accession numbers referred as gse60333 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60333 ) . this superseries record provides access to two linked subseries that are gse60332 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60332 ) and gse60093 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60093 ) , along with platform gpl19072 for agilent-052909 cbc_lncrnamrna_v3 ( probe name version ) , which is initially submitted by us ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gpl19072 ) . summary of microarray analysis results * significant differential expression was defined as probes with p0.05 and absolute fold - change 2 . overall profiles of the glucocorticoid versus control transcriptomes of bone microvascular endothelial cells ( bmecs ) . ( a ) relative distribution of expressed lncrnas ( expressed probes / total probes ) derived from each chromosome ; ( b ) relative chromosomal distribution of differentially regulated ( up- or down - regulated probe number / total probes number ) lncrnas ; ( c ) annotation of genomic context of differentially expressed lncrnas ; ( d ) validation of microarray results by quantitative real - time polymerase chain reaction . to validate the microarray data , we investigated seven randomly selected rnas ( 2 micrornas , 2 lncrnas and 3 mrnas ) which were identified to be differentially expressed after gcs administration using quantitative real - time pcr ( qrt - pcr ) . u6 served as an internal control for microrna and gapdh served as an internal control for lncrna and mrna detection , respectively . the qrt - pcr results showed that expression levels of ensg00000259007.1 ( probe name , p4493 ) , xloc_011117 ( probe name , p19376 ) , peroxisome proliferator - activated receptor gamma , bmp binding endothelial regulator and smad family member 3 were concordant with microarray data with a strong correlation [ figure 1d ] . as for micrornas , the percentages for above - background expression and the differential expression were 18% and 1.4% , respectively [ table 1 ] . hierarchical clustering analysis for the 16 sets of paired data ( two data each patient ) was summarized in a dendrogram [ figure 2a ] . eight sets of data from each group were combined for significance analysis of microarrays . after filtering out micrornas that were undetectable , of them , 5 micrornas were up - regulated by more than 2.5-fold , while 11 micrornas were down - regulated by more than 2.5-fold after 24-h treatment . only 5 micrornas among them were consistently up / down - regulated at least 2.5-fold at all 8 samples after treatment with corrected p 0.05 [ figure 2b ] . except for hsa - mir-339 - 5p that was up - regulated , ( a ) hierarchical clustered heat maps showing the log2 transformed expression values for differentially expressed micrornas between glucocorticoids ( gcs)-treated and untreated bone microvascular endothelial cells of eight patients ; ( b ) scatter and volcano plots of micrornas with significant differential expression induced by gcs - treatment . summary of micrornas with significantly differentially expressed genes in gcs - treated versus control bmec cells * * significant differential expression was defined as p0.05 and absolute fold - change 2.5 . bmec : bone microvascular endothelial cell ; gcs : glucocorticoids ; fc : fold - change . the microrna target prediction was performed for all 5 micrornas that were significantly influenced by gcs - treatment . we found a total of 55 target genes were predicted to be targeted by these micrornas . we further expanded our analysis to identify protein - coding genes whose expression inversely correlated to these micrornas . some lncrnas were also implicated in specific diseases by significantly correlating with genes participating in graft - versus - host disease , type 1 diabetes , arthritis , asthma , periodontitis , systemic lupus erythematosus , graves disease , etc . , based on this dataset , we performed the enrichment analysis for go terms , functionally annotating the genes by a conditional hypergeometric test . gene functional annotations were classified into three categories : biological process , molecular function ( mf ) and cellular component . because micrornas and lncrnas were prone to exert influence on associated protein - coding genes in transcription - related processes , we submitted a list of protein - coding genes which were significantly correlated with differentially expressed noncoding rnas that is , 172 mrnas correlated with lncrnas along with a list of 214 mrnas correlated with differentially expressed micrornas , and 518 statistically differentially expressed mrnas to mas 3.0 ( http://bioinfo.capitalbio.com/mas3/ ) for go term enrichment analysis . some pivotal roles involving both mrnas and mrnas were significantly correlated with noncoding rnas . differentially expressed mrnas and their counterparts statistically correlated with noncoding rnas that were composed of micrornas and lncrnas were classified according to their go annotation terms , respectively [ figure 3a and b ] . there were similar portfolios of the highest enriched go terms that were presented by both mrnas and mrnas that significantly correlated with noncoding rnas . the gene ontology ( go ) enrichment analysis provides a controlled vocabulary to describe differentially expressed transcriptomes . the most significantly enriched terms for mrnas ( a ) and the most enriched go terms for the genes significantly correlated to differentially expressed noncoding rnas ( b ) are displayed here . for further analyses of protein - coding genes correlated with micrornas , we constructed a gene interaction network [ figure 4a ] . for example , enst00000553603.1 ( probed by p4493 ) not only correlated with many protein - coding genes , but also had positive interrelated relationships to tcons_00024496 ( probed by p20100 ) and enst00000585190.1 ( probed by p6957 ) , suggesting the complex regulatory network which p4493 could be implicated [ figure 4b ] . notwithstanding the intricate interrelationship among some lncrnas , no differentially expressed lncrnas overlapped with any annotated micrornas as far as our study is concerned . through significantly correlating key genes , noncoding rnas participate in some important processes and functions . in addition , some signaling pathways were conspicuously modulated by noncoding rnas . among these pathways , compensatory increased foxo signaling pathway should be involved in the development of sinfh [ figure 4c ] . ( a ) co - expression network composed with differentially expressed micrornas and their correlated genes ( pearson 's correlation coefficient minimum of 0.99 ) . ( b ) co - expression network composed with differentially expressed lncrnas and their correlated genes ( pearson 's correlation coefficient minimum of 0.99 ) . ( c ) kegg pathway analysis is a functional analysis that maps genes to pathways . the foxo signaling pathway shows modulation in apoptotic signatures and may associate with onset of avascular osteonecrosis . they are believed to interfere with their target gene - coding mrnas by base - pairing and binding to the 3-utrs , 5-utrs or coding regions . a large number of target genes involved in enriched go terms were targeted by hsa - mir-100 - 3p in the present study . for example , by targeting interferon receptor 1 , hsa - mir-100 - 3p is believed to be involved in periodontitis . in addition , hsa - mir-339 - 5p was believed to participate in osteoporosis , periodontitis and rheumatoid arthritis by targeting interleukin 11 , while , hsa - mir-933 was related to osteoporosis via targeting cannabinoid receptor 1 . although few reports have proven their involvement in sinfh , these results highlight the need to further elucidate the role of micrornas in sinfh . in some case these noncoding transcripts act as natural microrna sponges by competing for binding to shared micrornas . to explore the possibility that some lncrnas , which are differentially expressed after gcs - treatment , might also act as primary transcripts for micrornas , we systematically searched for genomic overlap between differentially expressed lncrnas and known micrornas . unfortunately , our study was unable to identify lncrnas overlapping annotated micrornas . this result suggested that there might not possibly be any primary precursor or endogenous competitor for micrornas being significantly influenced by gcs . the lncrnas are considerably different from coding rnas with regards to their pattern of splicing and the narrow distribution of gc content . they typically resided at genomic loci within 10 kb of a protein - coding gene along the genome . the lncrnas often participated in important , and critical cellular and biological functions regulating gene expression via epigenetic modification or transcriptional co - activation / repression . numerous studies have demonstrated that noncoding rnas can regulate the expression of their associated protein - coding genes via a range of systems , including chromatin remodeling , alternative splicing , translational interference and promoter targeting by cis - acting ( the same locus ) regulatory mechanisms , which is an antisense - mediated regulation by lncrnas of their neighboring protein - coding counterparts . the high ratio of lncrnas derived from the mitochondrial genome may be related to the high abundance of mitochondrial lncrnas in certain tissues . as for their genomic context , antisense transcripts are prevalent throughout the mammalian genome . among the antisense transcripts that were expressed above background , 51 of them were found to be differentially expressed . intronic lncrnas comprise a large class of transcriptional units transcribed from intronic regions of mammalian protein - coding genes in a tissue - specific pattern . previous studies indicated that intronic lncrnas could regulate the expression of their host or neighboring genes . long intergenic noncoding rnas ( lincrnas ) rapidly evolved their sequences despite conserved functions across species . by modulating the expression of their neighboring protein - coding genes or other target genes scattered across the genome via the recruitment of histone - modifying enzymes to chromatin , lincrnas play important roles in the development of certain diseases . most lncrnas within our datasets could fit into this category that is , 126 lincrnas that approximately made up 58% of the entire differentially expressed lncrnas . except for the housekeeping mfs such as protein binding , the most relevant go terms were enriched in transcription - related processes and some well - annotated transcriptional regulators belonged to the cell cycle , apoptosis and cell proliferation gene lists , including gadd45b , nav1 , fgf5 , lifr , stat5a , il11 , cdk10 , il6 , rgs2 and gas1 . it indicated that lncrnas were likely to perform their function at transcriptional levels by regulating transcription - related genes . gc receptor binding were poorly enriched , which could be caused by the insufficient annotations for gcs - related go terms . it is noteworthy that the search results for gc and bone in the geo datasets database are scarce ( http://www.ncbi.nlm.nih.gov/gds/?term=(glucocorticoid+and+bone ) ) . although there was few go term enrichment for gcs - related genes , we still found that 2 of genes had associated go terms relating to degenerative disorders , which were fkbp4 and il6 . in conclusion , we postulated that noncoding rnas could be involved in microvascular events and related to endothelial dysfunction , hence diffusing apoptosis throughout the femoral head . sinfh impacts such a large population that preventing its attack is of critical significance to gcs users . in addition to gc - induced transcriptional changes , hereditary factors also predispose people to sinfh . combining the hierarchical clustering data with clinical meta - analysis we identified significantly differentially expressed transcripts but nonetheless we are still short of a reliable clinical method to control this disorder . the current study outlined the differential expression profiles induced by gcs but did nt specify any intracellular signaling pathway that is solely responsible for the pathogenesis of sinfh . all the aforementioned propel us to perform further in - depth investigation in order to enhance our understanding of sinfh . intensive investigations of the signaling pathways screened out in this study should be carried out to verify the pivotal one underlying pathogenesis of sinfh .

background : appropriate expression and regulation of the transcriptome , which mainly comprise of mrnas and lncrnas , are important for all biological and cellular processes including the physiological activities of bone microvascular endothelial cells ( bmecs ) . through an intricate intracellular signaling systems , the transcriptome regulates the pharmacological response of the cells . although studies have elucidated the impact of glucocorticoids ( gcs ) cell - specific gene expression signatures , it remains necessary to comprehensively characterize the impact of lncrnas to transcriptional changes.methods:bmecs were divided into two groups . one was treated with gcs and the other left untreated as a paired control . differential expression was analyzed with genespring software v12.0 ( agilent , santa clara , ca , usa ) and hierarchical clustering was conducted using cluster 3.0 software . the gene ontology ( go ) analysis was performed with molecular annotation system provided by capitalbio corporation.results:our results highlight the involvement of genes implicated in development , differentiation and apoptosis following gc stimulation . elucidation of differential gene expression emphasizes the importance of regulatory gene networks induced by gcs . we identified 73 up - regulated and 166 down - regulated long noncoding rnas , the expression of 107 of which significantly correlated with 172 mrnas induced by hydrocortisone.conclusions:transcriptome analysis of bmecs from human samples was performed to identify specific gene networks induced by gcs . our results identified complex rna crosstalk underlying the pathogenesis of steroid - induced necrosis of femoral head .

I M Ethics statement and patients Primary cell culture and phenotyping Microarrays Data analysis Quantitative real-time polymerase chain reaction R Expression profiles of mRNAs and lncRNAs in glucocorticoids-treated bone microvascular endothelial cells The microRNA transcriptome in bone microvascular endothelial cells after glucocorticoids treatment Correlation among differentially expressed microRNAs and lncRNAs with protein-coding genes Correlated expression among different transcriptomes D

osteonecrosis of the femoral head ( onfh ) , of which avascular necrosis comprises the principal part , is a progressive and disabling condition in young and middle - aged patients . it has been reported that erythropoietin protects the femoral head against glucocorticoids ( gcs)-induced osteonecrosis in part by stimulating the expression of vegf . besides thrombophilia and other genetic propensities , gcs have been proven to be crucial in early stages of steroid - induced necrosis of femoral head ( sinfh ) . because their treatment results in reduced blood flow and hypercoagulability , gcs damage the bone microvascular endothelial cells ( bmecs ) of the femoral head . among several pathogenic extrapolations for sinfh , the vascular hypothesis - in that local microvascular dysfunction leads to decreased blood flow in the femoral head has become the most widely supported . based on the specific microstructures of the femoral head those effects that gcs particularly have on bmecs would destroy the prop of osseous microcirculation , hereby leading to sinfh . in the current study , we assumed that gcs significantly impact the transcriptome of bmecs during the early stage of sinfh . , osteonecrosis , arthritis or osteoarthritis ) or secondary fracture related to precipitating factors , such as osteoporosis , the current study was performed in beijing key lab for immune - mediated inflammatory diseases , which is affiliated to china - japan friendship hospital , china . to harvest bmecs for microarray analysis , cancellous bone of femoral head the crunched cancellous bones were promptly shipped to the cell culture facility , where they were digested overnight with 0.2% of type one collagenase and subsequent trypsinized of 20 min . once the bmecs in each culture had grown to 80% confluence , one of the duplicate cultures was treated with hydrocortisone ( tianjin kingyork group co. ltd . the paired - control culture , which was from the same patient , was left untreated . for analysis of cell surface markers , the cells were grown on sterile coverslips in a basal culture medium for 48 h , then washed and fixed in 4% paraformaldehyde following permeabilization with 0.1% triton x-100 . after blocking with 5% bovine serum albumin , cells were incubated overnight at 4c with specific primary antibody , which was one of cd31 ( pecam-1 ) , cd54 ( icam-1 ) , cd106 ( vcam-1 ) and cd144 ( ve - cadherin ) . for negative controls , total rna was extracted from cells using the trizol reagent and purified with mirvana mirna isolation kit ( ambion , austin , tx , usa ) . rna integrity was determined by capillary electrophoresis using the rna 6000 nano lab - on - a - chip kit and the bioanalyzer 2100 ( agilent technologies , santa clara , ca , usa ) . the labeled crnas from lncrnas and mrnas were purified and hybridized to agilent human lncrna + mrna array v3.0 ( agilent ) . the raw data were summarized and normalized using the genespring software v12.0 ( agilent ) . the data were log2 transformed and median centered by genes using the adjust data function of cluster 3.0 software ( open source ) . these genes were classified according to the go analysis provided by molecular annotation system 3.0 ( http://bioinfo.capitalbio.com/mas3/ ) . the trans - prediction was conducted using blat tools ( standalone blat v. 35 1 fast sequence search command line tool ) to compare the full sequence of the lncrna with the 3-untranslated region ( 3 utr ) of its co - expression mrnas . the relative quantity of each microrna in each sample , normalized to u6 rna and relative to the expression in control samples , was calculated using the comparative ct ( ct ) method , with the equation of rq = 2 , where ct = ( ctmicrorna ctu6 ) exp ( ctmicrorna ctu6 ) control the protocol described herein were approved by the ethics reviewing council of china - japan friendship hospital , which abides by the declaration of helsinki on ethical principles for medical research involving human subjects ( institutional clinical trials register number is 2014 - 34 , beijing 100029 , china ) . , osteonecrosis , arthritis or osteoarthritis ) or secondary fracture related to precipitating factors , such as osteoporosis , the current study was performed in beijing key lab for immune - mediated inflammatory diseases , which is affiliated to china - japan friendship hospital , china . to harvest bmecs for microarray analysis , cancellous bone of femoral head the crunched cancellous bones were promptly shipped to the cell culture facility , where they were digested overnight with 0.2% of type one collagenase and subsequent trypsinized of 20 min . complete culture medium was m199 medium ( thermo fisher scientific , waltham , ma , usa ) supplemented with 2 mmol / l l - glutamine , 100 g / ml streptomycin and 100 iu / ml penicillin ( sigma - aldrich ) , 20% fetal bovine serum , and recombinant human vegf 165 ( rhvegf 165 , sino biological inc . once the bmecs in each culture had grown to 80% confluence , one of the duplicate cultures was treated with hydrocortisone ( tianjin kingyork group co. ltd . the paired - control culture , which was from the same patient , was left untreated . for analysis of cell surface markers , the cells were grown on sterile coverslips in a basal culture medium for 48 h , then washed and fixed in 4% paraformaldehyde following permeabilization with 0.1% triton x-100 . after blocking with 5% bovine serum albumin , cells were incubated overnight at 4c with specific primary antibody , which was one of cd31 ( pecam-1 ) , cd54 ( icam-1 ) , cd106 ( vcam-1 ) and cd144 ( ve - cadherin ) . for negative controls , total rna was extracted from cells using the trizol reagent and purified with mirvana mirna isolation kit ( ambion , austin , tx , usa ) . rna integrity was determined by capillary electrophoresis using the rna 6000 nano lab - on - a - chip kit and the bioanalyzer 2100 ( agilent technologies , santa clara , ca , usa ) . the labeled crnas from lncrnas and mrnas were purified and hybridized to agilent human lncrna + mrna array v3.0 ( agilent ) . the raw data were summarized and normalized using the genespring software v12.0 ( agilent ) . the data were log2 transformed and median centered by genes using the adjust data function of cluster 3.0 software ( open source ) . these genes were classified according to the go analysis provided by molecular annotation system 3.0 ( http://bioinfo.capitalbio.com/mas3/ ) . signaling pathway analysis of these genes was performed with kegg pathway database ( http://www.kegg.jp/kegg/pathway.html ) . the trans - prediction was conducted using blat tools ( standalone blat v. 35 1 fast sequence search command line tool ) to compare the full sequence of the lncrna with the 3-untranslated region ( 3 utr ) of its co - expression mrnas . when we evaluated the expression levels of lncrnas in paired samples ( gcs - treated to untreated ratio ) by log fold - change , 166 lncrnas were found to be significantly down - regulated , and 73 lncrnas were significantly up - regulated in the gcs - treated bmecs . there were more than twice as many lncrnas down - regulated as there were lncrnas up - regulated [ figure 1b ] . conversely , differentially up - regulated mrnas ( 336 out of 518 ) were more common than significantly down - regulated mrnas ( 182 out of 518 ) . this superseries record provides access to two linked subseries that are gse60332 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60332 ) and gse60093 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60093 ) , along with platform gpl19072 for agilent-052909 cbc_lncrnamrna_v3 ( probe name version ) , which is initially submitted by us ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gpl19072 ) . summary of microarray analysis results * significant differential expression was defined as probes with p0.05 and absolute fold - change 2 . overall profiles of the glucocorticoid versus control transcriptomes of bone microvascular endothelial cells ( bmecs ) . ( a ) relative distribution of expressed lncrnas ( expressed probes / total probes ) derived from each chromosome ; ( b ) relative chromosomal distribution of differentially regulated ( up- or down - regulated probe number / total probes number ) lncrnas ; ( c ) annotation of genomic context of differentially expressed lncrnas ; ( d ) validation of microarray results by quantitative real - time polymerase chain reaction . as for micrornas , the percentages for above - background expression and the differential expression were 18% and 1.4% , respectively [ table 1 ] . after filtering out micrornas that were undetectable , of them , 5 micrornas were up - regulated by more than 2.5-fold , while 11 micrornas were down - regulated by more than 2.5-fold after 24-h treatment . only 5 micrornas among them were consistently up / down - regulated at least 2.5-fold at all 8 samples after treatment with corrected p 0.05 [ figure 2b ] . except for hsa - mir-339 - 5p that was up - regulated , ( a ) hierarchical clustered heat maps showing the log2 transformed expression values for differentially expressed micrornas between glucocorticoids ( gcs)-treated and untreated bone microvascular endothelial cells of eight patients ; ( b ) scatter and volcano plots of micrornas with significant differential expression induced by gcs - treatment . summary of micrornas with significantly differentially expressed genes in gcs - treated versus control bmec cells * * significant differential expression was defined as p0.05 and absolute fold - change 2.5 . bmec : bone microvascular endothelial cell ; gcs : glucocorticoids ; fc : fold - change . the microrna target prediction was performed for all 5 micrornas that were significantly influenced by gcs - treatment . gene functional annotations were classified into three categories : biological process , molecular function ( mf ) and cellular component . because micrornas and lncrnas were prone to exert influence on associated protein - coding genes in transcription - related processes , we submitted a list of protein - coding genes which were significantly correlated with differentially expressed noncoding rnas that is , 172 mrnas correlated with lncrnas along with a list of 214 mrnas correlated with differentially expressed micrornas , and 518 statistically differentially expressed mrnas to mas 3.0 ( http://bioinfo.capitalbio.com/mas3/ ) for go term enrichment analysis . some pivotal roles involving both mrnas and mrnas were significantly correlated with noncoding rnas . differentially expressed mrnas and their counterparts statistically correlated with noncoding rnas that were composed of micrornas and lncrnas were classified according to their go annotation terms , respectively [ figure 3a and b ] . there were similar portfolios of the highest enriched go terms that were presented by both mrnas and mrnas that significantly correlated with noncoding rnas . the gene ontology ( go ) enrichment analysis provides a controlled vocabulary to describe differentially expressed transcriptomes . the most significantly enriched terms for mrnas ( a ) and the most enriched go terms for the genes significantly correlated to differentially expressed noncoding rnas ( b ) are displayed here . when we evaluated the expression levels of lncrnas in paired samples ( gcs - treated to untreated ratio ) by log fold - change , 166 lncrnas were found to be significantly down - regulated , and 73 lncrnas were significantly up - regulated in the gcs - treated bmecs . there were more than twice as many lncrnas down - regulated as there were lncrnas up - regulated [ figure 1b ] . conversely , differentially up - regulated mrnas ( 336 out of 518 ) were more common than significantly down - regulated mrnas ( 182 out of 518 ) . this superseries record provides access to two linked subseries that are gse60332 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60332 ) and gse60093 ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gse60093 ) , along with platform gpl19072 for agilent-052909 cbc_lncrnamrna_v3 ( probe name version ) , which is initially submitted by us ( http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gpl19072 ) . summary of microarray analysis results * significant differential expression was defined as probes with p0.05 and absolute fold - change 2 . overall profiles of the glucocorticoid versus control transcriptomes of bone microvascular endothelial cells ( bmecs ) . as for micrornas , the percentages for above - background expression and the differential expression were 18% and 1.4% , respectively [ table 1 ] . after filtering out micrornas that were undetectable , of them , 5 micrornas were up - regulated by more than 2.5-fold , while 11 micrornas were down - regulated by more than 2.5-fold after 24-h treatment . only 5 micrornas among them were consistently up / down - regulated at least 2.5-fold at all 8 samples after treatment with corrected p 0.05 [ figure 2b ] . except for hsa - mir-339 - 5p that was up - regulated , ( a ) hierarchical clustered heat maps showing the log2 transformed expression values for differentially expressed micrornas between glucocorticoids ( gcs)-treated and untreated bone microvascular endothelial cells of eight patients ; ( b ) scatter and volcano plots of micrornas with significant differential expression induced by gcs - treatment . summary of micrornas with significantly differentially expressed genes in gcs - treated versus control bmec cells * * significant differential expression was defined as p0.05 and absolute fold - change 2.5 . bmec : bone microvascular endothelial cell ; gcs : glucocorticoids ; fc : fold - change . the microrna target prediction was performed for all 5 micrornas that were significantly influenced by gcs - treatment . because micrornas and lncrnas were prone to exert influence on associated protein - coding genes in transcription - related processes , we submitted a list of protein - coding genes which were significantly correlated with differentially expressed noncoding rnas that is , 172 mrnas correlated with lncrnas along with a list of 214 mrnas correlated with differentially expressed micrornas , and 518 statistically differentially expressed mrnas to mas 3.0 ( http://bioinfo.capitalbio.com/mas3/ ) for go term enrichment analysis . some pivotal roles involving both mrnas and mrnas were significantly correlated with noncoding rnas . differentially expressed mrnas and their counterparts statistically correlated with noncoding rnas that were composed of micrornas and lncrnas were classified according to their go annotation terms , respectively [ figure 3a and b ] . there were similar portfolios of the highest enriched go terms that were presented by both mrnas and mrnas that significantly correlated with noncoding rnas . the gene ontology ( go ) enrichment analysis provides a controlled vocabulary to describe differentially expressed transcriptomes . the most significantly enriched terms for mrnas ( a ) and the most enriched go terms for the genes significantly correlated to differentially expressed noncoding rnas ( b ) are displayed here . for example , enst00000553603.1 ( probed by p4493 ) not only correlated with many protein - coding genes , but also had positive interrelated relationships to tcons_00024496 ( probed by p20100 ) and enst00000585190.1 ( probed by p6957 ) , suggesting the complex regulatory network which p4493 could be implicated [ figure 4b ] . although few reports have proven their involvement in sinfh , these results highlight the need to further elucidate the role of micrornas in sinfh . to explore the possibility that some lncrnas , which are differentially expressed after gcs - treatment , might also act as primary transcripts for micrornas , we systematically searched for genomic overlap between differentially expressed lncrnas and known micrornas . numerous studies have demonstrated that noncoding rnas can regulate the expression of their associated protein - coding genes via a range of systems , including chromatin remodeling , alternative splicing , translational interference and promoter targeting by cis - acting ( the same locus ) regulatory mechanisms , which is an antisense - mediated regulation by lncrnas of their neighboring protein - coding counterparts . previous studies indicated that intronic lncrnas could regulate the expression of their host or neighboring genes . in conclusion , we postulated that noncoding rnas could be involved in microvascular events and related to endothelial dysfunction , hence diffusing apoptosis throughout the femoral head . in addition to gc - induced transcriptional changes , hereditary factors also predispose people to sinfh . combining the hierarchical clustering data with clinical meta - analysis we identified significantly differentially expressed transcripts but nonetheless we are still short of a reliable clinical method to control this disorder . the current study outlined the differential expression profiles induced by gcs but did nt specify any intracellular signaling pathway that is solely responsible for the pathogenesis of sinfh . intensive investigations of the signaling pathways screened out in this study should be carried out to verify the pivotal one underlying pathogenesis of sinfh .

genomic dna exists in a crowded space where a cell must juggle many tasks , and where problems can easily arise . during s phase , the dna replication machinery known as the replisome competes for access to template dna with numerous dna - binding proteins involved in various nuclear processes , including transcription , chromatin remodeling , sister chromatid cohesion ( scc ) and dna repair . proteins involved in these nuclear transactions can block replisome progression , leading to replication fork arrest or even breakage , which , in turn , causes genomic instability , including mutations and chromosome rearrangements . to prevent these problems , such accommodation requires specialized replisome components that interact with chromatin itself as well as other chromatin - associated proteins . the fpc is comprised of two proteins : timeless ( also called tim ) and tipin in metazoans , swi1 and swi3 in the fission yeast schizosaccharomyces pombe , and tof1 and csm3 in the budding yeast saccharomyces cerevisiae ( table 1 ) . timeless and tipin protein levels are mutually dependent ; timeless downregulation leads to depletion of tipin , and vice versa , indicating that timeless and tipin form an obligate heterodimeric complex . the fpc interacts with chromatin as well as many replisome components , including mini - chromosome maintenance ( mcm ) dna helicase subunits , dna polymerases and , replication protein a ( rpa ) and other ancillary factors . in both yeast and human cells , the fpc associates with replication origins at the onset of s phase and travels with the replisome during dna replication . the functions of the fpc are far - reaching compared with other ancillary replisome components . compelling evidence suggests the roles of the fpc in normal dna replication , replication at difficult - to - replicate genomic regions , replication fork pausing / stalling , the dna replication checkpoint and scc . neither protein has been characterized as an enzyme or has significant sequence homology to other proteins to inform us of their functions . therefore , much of what is known about fpc functions comes from deletion / depletion experiments in which the consequences of fpc loss are observed . in this review , we summarize the current understanding of the roles of the fpc in the diverse pathways coordinated at the replication fork . in addition , we discuss what functions of the fpc make it central for a variety of processes at the replication fork in order to promote genome stability . the fpc proteins were first functionally characterized in fission yeast , where swi1 and swi3 were isolated in a screen for mutants unable to generate a double - strand break during mating - type switching . later , budding yeast fpc protein tof1 was implicated in processes seemingly unrelated to mating - type switching . tof1 ( topoisomerase i - interacting factor 1 ) was named for its interactions with topoisomerase i , both in the yeast two - hybrid assay and in vitro . this interaction provided clues that the fpc proteins may be involved in general genome maintenance mechanisms . the following studies highlighted the roles of the fpc proteins in the dna replication checkpoint , the master signaling pathway responsible for recognizing problems during dna replication at the fork , in order to arrest the cell cycle and allow time for dna repair . to identify novel genes in the s - phase - specific checkpoint pathway , a synthetic lethal screen was performed in budding yeast , isolating mutants that conferred additional sensitivity to a rad9 mutant in response to the dna alkylating agent , methyl methanesulfonate ( mms ) . this synthetic lethal screen was performed based on the observation that cells missing both cell cycle - wide and s - phase - specific pathways have stronger defects in checkpoint activation . importantly , this screen identified tof1 mutations , which rendered cells highly sensitive to mms when combined with a rad9 mutation . tof1 mutants also showed sensitivity to other s - phase stressing agents , including hydroxyurea ( hu ) and uv light ( uv ) , suggesting the role of tof1 in the s - phase - specific checkpoint pathway . indeed , the same study showed that loss of both rad9 and tof1 ( but not the single mutations ) inhibits hu - dependent phosphorylation and activation of rad53 , the budding yeast master checkpoint kinase . similarly , swi1 , an s. pombe fpc subunit , has a role in activation of the dna replication checkpoint . in the absence of swi1 , cells are defective for full activation of the replication checkpoint kinase cds1 , the s. pombe ortholog of rad53 . . swi3 cells also have decreased cds1 kinase activity in response to hu , indicating that both fpc proteins are required for checkpoint activation in response to replication stress . although the dna replication checkpoint pathway is somewhat different in metazoans , the role of fpc in supporting checkpoint signaling is similar to what is known in yeast systems . in metazoans , the atr ( ataxia - telangiectasia mutated and rad3-raleted)-atrip ( atr - interacting protein ) kinase phosphorylates and activates the chk1 kinase to arrest the cell cycle in response to replication stress . in studies using human cells , cellular sensitivity to genotoxic agents ( such as hu , uv , gamma irradiation and camptothecin ) increases in the absence of the fpc . when timeless or tipin protein levels are knocked down using sirna , chk1 phosphorylation ( and thus its activation ) is reduced in response to hu or uv . in xenopus laevis , depletion of tipin from cell extracts also reduces chk1 phosphorylation in response to aphidicolin , a dna replication inhibitor . however , some increases of chk1 phosphorylation occur after loss of timeless in the absence of exogenous genotoxic agents . these results suggest that while fpc is required for full activation of replication stress - induced chk1 activation ; the loss of fpc results in accumulation of unusual dna structures that promote an increase of basal atr - dependent chk1 activity in the absence of exogenously provoked replication stress . these results suggest a role for the fpc in mediating checkpoint signaling in response to replication stress ( fig . 1 ) . it is known that single - stranded dna ( ssdna ) generated at the replication fork recruits atr - atrip to phosphorylate chk1 . it is also understood that claspin , a checkpoint mediator , is required for chk1 phosphorylation . therefore , to efficiently activate checkpoint signaling , it is important to recruit all the required proteins to ssdna at the replication fork . this interaction is important for localization of the fpc to ssdna at the replication fork . in addition , tipin - claspin complexes have been detected by immunoprecipitation , and a tipin mutant unable to bind rpa is no longer able to recruit claspin to rpa , indicating that the tipin also recruits claspin to the replication fork . consistently , claspin localization to the nucleus is severely reduced in the absence of the fpc proteins in human cells , further suggesting that the fpc is required for claspin recruitment . therefore , it appears that the role of tipin in mediating checkpoint activation is to act as a scaffold at the replication fork , recruiting all players at the rpa - ssdna complexes to promote chk1 activation ( fig . 1 ) . model for the roles of the fork protection complex ( fpc ) in checkpoint activation , dna replication and sister chromatid cohesion . the replication fork can pause or stall at a variety of impediments throughout the genome . these impediments may include dna damage , fork barriers , dna secondary structures , proteins bound on dna and even cohesin complexes . rpa is also bound by tipin , which then recruits the checkpoint mediator claspin , resulting in activation of the atr - chk1-dependent checkpoint . timeless interacts with chlr1 dna helicase , which stimulates fen1 activity for okazaki fragment processing . this promotes efficient lagging - strand dna synthesis and minimizes the size of the replication loop at the lagging strand , thereby promoting efficient progression of replication forks through cohesin rings . timeless is also required for chk1 activation in response to replication stress . because timeless depletion causes tipin downregulation , however , once recruited to the replication fork , timeless may have tipin - independent roles , as timeless depletion causes defects in a variety of genome maintenance processes , as discussed later in this review . another important activity of s - phase checkpoints is to inhibit firing of late replication origins in response to replication stress . in budding yeast , rad53-dependent phosphorylation of dbf4 blocks late - origin firing after dna damage . dbf4 interacts with the cdc7 kinase to form the ddk complex , which is required for loading of cdc45 and other replication factors just before an individual replication origin fires . interestingly , the fission yeast fpc functionally interacts with the hsk1-dfp1 complex , the fission yeast ortholog of the ddk complex . loss of functional hsk1 sensitizes cells to s - phase - stressing agents including hu and mms . in addition , hsk1 mutant cells have defects in arresting s - phase progression in response to mms , and this effect is epistatic with fpc mutations . these data suggest that fpc - ddk interaction mediates origin inhibition in response to dna damage , although further investigation is necessary to mechanistically understand the role of fpc in origin inhibition . studies have indicated that replication fork progression is generally compromised in the absence of the fpc . dna combing method , which allows for uniformly stretching dna fibers that are then examined by fluorescent microscopy . using this method , budding yeast tof1 cells were found to contain shorter replication tracks when compared with wild type cells . in addition , tof1 cells take approximately 20 min longer to complete dna replication than wild type cells , suggesting the slower rate of replication in tof1 cells . similarly , dna fiber analysis using human cells demonstrated that timeless depletion also generates shorter tracks of nascent dna synthesis . these results suggest that the fpc proteins promote efficient replisome progression by stabilizing replication fork structures and replisome components during dna replication throughout the genome . however , numerous genomic regions are difficult to replicate , which causes pausing of replication forks . as described in the following section , the fpc has a critical role in stabilizing replication forks and suppressing dna damage at these difficult - to - replicate regions during normal dna replication . therefore , we suggest that fpc depletion causes more frequent dna breakage at difficult - to - replicate regions , resulting in the appearance of shorter replication tracks . indeed , the fpc is required for stable fork pausing at rdna fork block sites in budding yeast ( fig . tof1 and csm3 remain with the replisome at the fork block sites , maintaining the replisome complex . fpc - dependent fork pausing and stabilization is independent of checkpoints , as rad53 ( chk1/chk2 ) and mec1 ( atr ) are dispensable for replisome pausing . these data suggest that continuous fpc association with the replisome during replication fork pausing is required to stabilize the replisome . the data also suggest that the fpc stabilizes the forked dna to prevent the formation of unusual dna structures that induce dna repair pathways for resolution . pausing defects could lead to a temporal increase of replication fork velocity in the presence of fork - blocking agents including hu . indeed , chip - on - chip analysis ( chromatin immunoprecipitation with microarray analysis ) of replication proteins suggests that the replication fork migrates faster in fpc - deficient cells than in wild type cells upon hu treatment . this study showed that cdc45 localization was coupled with replicated regions represented by brdu incorporation in hu - treated wild type budding yeast cells . however , in the absence of tof1 , cdc45 moved 2.5 to 3.0 kb further from replicated regions in response to hu . cdc45 complexes with gins ( go ichi ni san ) and mcm subunits ( mcm2 to 7 ) , which unwinds duplex dna at the replication fork . therefore , the chip - on - chip results indicate that dna unwinding is uncoupled from the site of dna synthesis in tof1 cells . in addition , this uncoupling leads to extensive exposure of ssdna at the replication fork and subsequent activation of dna damage checkpoints . accordingly , a checkpoint mediator rad9 was recruited to replicating chromatin in tof1 cells but not in wild type cells . roles of the fork protection complex ( fpc ) at various fork barriers in the genome . ( a ) the s. pombe mating - type ( mat1 ) locus contains two distinct polar fork block sites . a myb factor , rtf1 cooperates with the fpc to promote polar fork arrest at rts1 . fpc - dependent fork pausing at mps1 is required to generate an imprint to initiate a gene conversion event that leads to mating - type switching . ( b ) the s. cerevisiae rdna locus contains an rfb that is bound by fob1 . fob1 functions together with the fpc to inhibit fork progression in the direction that is opposite to the transcriptional direction of the 35s rrna gene . this polar fork block allows the replication fork to move only in the direction of transcription . ( c ) the s. pombe rdna locus contains three fork block sites that are dependent on the fpc . the myb factor reb1 binds ter2 and ter3 to promote polar fork arrest , while the sap1 protein binds ter1 for fork arrest . fork arrest at rfp4 is not dependent on the fpc and is regulated by transcription . ( d ) metazoan telomeres contain myb factors , trf1 and trf2 , which protect telomeres . timeless interacts with trf1 and trf2 to promote efficient dna replication through telomeres in human cells . in fission yeast , swi1 deletion results in replication fork collapse near the rdna replication pausing site , leading to accumulation of recombination structures , including holliday junctions , as visualized by two - dimensional gel electrophoresis ( 2d - gel ) of chromosomal dna . the formation of holliday junction structures are indicative of replication fork collapse and rearrangement , and these collapsed replication structures correlate with an increase in rad22 dna repair foci in swi1 and swi3 nuclei , even in the absence of exogenous dna - damaging agents . rad22 is the s. pombe ortholog of rad52 recombinase that localizes to sites of dna damage . elevated levels of rad22 foci indicate a higher level of dna damage , indicating that swi1 prevents replication fork collapse . similarly , timeless - depleted mouse cells accumulate ssdna at the fork and dna damage foci containing rad51 and rad52 recombinases . the increase of dna damage foci correlated with elevated levels of sister chromatid exchange , which is an indication of a dna repair process that utilizes sister - chromatids for homologous recombination . taken together , these finding suggest that the fpc has a conserved role in preventing recombinogenic dna damage at the replication fork during genotoxic stress and unperturbed dna replication . replication checkpoint studies have typically used chemical agents to stall replication forks . however , there are a number of chromosome regions that present obstacles for dna replication . these include replication fork blocking sites , dna secondary structures caused by repeat sequences ( such as telomeres ) and dna - binding proteins ( such as the transcription machinery ) . these sites are considered to be difficult to replicate , causing replication fork arrest or even breakage during normal dna replication . the first class includes protein - dna barriers , where the replication fork pauses to control the direction of fork progression and to coordinate replication pausing with other processes on chromatin . the second type of replication stoppage occurs at repetitive dna , where fork stalling occurs as a result of the inherent properties of dna repeats . stalling at these sites is induced by the higher propensity of short repeat sequences to form hairpins and other secondary structures . such dna structures can cause repeat contraction and expansion , resulting in negative consequences , including human neurodegenerative diseases . therefore , cells must possess mechanisms to ensure proper replication fork pausing and the smooth passage of replication forks through difficult - to - replicate regions . one well - characterized fork - pausing event involves mating - type switching in fission yeast ( fig . two genetically programmed fork pausing sites are found near the mating - type ( mat1 ) locus : the mat1 replication pause site 1 ( mps1 ) and the replication termination site 1 ( rts1 ) . a strong polar replication pausing at rts1 prevents one replication fork from migrating into the mat1 locus . this fork pauses at mps1 and generates an imprint , probably a dna strand discontinuity , which initiates a specific recombination event , resulting in mating - type switching ( fig . , the replication fork fails to pause at these sites ; consequently , swi1 and swi3 cells are defective for mating - type switching . interestingly , rts1 is bound by rtf1 , a protein containing the myb dna - binding domain , and rtf1 deletion also causes loss of fork pausing at rts1 ( fig . 2a ) , suggesting that the fpc works together with rtf1 to pause the fork at rts1 . another well - studied replication pausing occurs at rdna arrays and is dependent on the fpc ( fig . budding yeast chromosome xii contains approximately 150 repeats of the rdna unit , each of which contains two transcribed regions : 35s and 5s rrna genes . each repeat also contains a replication origin and a replication fork barrier ( rfb ) upstream and downstream of the 35s rrna gene , respectively . a site - specific dna - binding protein fob1 binds rfb and blocks the fork that moves in the direction opposite to 35s rrna transcription ; in addition , fpc subunits tof1 and csm3 allows for stable replication fork pausing at rfbs by protecting fob1 from rrm3 helicase , which displaces fob1 to promote fork progression . this polar fork block arrangement prevents replication fork progression in the direction opposite to the direction of transcription , preventing head - on collisions of the replisome and the transcription machinery ( fig . a fob1-like protein has not been identified . however , similar fork blocking strategies are used to coordinate the directionality of replication and transcription at rdna repeats ( fig . the s. pombe rdna unit has four polar fork barriers , three of which are dependent on the fpc . the sap1 protein , which recognizes the switch - activating site ( sas1 ) near the mating - type locus , binds the ter1 sequence to cause polar fork arrest in collaboration with the fpc . interestingly , the ter1 sequence is highly similar to the sas1 sequence , indicating that sap1 is a sequence - specific dna - binding protein . two additional fpc - dependent fork barriers within the s. pombe rdna repeat are ter2 and ter3 . reb1 , which is the s. pombe ortholog of the mammalian transcription termination factor-1 ( ttf-1 ) , binds ter2 and ter3 and causes polar fork arrest ( fig . reb1 contains the myb dna - binding domain , and a similar fork block mechanism is reported in mammalian cells ; hela cell extracts depleted of ttf-1 fail to exhibit fork block activity at the sal boxes of the rdna repeat . interestingly , mammalian ttf-1 and fission yeast reb1 also mediate termination of transcription by rna polymerase i ( rna pol i ) at the sal boxes and ter2/3 sites , respectively , although budding yeast reb1 has no apparent role in fork blocking activity . although whether the ttf-1-dependent fork block requires the fpc is unknown , fork arrest at the s. pombe ter2 and ter3 sites is dependent on the fpc proteins ( fig . therefore , cooperation of the fpc and myb factors likely promotes replication fork pausing at specific sites in the genome . other fpc - mediated replication fork pausing events , which are also associated with protein - dna interaction , also occur frequently during dna replication . in s. cerevisiae , replication forks pause at trna and highly transcribed rna polymerase ii ( pol ii ) genes . such pausing events appeared to be dependent on the fpc , because 2d - gel analyses detected pausing events at trna sites only in the presence of tof1 . researchers have suggested that high rates of transcription and the presence of the transcription machinery increase the chances of the replisome encountering an rna transcription fork . analyses of replication through budding yeast trna genes indicate that replication forks pause at these sites for approximately 10 sec , roughly four times longer than replisome transit of a similarly sized region . owing to the abundance of pausing sites , the pfh1 dna helicase is required for the replisome to replicate through highly transcribed rna pol ii and rna pol iii genes . in pfh1 mutants , paused replication forks accumulate at these genes , which would then require the function of the fpc to stabilize them . these results indicate that highly transcribed genomic regions are difficult to replicate , and that the fpc plays a critical role in stabilization of paused replication forks at these genomic regions ( fig . the fpc travels with the replication fork , monitoring and detecting problems during dna replication . the fpc coordinates dna replication at a variety of difficult - to - replicate regions , including cohesin sites , highly transcribed regions , fork block sites , secondary structures and telomeres . in addition to dna - protein complexes , dna structures themselves can cause fork pausing or arrest . it is widely thought that repeat dna sequences form secondary structures that are associated with genomic instability . emerging evidence indicates that fpc proteins prevent genomic instability at these non - programmed sites . however , at these sites , the fpc proteins play a different role in regulating stalled forks . instead of allowing for stable pausing , the fpc appears to counteract replication fork stalling mediated by dna structures , such as hairpins . when inverted alu repeats are inserted into the budding yeast genome , replication fork stalling at the alu repeats nearly doubles in the absence of tof1 . this result is consistent with the notion that the fpc helps to move replication forks forward by minimizing stalling events at dna structure - mediated fork block sites . increases in replication fork stalling is thought to contribute to expansion of trinucleotide repeats , and a number of human diseases are associated with abnormal replication of repeat regions . in human cells , cgg repeat expansion leads to an increased propensity to form secondary structures , which is thought to lead to dna replication mediated breakage , causing fragile x syndrome . in budding yeast tof1 cells , the rate of fork stalling at inserted cgg repeats increases compared with wild type strains . importantly , the lagging - strand folds back to form a loop structure at the fork ( fig . 1 ) . owing to the presence of repeat dna sequences , once the replisome stalls at repeat regions , the replisome might continue dna synthesis by switching the template to the lagging - strand loop , which is located upstream of the stalled site . consistently , when gaa repeats were inserted into the budding yeast genome , the repeats expanded at a rate corresponding to their original length in a manner dependent on dna replication ( the longer the repeats , the faster the expansion ) . similar genomic instability in the absence of the fpc is reported in human cells . in humans , the expansion of a particular ( ctg)n(cag)n repeat tract on chromosome 19 is associated with myotonic dystrophy type 1 ( dm1 ) , a form of muscular dystrophy . in a tissue culture model , fpc proteins appear to prevent or reduce replication fork stalling when the replication fork is blocked by dna secondary structure . fork stalling could result in elevated levels of ssdna exposed at the replication fork upon fpc dysfunction . existence of longer ssdna may further increase the risk of secondary structure formation and fork stalling . currently , whether the fpc promotes replication of repeat regions by interacting with other proteins is unknown . however , the mechanism of the fpc - dependent fork regulation at repeat regions appeared to be different from that at protein - dna complex - mediated fork barriers . an interesting example of fpc - dependent fork protection against site - specific dna damage is the telomere . at the telomere , the replication fork encounters protein - dna complexes as well as repetitive dna regions . for that reason , the replisome must deal with both protein - dna complex - mediated fork barriers and dna structure - based impediments . indeed , telomeres are difficult to replicate , and replication forks stall at telomere repeats . interestingly , telomeres also recruit proteins that contain the myb dna - binding domain . in fission yeast , a myb factor , taz1 , binds specifically to telomere repeats and promotes efficient replication through telomeres . in metazoans , taz1 orthologs , trf1 and trf2 , also contain the myb dna - binding domain and bind telomere repeat dna sequences ( fig . trf1 prevents telomere damage foci generated during s - phase , and trf1 deletion lowers the efficiency of dna replication , as visualized by dna combing . conversely , ohki and ishikawa showed that overexpression of trf1 or trf2 increases fork stalling specifically at telomere repeats in vivo and in vitro . although these results contradict each other , they suggest that trf1 and trf2 prevent replication fork collapse at telomeres , and that maintaining the quantity of these proteins at telomeres is required for efficient telomere replication . considering that taz1 and trf1/2 are myb - related proteins , molecular mechanisms required for replication of telomeres may be similar to those required for replication through other protein - dna barriers , including the fission yeast mat1 locus and rdna barriers in fission yeast and mammalian cells ( fig . myb - related proteins associate tightly with barrier regions , and the fpc is required for stable fork pausing and prevention of fork collapse at the barrier regions . compelling evidence suggests that the fpc is required for efficient replication of telomeres . in yeast , loss of timeless orthologs leads to contrasting telomere length phenotypes ; budding yeast tof1 cells contain extended telomeres while swi1 cells in fission yeast undergo telomere shortening . the differences may be attributed to the fact that fission yeasts express a myb - dna binding domain - containing protein , taz1 , whereas s. cerevisiae has no apparent taz1 ortholog . indeed , in human cells , in which taz1 orthologs ( trf1/2 ) exist , long - term knockdown of timeless causes telomere shortening . these findings suggest that the fpc regulates replication of telomeric dna sequences that are bound by myb factors . consistently , immunoprecipitation studies in human cells demonstrated that timeless interacts with trf1 and trf2 . studies have also shown that timeless - depleted cells fail to maintain trf1-mediated replication fork arrest . . however , timeless - trf1 interaction may not be the only fpc role in telomere replication . in the same study , in vitro replication assays also demonstrated that the loss of timeless reduces replication efficiency on naked telomeric dna , suggesting that the fpc may be involved in replisome stability through repeats independent of interaction with trf1 . therefore , timeless may have two roles during telomere replication : ( 1 ) stabilization of the replisome through dna structure - based impediments , and ( 2 ) specific interaction with trf1 to promote proper fork pausing where trf1 binds telomeres . therefore , telomeres may be unique loci where two distinct fpc - dependent stabilization mechanisms are required . seemingly disparate from its role in checkpoint activation , the fpc plays a role in the establishment of scc during dna replication . the establishment of scc requires dna replication in s - phase . during dna replication , newly copied chromosomes are held in close association by a functional complex of proteins known as cohesins . the cohesin complex ensures that sister chromosomes are paired until they can be separated equally during mitosis . smc1 and smc3 ( structural maintenance of chromosomes 1 and 3 ) are conserved cohesin proteins with long flexible linker domains thought to encircle one or both copies of dna . they are held in a ring - like complex by the non - smc subunits , scc1 and scc3 proteins ( fig . 1 ) . although the mechanism by which the cohesins promote scc is unclear , stable loading of cohesins onto dna evidently requires acetylation of smc3 by the conserved eco1/ctf7 acetyltransferase ( known as esco1/2 in humans ) . proper scc is important for cellular health and viability , as mutations in cohesion pathways cause cohesinopathies such as cornelia de lange and roberts syndromes characterized by multisystem developmental defects . earlier studies implicated the role of fpc proteins in chromosome cohesion and segregation . in an effort to understand the mechanism of meiotic chromosome segregation this screen identified csm3 , the budding yeast tipin ortholog , as a factor required for proper chromosome segregation in meiosis . csm3 deletion caused mild chromosome missegregation and reduced spore viability , suggesting a possible role of csm3 in chromatid pairing . in caenorhabditis elegans , meyer and colleagues used a biochemical approach to understand the cohesion mechanism . they performed immunoprecipitation of smc-1 ( an smc1 ortholog ) using c. elegans embryonic extracts . tim-1 depletion or mutation was found to cause defects in meiotic chromosome cohesion and segregation . this study also reported that tim-1 is required for the loading or stability of non - smc subunits of the cohesin complex . several replication factors including ctf4 ( dna polymerase -binding factor ) and rfc ( alternative replication factor c - like complex ) are involved in efficient scc in budding yeast . to identify additional cohesion factors , synthetic lethal screens were performed using ctf4 and ctf8 ( a subunit of rfc ) mutations . these studies revealed that both tof1 and csm3 were synthetically lethal with ctf4 or ctf8. they also found that tof1 and csm3 were required for scc and that tof1 co - purifies with csm3 from budding yeast cell extracts . in fission yeast , a synthetic lethal screen was performed using swi1 mutation to identify additional factors involved in replication fork maintenance . consistently , swi1 and swi3 cells displayed premature centromere dissociation during metaphase . considering that fpc subunits ( tof1/swi1csm3/swi3 ) are replication fork constituents , these genetic data confirmed the role of replication fork processing in the establishment of scc . furthermore , because the fpc is involved in stabilization of replication forks , it is possible that fpc proteins coordinate dna synthesis with scc established at the replication fork . using molecular biology approaches in vertebrate model systems , xenopus egg extracts depleted of timeless or tipin fail to pair sister chromatids in a manner similar to smc3 depleted extracts , indicating that loss of xenopus timeless - tipin induces an scc defect . interestingly , in xenopus egg extracts , timeless interacts with and1 , a ctf4 homolog and supports stable chromatin binding of dna polymerase and dna replication under minimal licensing conditions . in human epithelial cells , timeless or tipin depletion via rnai also causes a substantial increase in scc defects . a recent study using human fibroblasts found that scc defects caused by timeless depletion are 10 times greater than those caused by tipin deletion , suggesting that timeless but not tipin has a specific role in scc establishment . mechanistically , cohesin subunits , including smc1 and smc3 , coimmunoprecipitate with timeless or tipin in human cell extracts . timeless - cohesins interaction was maintained after dnase treatment , whereas tipin - cohesins interaction was disrupted . this is consistent with the notion that timeless is closely associated with the cohesin complex ( fig . 1 ) . moreover , timeless depletion led to dissociation of cohesin subunits from chromatin , suggesting that timeless is required for stable association of cohesins with chromatin . additional evidence supports the notion that the fpc facilitates scc through protein - protein interactions . the aforementioned synthetic lethal screens revealed that deletion of the chl1 dna helicase is synthetically lethal with ctf8 deletion . chl1 ( chlr1 in humans ) is a deah / dead box containing dna helicase belonging to the fancj - like dna helicase family . chl1/chlr1 downregulation causes scc defects , and a mutation in the human gene causes a cohesinopathy - related disease termed warsaw breakage syndrome . compelling evidence suggests that the fpc cooperates with chl1/chlr1 to promote scc . in human cells , timeless coimmunoprecipitates with chlr1 ; timeless is involved in loading of chlr1 onto chromatin ; and chlr1 overexpression partially rescues scc defects due to timeless or tipin depletion . in fission yeast , chl1 overexpression rescues the hu and mms sensitivity of swi1 , while genetic studies in both budding and fission yeast suggest that swi1 and chl1 function in the same pathway . fen1 is a flap endonuclease required for okazaki fragment processing during lagging - strand dna synthesis . importantly , chlr1 enhances fen1 activity in vitro , suggesting that lagging - strand processing may affect the efficiency of cohesion . these results support the idea that chlr1 promotes scc by facilitating efficient lagging - strand processing . considering that the level of chromatin - bound chlr1 is reduced in timeless depleted cells , it is possible that timeless effectively recruits chlr1 to promote lagging - strand synthesis ( fig . 1 ) . consistently , timeless downregulation leads to ssdna accumulation in human cells , and yeast genetic studies have suggested the role of the timeless ortholog swi1 in coordination of leading- and lagging - strand dna synthesis to prevent accumulation of ssdna . delayed lagging - strand synthesis causes a long stretch of ssdna , generating a large loop structure at the replication fork , which may hamper replication fork progression through the cohesin ring ( fig therefore , efficient lagging - strand processing may have a critical role in scc establishment . interestingly , a recent study showed that fen1 interacts with chl1 and the cohesin aceyltransferase eco1/ctf7 in budding yeast , suggesting that scc may be coupled with lagging - strand synthesis . this pausing would generate ssdna at the lagging strand of the fork , leading to recruitment of rpa . tipin then binds rpa , tethering timeless to the lagging strand at the replication fork ( fig . 1 ) . we suggest that a division of labor of timeless and tipin occurs once the timeless - tipin complex is localized to the replication fork . timeless modulates chlr1 and fen1 functions to facilitate lagging - strand synthesis , while timeless also holds cohesin subunits on chromatin via protein - protein interaction , which , in turn , coordinates lagging - strand synthesis with scc ( fig . 1 ) . it is also possible that timeless - dependent stabilization of cohesin subunits creates a favorable environment for smc3 acetylation by eco1/ctf7 , although further studies are needed to address this question . nevertheless , current evidence suggests a critical link between lagging - strand synthesis and cohesion processes . the pleiotropic phenotypes that are caused by fpc deficiency are all associated with the functions of the fpc at the replication fork . the fpc interacts with a number of proteins , including rpa , claspin , trf1/2 , fob1 , chlr1 , cohesin and core replisome components . these findings suggest that the fpc acts as a platform for multiple proteins to regulate a variety of transactions at the replication fork . during dna replication , the replisome encounters numerous impediments , where dna resident proteins are localized ( fig . 3 ) . many such dna resident proteins may contain the myb dna - binding domain and regulate stable pausing of the replisome to coordinate dna replication with other processes that take place on chromatin . however , the replisome must contain factors that interact with the myb - containing proteins and recognize the problems at various challenging sites throughout the genome . the fpc appears to be the perfect candidate for this job . in this model , the fpc moves with the fork and detects and reconciles problems during dna replication throughout the genome , stabilizing the replisome while also facilitating proper checkpoint signaling if necessary ( figs . 1 and 3 ) . in the future , it will be interesting to see how genome - wide studies identify dna damage sites in the absence of the fpc . we anticipate that many of dna damage sites contain resident proteins that interact with the fpc . from the findings described above , it is also anticipated that many of these resident proteins will contain myb dna - binding domains . future molecular and proteomic studies may identify various interactions between the fpc and other dna - binding proteins . these interactions can be interrupted genetically or pharmacologically , and loci - specific phenotypes can then be assessed . however , because a vast number of genomic sites require the fpc , we deduce that the fpc controls dna replication globally .

the eukaryotic cell replicates its chromosomal dna with almost absolute fidelity in the course of every cell cycle . this accomplishment is remarkable considering that the conditions for dna replication are rarely ideal . the replication machinery encounters a variety of obstacles on the chromosome , including damaged template dna . in addition , a number of chromosome regions are considered to be difficult to replicate owing to dna secondary structures and dna binding proteins required for various transactions on the chromosome . under these conditions , replication forks stall or break , posing grave threats to genomic integrity . how does the cell combat such stressful conditions during dna replication ? the replication fork protection complex ( fpc ) may help answer this question . recent studies have demonstrated that the fpc is required for the smooth passage of replication forks at difficult - to - replicate genomic regions and plays a critical role in coordinating multiple genome maintenance processes at the replication fork .

Introduction The FPC Supports Checkpoint Activities Roles of the FPC in Replisome Progression Roles of the FPC at Replication Fork Barriers The FPC Stabilizes Replication Forks at DNA Repeats Telomere Replication and the FPC Coordinating Cohesion Establishment with Replication Concluding Remarks and Future Directions

during s phase , the dna replication machinery known as the replisome competes for access to template dna with numerous dna - binding proteins involved in various nuclear processes , including transcription , chromatin remodeling , sister chromatid cohesion ( scc ) and dna repair . proteins involved in these nuclear transactions can block replisome progression , leading to replication fork arrest or even breakage , which , in turn , causes genomic instability , including mutations and chromosome rearrangements . the fpc is comprised of two proteins : timeless ( also called tim ) and tipin in metazoans , swi1 and swi3 in the fission yeast schizosaccharomyces pombe , and tof1 and csm3 in the budding yeast saccharomyces cerevisiae ( table 1 ) . the fpc interacts with chromatin as well as many replisome components , including mini - chromosome maintenance ( mcm ) dna helicase subunits , dna polymerases and , replication protein a ( rpa ) and other ancillary factors . in both yeast and human cells , the fpc associates with replication origins at the onset of s phase and travels with the replisome during dna replication . compelling evidence suggests the roles of the fpc in normal dna replication , replication at difficult - to - replicate genomic regions , replication fork pausing / stalling , the dna replication checkpoint and scc . in this review , we summarize the current understanding of the roles of the fpc in the diverse pathways coordinated at the replication fork . in addition , we discuss what functions of the fpc make it central for a variety of processes at the replication fork in order to promote genome stability . this interaction provided clues that the fpc proteins may be involved in general genome maintenance mechanisms . the following studies highlighted the roles of the fpc proteins in the dna replication checkpoint , the master signaling pathway responsible for recognizing problems during dna replication at the fork , in order to arrest the cell cycle and allow time for dna repair . to identify novel genes in the s - phase - specific checkpoint pathway , a synthetic lethal screen was performed in budding yeast , isolating mutants that conferred additional sensitivity to a rad9 mutant in response to the dna alkylating agent , methyl methanesulfonate ( mms ) . this synthetic lethal screen was performed based on the observation that cells missing both cell cycle - wide and s - phase - specific pathways have stronger defects in checkpoint activation . tof1 mutants also showed sensitivity to other s - phase stressing agents , including hydroxyurea ( hu ) and uv light ( uv ) , suggesting the role of tof1 in the s - phase - specific checkpoint pathway . similarly , swi1 , an s. pombe fpc subunit , has a role in activation of the dna replication checkpoint . in the absence of swi1 , cells are defective for full activation of the replication checkpoint kinase cds1 , the s. pombe ortholog of rad53 . in metazoans , the atr ( ataxia - telangiectasia mutated and rad3-raleted)-atrip ( atr - interacting protein ) kinase phosphorylates and activates the chk1 kinase to arrest the cell cycle in response to replication stress . in studies using human cells , cellular sensitivity to genotoxic agents ( such as hu , uv , gamma irradiation and camptothecin ) increases in the absence of the fpc . in xenopus laevis , depletion of tipin from cell extracts also reduces chk1 phosphorylation in response to aphidicolin , a dna replication inhibitor . these results suggest that while fpc is required for full activation of replication stress - induced chk1 activation ; the loss of fpc results in accumulation of unusual dna structures that promote an increase of basal atr - dependent chk1 activity in the absence of exogenously provoked replication stress . these results suggest a role for the fpc in mediating checkpoint signaling in response to replication stress ( fig . it is known that single - stranded dna ( ssdna ) generated at the replication fork recruits atr - atrip to phosphorylate chk1 . it is also understood that claspin , a checkpoint mediator , is required for chk1 phosphorylation . therefore , to efficiently activate checkpoint signaling , it is important to recruit all the required proteins to ssdna at the replication fork . this interaction is important for localization of the fpc to ssdna at the replication fork . in addition , tipin - claspin complexes have been detected by immunoprecipitation , and a tipin mutant unable to bind rpa is no longer able to recruit claspin to rpa , indicating that the tipin also recruits claspin to the replication fork . consistently , claspin localization to the nucleus is severely reduced in the absence of the fpc proteins in human cells , further suggesting that the fpc is required for claspin recruitment . therefore , it appears that the role of tipin in mediating checkpoint activation is to act as a scaffold at the replication fork , recruiting all players at the rpa - ssdna complexes to promote chk1 activation ( fig . model for the roles of the fork protection complex ( fpc ) in checkpoint activation , dna replication and sister chromatid cohesion . the replication fork can pause or stall at a variety of impediments throughout the genome . this promotes efficient lagging - strand dna synthesis and minimizes the size of the replication loop at the lagging strand , thereby promoting efficient progression of replication forks through cohesin rings . because timeless depletion causes tipin downregulation , however , once recruited to the replication fork , timeless may have tipin - independent roles , as timeless depletion causes defects in a variety of genome maintenance processes , as discussed later in this review . dbf4 interacts with the cdc7 kinase to form the ddk complex , which is required for loading of cdc45 and other replication factors just before an individual replication origin fires . in addition , hsk1 mutant cells have defects in arresting s - phase progression in response to mms , and this effect is epistatic with fpc mutations . studies have indicated that replication fork progression is generally compromised in the absence of the fpc . in addition , tof1 cells take approximately 20 min longer to complete dna replication than wild type cells , suggesting the slower rate of replication in tof1 cells . these results suggest that the fpc proteins promote efficient replisome progression by stabilizing replication fork structures and replisome components during dna replication throughout the genome . however , numerous genomic regions are difficult to replicate , which causes pausing of replication forks . as described in the following section , the fpc has a critical role in stabilizing replication forks and suppressing dna damage at these difficult - to - replicate regions during normal dna replication . therefore , we suggest that fpc depletion causes more frequent dna breakage at difficult - to - replicate regions , resulting in the appearance of shorter replication tracks . indeed , the fpc is required for stable fork pausing at rdna fork block sites in budding yeast ( fig . these data suggest that continuous fpc association with the replisome during replication fork pausing is required to stabilize the replisome . the data also suggest that the fpc stabilizes the forked dna to prevent the formation of unusual dna structures that induce dna repair pathways for resolution . pausing defects could lead to a temporal increase of replication fork velocity in the presence of fork - blocking agents including hu . indeed , chip - on - chip analysis ( chromatin immunoprecipitation with microarray analysis ) of replication proteins suggests that the replication fork migrates faster in fpc - deficient cells than in wild type cells upon hu treatment . cdc45 complexes with gins ( go ichi ni san ) and mcm subunits ( mcm2 to 7 ) , which unwinds duplex dna at the replication fork . in addition , this uncoupling leads to extensive exposure of ssdna at the replication fork and subsequent activation of dna damage checkpoints . roles of the fork protection complex ( fpc ) at various fork barriers in the genome . fob1 functions together with the fpc to inhibit fork progression in the direction that is opposite to the transcriptional direction of the 35s rrna gene . this polar fork block allows the replication fork to move only in the direction of transcription . ( c ) the s. pombe rdna locus contains three fork block sites that are dependent on the fpc . fork arrest at rfp4 is not dependent on the fpc and is regulated by transcription . in fission yeast , swi1 deletion results in replication fork collapse near the rdna replication pausing site , leading to accumulation of recombination structures , including holliday junctions , as visualized by two - dimensional gel electrophoresis ( 2d - gel ) of chromosomal dna . the formation of holliday junction structures are indicative of replication fork collapse and rearrangement , and these collapsed replication structures correlate with an increase in rad22 dna repair foci in swi1 and swi3 nuclei , even in the absence of exogenous dna - damaging agents . similarly , timeless - depleted mouse cells accumulate ssdna at the fork and dna damage foci containing rad51 and rad52 recombinases . taken together , these finding suggest that the fpc has a conserved role in preventing recombinogenic dna damage at the replication fork during genotoxic stress and unperturbed dna replication . replication checkpoint studies have typically used chemical agents to stall replication forks . however , there are a number of chromosome regions that present obstacles for dna replication . these include replication fork blocking sites , dna secondary structures caused by repeat sequences ( such as telomeres ) and dna - binding proteins ( such as the transcription machinery ) . these sites are considered to be difficult to replicate , causing replication fork arrest or even breakage during normal dna replication . the first class includes protein - dna barriers , where the replication fork pauses to control the direction of fork progression and to coordinate replication pausing with other processes on chromatin . therefore , cells must possess mechanisms to ensure proper replication fork pausing and the smooth passage of replication forks through difficult - to - replicate regions . , the replication fork fails to pause at these sites ; consequently , swi1 and swi3 cells are defective for mating - type switching . 2a ) , suggesting that the fpc works together with rtf1 to pause the fork at rts1 . another well - studied replication pausing occurs at rdna arrays and is dependent on the fpc ( fig . a site - specific dna - binding protein fob1 binds rfb and blocks the fork that moves in the direction opposite to 35s rrna transcription ; in addition , fpc subunits tof1 and csm3 allows for stable replication fork pausing at rfbs by protecting fob1 from rrm3 helicase , which displaces fob1 to promote fork progression . this polar fork block arrangement prevents replication fork progression in the direction opposite to the direction of transcription , preventing head - on collisions of the replisome and the transcription machinery ( fig . the s. pombe rdna unit has four polar fork barriers , three of which are dependent on the fpc . interestingly , mammalian ttf-1 and fission yeast reb1 also mediate termination of transcription by rna polymerase i ( rna pol i ) at the sal boxes and ter2/3 sites , respectively , although budding yeast reb1 has no apparent role in fork blocking activity . although whether the ttf-1-dependent fork block requires the fpc is unknown , fork arrest at the s. pombe ter2 and ter3 sites is dependent on the fpc proteins ( fig . therefore , cooperation of the fpc and myb factors likely promotes replication fork pausing at specific sites in the genome . other fpc - mediated replication fork pausing events , which are also associated with protein - dna interaction , also occur frequently during dna replication . in s. cerevisiae , replication forks pause at trna and highly transcribed rna polymerase ii ( pol ii ) genes . such pausing events appeared to be dependent on the fpc , because 2d - gel analyses detected pausing events at trna sites only in the presence of tof1 . analyses of replication through budding yeast trna genes indicate that replication forks pause at these sites for approximately 10 sec , roughly four times longer than replisome transit of a similarly sized region . owing to the abundance of pausing sites , the pfh1 dna helicase is required for the replisome to replicate through highly transcribed rna pol ii and rna pol iii genes . in pfh1 mutants , paused replication forks accumulate at these genes , which would then require the function of the fpc to stabilize them . these results indicate that highly transcribed genomic regions are difficult to replicate , and that the fpc plays a critical role in stabilization of paused replication forks at these genomic regions ( fig . the fpc travels with the replication fork , monitoring and detecting problems during dna replication . the fpc coordinates dna replication at a variety of difficult - to - replicate regions , including cohesin sites , highly transcribed regions , fork block sites , secondary structures and telomeres . in addition to dna - protein complexes , dna structures themselves can cause fork pausing or arrest . however , at these sites , the fpc proteins play a different role in regulating stalled forks . instead of allowing for stable pausing , the fpc appears to counteract replication fork stalling mediated by dna structures , such as hairpins . when inverted alu repeats are inserted into the budding yeast genome , replication fork stalling at the alu repeats nearly doubles in the absence of tof1 . this result is consistent with the notion that the fpc helps to move replication forks forward by minimizing stalling events at dna structure - mediated fork block sites . increases in replication fork stalling is thought to contribute to expansion of trinucleotide repeats , and a number of human diseases are associated with abnormal replication of repeat regions . in human cells , cgg repeat expansion leads to an increased propensity to form secondary structures , which is thought to lead to dna replication mediated breakage , causing fragile x syndrome . similar genomic instability in the absence of the fpc is reported in human cells . in a tissue culture model , fpc proteins appear to prevent or reduce replication fork stalling when the replication fork is blocked by dna secondary structure . fork stalling could result in elevated levels of ssdna exposed at the replication fork upon fpc dysfunction . at the telomere , the replication fork encounters protein - dna complexes as well as repetitive dna regions . indeed , telomeres are difficult to replicate , and replication forks stall at telomere repeats . although these results contradict each other , they suggest that trf1 and trf2 prevent replication fork collapse at telomeres , and that maintaining the quantity of these proteins at telomeres is required for efficient telomere replication . considering that taz1 and trf1/2 are myb - related proteins , molecular mechanisms required for replication of telomeres may be similar to those required for replication through other protein - dna barriers , including the fission yeast mat1 locus and rdna barriers in fission yeast and mammalian cells ( fig . myb - related proteins associate tightly with barrier regions , and the fpc is required for stable fork pausing and prevention of fork collapse at the barrier regions . compelling evidence suggests that the fpc is required for efficient replication of telomeres . these findings suggest that the fpc regulates replication of telomeric dna sequences that are bound by myb factors . studies have also shown that timeless - depleted cells fail to maintain trf1-mediated replication fork arrest . in the same study , in vitro replication assays also demonstrated that the loss of timeless reduces replication efficiency on naked telomeric dna , suggesting that the fpc may be involved in replisome stability through repeats independent of interaction with trf1 . seemingly disparate from its role in checkpoint activation , the fpc plays a role in the establishment of scc during dna replication . during dna replication , newly copied chromosomes are held in close association by a functional complex of proteins known as cohesins . this study also reported that tim-1 is required for the loading or stability of non - smc subunits of the cohesin complex . considering that fpc subunits ( tof1/swi1csm3/swi3 ) are replication fork constituents , these genetic data confirmed the role of replication fork processing in the establishment of scc . furthermore , because the fpc is involved in stabilization of replication forks , it is possible that fpc proteins coordinate dna synthesis with scc established at the replication fork . interestingly , in xenopus egg extracts , timeless interacts with and1 , a ctf4 homolog and supports stable chromatin binding of dna polymerase and dna replication under minimal licensing conditions . moreover , timeless depletion led to dissociation of cohesin subunits from chromatin , suggesting that timeless is required for stable association of cohesins with chromatin . additional evidence supports the notion that the fpc facilitates scc through protein - protein interactions . compelling evidence suggests that the fpc cooperates with chl1/chlr1 to promote scc . considering that the level of chromatin - bound chlr1 is reduced in timeless depleted cells , it is possible that timeless effectively recruits chlr1 to promote lagging - strand synthesis ( fig . delayed lagging - strand synthesis causes a long stretch of ssdna , generating a large loop structure at the replication fork , which may hamper replication fork progression through the cohesin ring ( fig therefore , efficient lagging - strand processing may have a critical role in scc establishment . tipin then binds rpa , tethering timeless to the lagging strand at the replication fork ( fig . we suggest that a division of labor of timeless and tipin occurs once the timeless - tipin complex is localized to the replication fork . the pleiotropic phenotypes that are caused by fpc deficiency are all associated with the functions of the fpc at the replication fork . the fpc interacts with a number of proteins , including rpa , claspin , trf1/2 , fob1 , chlr1 , cohesin and core replisome components . these findings suggest that the fpc acts as a platform for multiple proteins to regulate a variety of transactions at the replication fork . during dna replication , the replisome encounters numerous impediments , where dna resident proteins are localized ( fig . the fpc appears to be the perfect candidate for this job . in this model , the fpc moves with the fork and detects and reconciles problems during dna replication throughout the genome , stabilizing the replisome while also facilitating proper checkpoint signaling if necessary ( figs . in the future , it will be interesting to see how genome - wide studies identify dna damage sites in the absence of the fpc . future molecular and proteomic studies may identify various interactions between the fpc and other dna - binding proteins . however , because a vast number of genomic sites require the fpc , we deduce that the fpc controls dna replication globally .

first rank symptoms ( frss ) a group of intriguing symptoms characterized by a striking breach of self - versus nonself boundaries , first described by schneider , have had a critical influence on the diagnosis of schizophrenia over the past many decades . this symptom cluster comprises of audible thoughts , voices arguing , voices commenting on one 's action , influence playing on the body ( somatic passivity ) , thought withdrawal , thought insertion , broadcasting of thoughts , made feelings , made impulses , made volitional acts , and delusional perception . factor analytical studies have supported a two - dimensional structure of frs that is stable over time : symptoms characterized by ego - boundary disturbances ( ebds ; delusions of alien control , thought alienation phenomena and somatic passivity ) and those characterized by different auditory hallucinations . although frs lack diagnostic specificity , these symptoms provide a well - defined template to study the neurobiological basis of psychotic symptoms from a phenomenological perspective . the first symptom dimension characterized by ebds is of particular interest as it represents an alienated feature of the sense of one 's own mental or physical activity . it has been hypothesized that a dysfunctional mirror neuron network ( inferior frontal gyrus , ventral premotor cortex and inferior parietal lobule ) leads to functional dissociations between the action imagination , and action enactment , thus resulting in misattribution of agency , which is seen in symptoms of ebd . mirror neurons are specialized neurons that are active not only when performing an action oneself , but also while observing someone else perform that action . interestingly , there is emerging evidence for dysfunctional mirror neuron activity ( mna ) in patients with schizophrenia . transcranial magnetic stimulation ( tms ) is a unique , noninvasive , tool having both investigative and therapeutic applications . it has been extensively used to derive a putative measure of mna ( henceforth referred to as mna ) in humans by detecting changes in the motor evoked potentials ( mep ) of hand muscles recorded using electromyography . enhancement in the mep amplitude during action observation relative to rest states gives a measure of putative mna . when compared to functional neuroimaging , tms has poorer spatial resolution , but better temporal resolution to measure mna . in this study , we aim to compare putative mna , measured using tms between schizophrenia patients with and without symptoms of ebds . a total of 50 right - handed schizophrenia patients ( 18 with frs and 32 without frs ) from the inpatient and outpatient services of the national institute of mental health and neurosciences , bengaluru who were recruited as part of a larger study assessing mna in schizophrenia were recruited for this analysis . they were diagnosed independently by two qualified psychiatrists according to diagnostic and statistical manual of mental disorders , fourth edition criteria , and confirmed using the mini - international neuropsychiatric interview ( mini ) . symptoms were assessed using positive and negative syndrome scale ( panss ) . presence or absence of ebd symptoms ( though withdrawal , broadcast and insertion , made volition , impulse and affect ; and somatic passivity ) was determined initially during interviews with panss and mini ; which was then confirmed by a comprehensive clinical interview and mental status examination , based on the definitions given by mellor . patients with substance dependence in the previous 6 months ( except nicotine ) , presence of co - morbid neurological or medical disorder , clinically diagnosable or self - reported visual or auditory impairment , current pregnant or postpartum state and a score of 19 on the hindi mental status examination were excluded from the study . all subjects were assessed with ( a ) the edinburgh inventory for handedness and ( b ) tms adult safety screen to screen for their potential to develop complications . the institute 's ethics committee approved the study and all subjects provided written informed consent . subjects underwent an experiment to assess motor cortical excitability during goal - directed action observation , relative to rest states , to give a putative measure of mna . they were seated comfortably in a chair , in a silent room , 50 cm from the presentation monitor , with their elbows flexed at 90 and hands rested on the armrest of the chair in a prone position . single pulse tms was applied using a 70-mm figure - of - eight coil ( magpro r30 with magoption ; magventure , farum , denmark ) positioned tangentially over the hand area of the left motor cortex , with the handle pointing posterolaterally at a 45 to the sagittal plane . for each subject , the optimal coil position was determined based on standard methods described in previous studies for localizing the scalp area to stimulate the right first dorsal interosseous ( fdi ) muscle . this site was marked with a skin marker pen to ensure uniformity of coil positioning throughout the experiment . magnetic pulses activate cortical pyramidal neurons , leading to corticospinal output that can be measured peripherally as an mep using electromyography . initially , all participants underwent a calibration session during which their motor thresholds were established . resting motor threshold ( rmt ) was defined as the minimum stimulation intensity ( measured in percentage of maximum machine output ) required , to evoke a > 50 v mep in the resting , right fdi muscle in at least 6 out of 10 consecutive trials , measured using electromyography . motor threshold of 1 mv ( mt1 ) was defined as the minimum stimulation intensity , evoking 1 mv peak - to - peak amplitude in the resting , right fdi muscle in at least 6 out of 10 successive recordings . both rmt and mt1 were calculated using progressive reduction of the stimulator intensity from supra - threshold levels in 1% decrements as described in previous studies . next , participants underwent the experiment session . four stimulus paradigms ( two single - pulse and two paired - pulse paradigms ) were used to study cortical excitability while the participants watched three different action related visual displays ( see below ) . 120% of rmt : meps obtained with stimulus intensity equal to 120% of rmt were recorded . this stimulus intensity has been the most commonly implemented in studying putative mirror mechanisms using tms.mt1 : meps obtained with stimulus intensity equal to mt1 were recorded . 120% of rmt : meps obtained with stimulus intensity equal to 120% of rmt were recorded . this stimulus intensity has been the most commonly implemented in studying putative mirror mechanisms using tms . both 120% rmt and mt1 are a measure of membrane excitability of pyramidal neurons , being influenced by voltage - gated sodium channels . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . the sub - threshold conditioning stimulus excites only the cortical interneurons and , therefore , inhibits the mep response to the test stimulus ( conditioned mep ) by 50%-90% . gabaa receptor agonists potentiate sici , thus suggesting that sici may be mediated by gabaa receptor - mediated neurotransmission.long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . the supra - threshold conditioning stimulus activates gabab receptor - mediated inhibitory postsynaptic potentials , thus inhibiting the mep response to the test stimulus ( conditioned mep ) . thus , lici reflects cortical inhibition mediated through gabab receptor - mediated neurotransmission , based on findings that baclofen , a specific gabab receptor agonist enhances lici . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . the sub - threshold conditioning stimulus excites only the cortical interneurons and , therefore , inhibits the mep response to the test stimulus ( conditioned mep ) by 50%-90% . gabaa receptor agonists potentiate sici , thus suggesting that sici may be mediated by gabaa receptor - mediated neurotransmission . long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . the supra - threshold conditioning stimulus activates gabab receptor - mediated inhibitory postsynaptic potentials , thus inhibiting the mep response to the test stimulus ( conditioned mep ) . thus , lici reflects cortical inhibition mediated through gabab receptor - mediated neurotransmission , based on findings that baclofen , a specific gabab receptor agonist enhances lici . cortical inhibition was expressed as a percentage of the ratio between the conditioned meps and the nonconditioned meps with stimulus intensity of mt1 ; that is , ( conditioned mep / nonconditioned mep ) 100 . ten mep recordings using each of these four stimulus paradigms ( total of 40 recordings ) were elicited in random sequence with 5-s intervals , while the subjects observed each of the following : actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . this action requires contraction of the fdi to abduct the index finger.virtual observation : the subjects observed a video showing the above action [ figure 1a].resting state : the subjects observed a still image of a hand and a lock displayed on the monitor [ figure 1b ] . actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . virtual observation : the subjects observed a video showing the above action [ figure 1a ] . resting state : the subjects observed a still image of a hand and a lock displayed on the monitor [ figure 1b ] . ( a ) screen - shot and ( b ) photograph of the video ( depicting a goal - directed action using the right first dorsal interosseous muscle ) and static images of a hand / lock and key , that were used in the experiment to assess putative mirror neuron activity during the virtual observation and rest states respectively the sequence of displaying these experimental states to each subject was randomized . the percent change of the mep or ci from resting to action observation states ( average of virtual and actual observation ) formed a measure of mna . it was calculated using the following formula , where cortical reactivity refers to mep ( in mv ) for single - pulse paradigms and ci ( % ) in paired - pulse paradigms : data acquisition and analysis were done using signal-4 software , ( cambridge electronic devices , cambridge , uk ) . univariate statistics ( independent samples t - test and chi - square tests ) were used to compare clinical and demographic parameters across the two groups ( patients with and without frs ) . all statistical tests were two - tailed , and significance was set at an error p 0.05 . subjects underwent an experiment to assess motor cortical excitability during goal - directed action observation , relative to rest states , to give a putative measure of mna . they were seated comfortably in a chair , in a silent room , 50 cm from the presentation monitor , with their elbows flexed at 90 and hands rested on the armrest of the chair in a prone position . single pulse tms was applied using a 70-mm figure - of - eight coil ( magpro r30 with magoption ; magventure , farum , denmark ) positioned tangentially over the hand area of the left motor cortex , with the handle pointing posterolaterally at a 45 to the sagittal plane . for each subject , the optimal coil position was determined based on standard methods described in previous studies for localizing the scalp area to stimulate the right first dorsal interosseous ( fdi ) muscle . this site was marked with a skin marker pen to ensure uniformity of coil positioning throughout the experiment . magnetic pulses activate cortical pyramidal neurons , leading to corticospinal output that can be measured peripherally as an mep using electromyography . initially , all participants underwent a calibration session during which their motor thresholds were established . resting motor threshold ( rmt ) was defined as the minimum stimulation intensity ( measured in percentage of maximum machine output ) required , to evoke a > 50 v mep in the resting , right fdi muscle in at least 6 out of 10 consecutive trials , measured using electromyography . motor threshold of 1 mv ( mt1 ) was defined as the minimum stimulation intensity , evoking 1 mv peak - to - peak amplitude in the resting , right fdi muscle in at least 6 out of 10 successive recordings . both rmt and mt1 were calculated using progressive reduction of the stimulator intensity from supra - threshold levels in 1% decrements as described in previous studies . four stimulus paradigms ( two single - pulse and two paired - pulse paradigms ) were used to study cortical excitability while the participants watched three different action related visual displays ( see below ) . 120% of rmt : meps obtained with stimulus intensity equal to 120% of rmt were recorded . this stimulus intensity has been the most commonly implemented in studying putative mirror mechanisms using tms.mt1 : meps obtained with stimulus intensity equal to mt1 were recorded . 120% of rmt : meps obtained with stimulus intensity equal to 120% of rmt were recorded . this stimulus intensity has been the most commonly implemented in studying putative mirror mechanisms using tms . both 120% rmt and mt1 are a measure of membrane excitability of pyramidal neurons , being influenced by voltage - gated sodium channels . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . the sub - threshold conditioning stimulus excites only the cortical interneurons and , therefore , inhibits the mep response to the test stimulus ( conditioned mep ) by 50%-90% . gabaa receptor agonists potentiate sici , thus suggesting that sici may be mediated by gabaa receptor - mediated neurotransmission.long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . the supra - threshold conditioning stimulus activates gabab receptor - mediated inhibitory postsynaptic potentials , thus inhibiting the mep response to the test stimulus ( conditioned mep ) . thus , lici reflects cortical inhibition mediated through gabab receptor - mediated neurotransmission , based on findings that baclofen , a specific gabab receptor agonist enhances lici . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . the sub - threshold conditioning stimulus excites only the cortical interneurons and , therefore , inhibits the mep response to the test stimulus ( conditioned mep ) by 50%-90% . gabaa receptor agonists potentiate sici , thus suggesting that sici may be mediated by gabaa receptor - mediated neurotransmission . long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . the supra - threshold conditioning stimulus activates gabab receptor - mediated inhibitory postsynaptic potentials , thus inhibiting the mep response to the test stimulus ( conditioned mep ) . thus , lici reflects cortical inhibition mediated through gabab receptor - mediated neurotransmission , based on findings that baclofen , a specific gabab receptor agonist enhances lici . cortical inhibition was expressed as a percentage of the ratio between the conditioned meps and the nonconditioned meps with stimulus intensity of mt1 ; that is , ( conditioned mep / nonconditioned mep ) 100 . ten mep recordings using each of these four stimulus paradigms ( total of 40 recordings ) were elicited in random sequence with 5-s intervals , while the subjects observed each of the following : actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . this action requires contraction of the fdi to abduct the index finger.virtual observation : the subjects observed a video showing the above action [ figure 1a].resting state : the subjects observed a still image of a hand and a lock displayed on the monitor [ figure 1b ] . actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . virtual observation : the subjects observed a video showing the above action [ figure 1a ] . resting state : the subjects observed a still image of a hand and a lock displayed on the monitor [ figure 1b ] . ( a ) screen - shot and ( b ) photograph of the video ( depicting a goal - directed action using the right first dorsal interosseous muscle ) and static images of a hand / lock and key , that were used in the experiment to assess putative mirror neuron activity during the virtual observation and rest states respectively the sequence of displaying these experimental states to each subject was randomized . the percent change of the mep or ci from resting to action observation states ( average of virtual and actual observation ) formed a measure of mna . it was calculated using the following formula , where cortical reactivity refers to mep ( in mv ) for single - pulse paradigms and ci ( % ) in paired - pulse paradigms : data acquisition and analysis were done using signal-4 software , ( cambridge electronic devices , cambridge , uk ) . 120% of rmt : meps obtained with stimulus intensity equal to 120% of rmt were recorded . this stimulus intensity has been the most commonly implemented in studying putative mirror mechanisms using tms.mt1 : meps obtained with stimulus intensity equal to mt1 were recorded . 120% of rmt : meps obtained with stimulus intensity equal to 120% of rmt were recorded . this stimulus intensity has been the most commonly implemented in studying putative mirror mechanisms using tms . both 120% rmt and mt1 are a measure of membrane excitability of pyramidal neurons , being influenced by voltage - gated sodium channels . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . the sub - threshold conditioning stimulus excites only the cortical interneurons and , therefore , inhibits the mep response to the test stimulus ( conditioned mep ) by 50%-90% . gabaa receptor agonists potentiate sici , thus suggesting that sici may be mediated by gabaa receptor - mediated neurotransmission.long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . the supra - threshold conditioning stimulus activates gabab receptor - mediated inhibitory postsynaptic potentials , thus inhibiting the mep response to the test stimulus ( conditioned mep ) . thus , lici reflects cortical inhibition mediated through gabab receptor - mediated neurotransmission , based on findings that baclofen , a specific gabab receptor agonist enhances lici . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . the sub - threshold conditioning stimulus excites only the cortical interneurons and , therefore , inhibits the mep response to the test stimulus ( conditioned mep ) by 50%-90% . gabaa receptor agonists potentiate sici , thus suggesting that sici may be mediated by gabaa receptor - mediated neurotransmission . long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . the supra - threshold conditioning stimulus activates gabab receptor - mediated inhibitory postsynaptic potentials , thus inhibiting the mep response to the test stimulus ( conditioned mep ) . thus , lici reflects cortical inhibition mediated through gabab receptor - mediated neurotransmission , based on findings that baclofen , a specific gabab receptor agonist enhances lici . cortical inhibition was expressed as a percentage of the ratio between the conditioned meps and the nonconditioned meps with stimulus intensity of mt1 ; that is , ( conditioned mep / nonconditioned mep ) 100 . ten mep recordings using each of these four stimulus paradigms ( total of 40 recordings ) were elicited in random sequence with 5-s intervals , while the subjects observed each of the following : actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . this action requires contraction of the fdi to abduct the index finger.virtual observation : the subjects observed a video showing the above action [ figure 1a].resting state : the subjects observed a still image of a hand and a lock displayed on the monitor [ figure 1b ] . actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . virtual observation : the subjects observed a video showing the above action [ figure 1a ] . resting state : the subjects observed a still image of a hand and a lock displayed on the monitor [ figure 1b ] . ( a ) screen - shot and ( b ) photograph of the video ( depicting a goal - directed action using the right first dorsal interosseous muscle ) and static images of a hand / lock and key , that were used in the experiment to assess putative mirror neuron activity during the virtual observation and rest states respectively the sequence of displaying these experimental states to each subject was randomized . the percent change of the mep or ci from resting to action observation states ( average of virtual and actual observation ) formed a measure of mna . it was calculated using the following formula , where cortical reactivity refers to mep ( in mv ) for single - pulse paradigms and ci ( % ) in paired - pulse paradigms : data acquisition and analysis were done using signal-4 software , ( cambridge electronic devices , cambridge , uk ) . univariate statistics ( independent samples t - test and chi - square tests ) were used to compare clinical and demographic parameters across the two groups ( patients with and without frs ) . all statistical tests were two - tailed , and significance was set at an error p 0.05 . as shown in table 1 , the two groups did not significantly differ from each other in terms of age , gender and education , illness duration , medication status ( drug - nave v / s on medication ) and symptom scores . twelve patients were receiving risperidone , 4 olanzapine , 3 risperidone + olanzapine , 1 olanzapine + amisulpride and 1 aripiprazole . median duration of treatment with anti - psychotics was 60 days and mean chlorpromazine equivalent was 413.43 226.95 mg / day . demographic and clinical variables a total of 18 ( 36% ) patients experienced symptoms of ebds as defined . thought broadcast was the most common symptom , being present in 72% of the patients who experienced symptoms of ebds [ table 2 ] . distribution of symptoms of ebds independent samples t - test on the two groups ( patients with and without ego - boundary disturbances ) revealed significantly greater mna in patients without ego - boundary disturbances than in patients with ego - boundary disturbances for mt1 and 120%rmt stimulus paradigms , indicating lesser mirror neuron activity in patients with symptoms of ego - boundary disturbances [ table 3 , figure 2 ] . there was no significant difference in mna measured using the two paired - pulse paradigms ( sici and lici ) across the 2 patient groups . putative mna in both groups : the mep difference between resting and action - observation states ( average across virtual and actual action - observation ) formed the measure of putative mna ( a ) error bars representing differences in means ( 95% confidence interval ) of mirror neuron activity ( y axis ) assessed using motor threshold-1 , ( b ) 120% resting motor threshold , ( c ) short interval intra - cortical inhibition and ( d ) long interval intra - cortical inhibition stimulus paradigms , across patients with and without symptoms of ego boundary disturbances ( x axis ) . as shown in table 1 , the two groups did not significantly differ from each other in terms of age , gender and education , illness duration , medication status ( drug - nave v / s on medication ) and symptom scores . twelve patients were receiving risperidone , 4 olanzapine , 3 risperidone + olanzapine , 1 olanzapine + amisulpride and 1 aripiprazole . median duration of treatment with anti - psychotics was 60 days and mean chlorpromazine equivalent was 413.43 226.95 mg / day . a total of 18 ( 36% ) patients experienced symptoms of ebds as defined . thought broadcast was the most common symptom , being present in 72% of the patients who experienced symptoms of ebds [ table 2 ] . independent samples t - test on the two groups ( patients with and without ego - boundary disturbances ) revealed significantly greater mna in patients without ego - boundary disturbances than in patients with ego - boundary disturbances for mt1 and 120%rmt stimulus paradigms , indicating lesser mirror neuron activity in patients with symptoms of ego - boundary disturbances [ table 3 , figure 2 ] . there was no significant difference in mna measured using the two paired - pulse paradigms ( sici and lici ) across the 2 patient groups . putative mna in both groups : the mep difference between resting and action - observation states ( average across virtual and actual action - observation ) formed the measure of putative mna ( a ) error bars representing differences in means ( 95% confidence interval ) of mirror neuron activity ( y axis ) assessed using motor threshold-1 , ( b ) 120% resting motor threshold , ( c ) short interval intra - cortical inhibition and ( d ) long interval intra - cortical inhibition stimulus paradigms , across patients with and without symptoms of ego boundary disturbances ( x axis ) . this study aimed to explore mna of a subset of schizophrenia patients with symptoms of ebds like thought alienation phenomena , made phenomena and somatic passivity . a common link across these symptoms is a breach in the barrier separating the self from one 's environment . a dysfunctional mirror neuron system has been proposed to underlie deficits in self - awareness and agency attribution , which may lead to symptoms of ebd . we used tms to derive a putative measure of mirror mechanisms of the brain as has been used in earlier studies . thrity - six percent of the patients experienced symptoms of ebds and thought broadcast was the most common symptom as has been reported in previous studies . we found greater enhancement of mep during action observation relative to rest states in schizophrenia patients without ebd than those with ebd . that is to say , schizophrenia patients with symptoms of ebd had significant deficits in mna when compared to patients without symptoms of ebd . the effect sizes ranged from 0.52 to 0.62 indicating moderate to good magnitude of difference across the two groups . the two patient groups did not differ in terms of their age , education , and illness duration or symptom scores assessed using panss . hence the observed differences in mna may be attributed to presence or absence of ebd symptoms . not surprisingly , more patients with symptoms of ebd had a diagnosis of paranoid sub - type of schizophrenia . results were consistent when mna was measured using single - pulse tms paradigms ( mt1 and 120% rmt ) . however , when mna was measured using the paired pulse paradigms ( sici and lici ) , there was no significant difference between the two groups , though the findings were in the expected direction ( i.e. , patients with ebd had poorer mna than those with ebd ) . this indicates a possible type-2 error as the sub - group with ebd had a small sample size ( n = 18 ) . while an aberrant hyperactive mna may underlie catatonic or affective symptoms , there is emerging evidence for mna deficits in patients with schizophrenia and its association with deficits in social information processing and negative symptoms . to the best of our knowledge this is the first study to demonstrate specific associations of premotor mna deficits with specific symptom dimensions ( ebd ) of schizophrenia . our findings indirectly support existing theoretical formulations of the neurobiological substrates of schneiderian frss of schizophrenia . aberrant hyperactivity , deficient cortical thickness and reduced volume of the inferior parietal lobule have been demonstrated in schizophrenia patients with frss . interestingly , the inferior parietal lobule is one of the brain regions most consistently shown to have mirror mechanisms in humans . our study showing deficient putative mna in schizophrenia patients with ebd indirectly supports these findings . whenever an action is intended an efference copy of the intended action diminishes responses to sensations of limb movements because of which the action is anticipated , the response does not come as a surprise to the person and his self - monitoring is , therefore , intact . when this attenuation does not happen , it leads to parietal cortex hyperactivity ; the person perceives the action as an event not under his / her control and goes further to confabulate the agency as external . mirror neurons could provide a physiological substrate of the efference copy , thus playing an integral role in self - monitoring . in schizophrenia patients with ebd , deficient mna may result in the absence of this attenuation of the responses to sensations of limb movements , thus resulting in a sense of alien control . it is crucial to note that tms is an indirect measurement of mirror neuron processes in the brain . intracranial depth electrodes give the most direct evidence of mna , but their use in humans , especially in those with psychiatric disorders , is extremely challenging . further , mna mediation using tms presumably reflects mirror properties of the premotor cortex , which through corticocortical connections to the motor cortex or cortico - spinal connections to the spinal cord , result in greater meps from the hand muscle during action observation when compared to rest states . measuring mna from the motor cortex limits the generalizability of these experiments to possible premotor / inferior frontal gyrus mirror properties . functional neuroimaging studies are better suited to produce an indirect measure of mna in other parts of the core mirror system ( inferior parietal lobule ) and the extended mirror system ( insular cortex , superior temporal sulcus , primary , and secondary motor and somatosensory cortices ) . our results need to be interpreted in the light of them being preliminary findings from a larger study . the results indicate possible mirror neuron dysfunction in schizophrenia patients with symptoms of ebd , and are not definitive . replication of these findings in studies with larger sample sizes is necessary to derive conclusive deductions . future studies should focus on assessing mna in a trans - diagnostic group of patients with symptoms of ebds using combined tms - functional neuroimaging methods to achieve optimal spatial and temporal resolution . frss of schizophrenia have been associated with a heightened social threat perception during emotion processing tasks . assessing the relationship between mirror neuron mechanisms , social cognition and ebds schizophrenia patients with ebd symptoms showed lower mep enhancement during action observation relative to rest states , than patients without ebd . this reflects a possible deficit in mirror neuron activation in schizophrenia patients who experience symptoms characterized by a breach in their ego - boundary . given the emerging evidence for the role of mirror neurons in psychiatric disorders in general , these findings contribute to the already existing understanding of the neurophysiology of frs in schizophrenia patients , thus providing novel treatment targets to be explored in future research .

background : ego - boundary disturbance ( ebd ) is a unique symptom cluster characterized by passivity experiences ( involving thoughts , actions , emotions and sensations ) attributed by patients to some external agency . the neurobiology of these first rank symptoms is poorly understood . aberrant mirror neuron activation may explain impaired self - monitoring and agency attribution underlying these symptoms . we aim to study mirror neuron activity ( mna ) in schizophrenia patients with and without ebd using transcranial magnetic stimulation ( tms).materials and methods:50 right - handed schizophrenia patients ( diagnostic and statistical manual of mental disorders , fourth edition ) were evaluated using the mini - international neuropsychiatric interview and the positive and negative syndrome scale . they completed a tms experiment to assess putative premotor mna . motor evoked potential ( mep ) was recorded in the right first dorsal interosseous muscle ( fdi ) with ( a ) 120% of resting motor threshold ( rmt ) , ( b ) stimulus intensity set to evoke mep of motor threshold 1 mv amplitude ( mt1 ) , ( c ) two paired pulse paradigms ( short- and long interval intra - cortical inhibition ) . these were done in three states : actual observation of an action using the fdi , virtual - observation ( video ) of this action and resting state . the percent change of mep from resting to action - observation states formed the measure of putative mna.results:mna measured using mt1 and 120% rmt paradigms was significantly lower in the 18 patients with ebd ( thought - broadcast / withdrawal / insertion , made - act / impulse / affect and somatic passivity ) than the 32 patients without ebd ( t = 2.431 , p = 0.020 ; t = 2.051 , p = 0.04 respectively for the two paradigms ) . the two groups did not differ on age , gender , education and total symptom scores.conclusion:schizophrenia patients with ebd have lower premotor mna . this highlights the role of mna dysfunction in the pathophysiology of this unique and intriguing symptom cluster in schizophrenia .

INTRODUCTION MATERIALS AND METHODS Transcranial magnetic stimulation experiment to assess putative mirror neuron activity Single-pulse paradigms Paired-pulse paradigms Statistical analyses RESULTS Demographic and clinical variables Symptoms of ego-boundary disturbances Mirror neuron activity in patients with and without symptoms of ego-boundary disturbances DISCUSSION CONCLUSION

first rank symptoms ( frss ) a group of intriguing symptoms characterized by a striking breach of self - versus nonself boundaries , first described by schneider , have had a critical influence on the diagnosis of schizophrenia over the past many decades . this symptom cluster comprises of audible thoughts , voices arguing , voices commenting on one 's action , influence playing on the body ( somatic passivity ) , thought withdrawal , thought insertion , broadcasting of thoughts , made feelings , made impulses , made volitional acts , and delusional perception . factor analytical studies have supported a two - dimensional structure of frs that is stable over time : symptoms characterized by ego - boundary disturbances ( ebds ; delusions of alien control , thought alienation phenomena and somatic passivity ) and those characterized by different auditory hallucinations . interestingly , there is emerging evidence for dysfunctional mirror neuron activity ( mna ) in patients with schizophrenia . transcranial magnetic stimulation ( tms ) is a unique , noninvasive , tool having both investigative and therapeutic applications . it has been extensively used to derive a putative measure of mna ( henceforth referred to as mna ) in humans by detecting changes in the motor evoked potentials ( mep ) of hand muscles recorded using electromyography . enhancement in the mep amplitude during action observation relative to rest states gives a measure of putative mna . in this study , we aim to compare putative mna , measured using tms between schizophrenia patients with and without symptoms of ebds . a total of 50 right - handed schizophrenia patients ( 18 with frs and 32 without frs ) from the inpatient and outpatient services of the national institute of mental health and neurosciences , bengaluru who were recruited as part of a larger study assessing mna in schizophrenia were recruited for this analysis . they were diagnosed independently by two qualified psychiatrists according to diagnostic and statistical manual of mental disorders , fourth edition criteria , and confirmed using the mini - international neuropsychiatric interview ( mini ) . symptoms were assessed using positive and negative syndrome scale ( panss ) . presence or absence of ebd symptoms ( though withdrawal , broadcast and insertion , made volition , impulse and affect ; and somatic passivity ) was determined initially during interviews with panss and mini ; which was then confirmed by a comprehensive clinical interview and mental status examination , based on the definitions given by mellor . patients with substance dependence in the previous 6 months ( except nicotine ) , presence of co - morbid neurological or medical disorder , clinically diagnosable or self - reported visual or auditory impairment , current pregnant or postpartum state and a score of 19 on the hindi mental status examination were excluded from the study . all subjects were assessed with ( a ) the edinburgh inventory for handedness and ( b ) tms adult safety screen to screen for their potential to develop complications . subjects underwent an experiment to assess motor cortical excitability during goal - directed action observation , relative to rest states , to give a putative measure of mna . for each subject , the optimal coil position was determined based on standard methods described in previous studies for localizing the scalp area to stimulate the right first dorsal interosseous ( fdi ) muscle . resting motor threshold ( rmt ) was defined as the minimum stimulation intensity ( measured in percentage of maximum machine output ) required , to evoke a > 50 v mep in the resting , right fdi muscle in at least 6 out of 10 consecutive trials , measured using electromyography . motor threshold of 1 mv ( mt1 ) was defined as the minimum stimulation intensity , evoking 1 mv peak - to - peak amplitude in the resting , right fdi muscle in at least 6 out of 10 successive recordings . four stimulus paradigms ( two single - pulse and two paired - pulse paradigms ) were used to study cortical excitability while the participants watched three different action related visual displays ( see below ) . 120% of rmt : meps obtained with stimulus intensity equal to 120% of rmt were recorded . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . gabaa receptor agonists potentiate sici , thus suggesting that sici may be mediated by gabaa receptor - mediated neurotransmission.long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . cortical inhibition was expressed as a percentage of the ratio between the conditioned meps and the nonconditioned meps with stimulus intensity of mt1 ; that is , ( conditioned mep / nonconditioned mep ) 100 . ten mep recordings using each of these four stimulus paradigms ( total of 40 recordings ) were elicited in random sequence with 5-s intervals , while the subjects observed each of the following : actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . this action requires contraction of the fdi to abduct the index finger.virtual observation : the subjects observed a video showing the above action [ figure 1a].resting state : the subjects observed a still image of a hand and a lock displayed on the monitor [ figure 1b ] . actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . ( a ) screen - shot and ( b ) photograph of the video ( depicting a goal - directed action using the right first dorsal interosseous muscle ) and static images of a hand / lock and key , that were used in the experiment to assess putative mirror neuron activity during the virtual observation and rest states respectively the sequence of displaying these experimental states to each subject was randomized . the percent change of the mep or ci from resting to action observation states ( average of virtual and actual observation ) formed a measure of mna . it was calculated using the following formula , where cortical reactivity refers to mep ( in mv ) for single - pulse paradigms and ci ( % ) in paired - pulse paradigms : data acquisition and analysis were done using signal-4 software , ( cambridge electronic devices , cambridge , uk ) . univariate statistics ( independent samples t - test and chi - square tests ) were used to compare clinical and demographic parameters across the two groups ( patients with and without frs ) . subjects underwent an experiment to assess motor cortical excitability during goal - directed action observation , relative to rest states , to give a putative measure of mna . for each subject , the optimal coil position was determined based on standard methods described in previous studies for localizing the scalp area to stimulate the right first dorsal interosseous ( fdi ) muscle . resting motor threshold ( rmt ) was defined as the minimum stimulation intensity ( measured in percentage of maximum machine output ) required , to evoke a > 50 v mep in the resting , right fdi muscle in at least 6 out of 10 consecutive trials , measured using electromyography . motor threshold of 1 mv ( mt1 ) was defined as the minimum stimulation intensity , evoking 1 mv peak - to - peak amplitude in the resting , right fdi muscle in at least 6 out of 10 successive recordings . four stimulus paradigms ( two single - pulse and two paired - pulse paradigms ) were used to study cortical excitability while the participants watched three different action related visual displays ( see below ) . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . gabaa receptor agonists potentiate sici , thus suggesting that sici may be mediated by gabaa receptor - mediated neurotransmission.long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . cortical inhibition was expressed as a percentage of the ratio between the conditioned meps and the nonconditioned meps with stimulus intensity of mt1 ; that is , ( conditioned mep / nonconditioned mep ) 100 . ten mep recordings using each of these four stimulus paradigms ( total of 40 recordings ) were elicited in random sequence with 5-s intervals , while the subjects observed each of the following : actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . this action requires contraction of the fdi to abduct the index finger.virtual observation : the subjects observed a video showing the above action [ figure 1a].resting state : the subjects observed a still image of a hand and a lock displayed on the monitor [ figure 1b ] . actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . ( a ) screen - shot and ( b ) photograph of the video ( depicting a goal - directed action using the right first dorsal interosseous muscle ) and static images of a hand / lock and key , that were used in the experiment to assess putative mirror neuron activity during the virtual observation and rest states respectively the sequence of displaying these experimental states to each subject was randomized . the percent change of the mep or ci from resting to action observation states ( average of virtual and actual observation ) formed a measure of mna . it was calculated using the following formula , where cortical reactivity refers to mep ( in mv ) for single - pulse paradigms and ci ( % ) in paired - pulse paradigms : data acquisition and analysis were done using signal-4 software , ( cambridge electronic devices , cambridge , uk ) . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . gabaa receptor agonists potentiate sici , thus suggesting that sici may be mediated by gabaa receptor - mediated neurotransmission.long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . short interval intra - cortical inhibition ( sici ) : a sub - threshold conditioning stimulus ( 80% of rmt ) was given 3 ms before a supra - threshold test stimulus ( mt1 ) with the right hand at rest . long interval intra - cortical inhibition ( lici ) : a supra - threshold conditioning stimulus ( mt1 ) is given 100 ms before a supra - threshold test stimulus ( mt1 ) . cortical inhibition was expressed as a percentage of the ratio between the conditioned meps and the nonconditioned meps with stimulus intensity of mt1 ; that is , ( conditioned mep / nonconditioned mep ) 100 . ten mep recordings using each of these four stimulus paradigms ( total of 40 recordings ) were elicited in random sequence with 5-s intervals , while the subjects observed each of the following : actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . this action requires contraction of the fdi to abduct the index finger.virtual observation : the subjects observed a video showing the above action [ figure 1a].resting state : the subjects observed a still image of a hand and a lock displayed on the monitor [ figure 1b ] . actual observation of an action with active fdi : the subjects observed the experimenter 's hand , holding a key in lateral pinch grip ( grasping objects between the side of the index finger and the thumb ) to perform locking / unlocking actions . ( a ) screen - shot and ( b ) photograph of the video ( depicting a goal - directed action using the right first dorsal interosseous muscle ) and static images of a hand / lock and key , that were used in the experiment to assess putative mirror neuron activity during the virtual observation and rest states respectively the sequence of displaying these experimental states to each subject was randomized . the percent change of the mep or ci from resting to action observation states ( average of virtual and actual observation ) formed a measure of mna . it was calculated using the following formula , where cortical reactivity refers to mep ( in mv ) for single - pulse paradigms and ci ( % ) in paired - pulse paradigms : data acquisition and analysis were done using signal-4 software , ( cambridge electronic devices , cambridge , uk ) . univariate statistics ( independent samples t - test and chi - square tests ) were used to compare clinical and demographic parameters across the two groups ( patients with and without frs ) . as shown in table 1 , the two groups did not significantly differ from each other in terms of age , gender and education , illness duration , medication status ( drug - nave v / s on medication ) and symptom scores . distribution of symptoms of ebds independent samples t - test on the two groups ( patients with and without ego - boundary disturbances ) revealed significantly greater mna in patients without ego - boundary disturbances than in patients with ego - boundary disturbances for mt1 and 120%rmt stimulus paradigms , indicating lesser mirror neuron activity in patients with symptoms of ego - boundary disturbances [ table 3 , figure 2 ] . there was no significant difference in mna measured using the two paired - pulse paradigms ( sici and lici ) across the 2 patient groups . putative mna in both groups : the mep difference between resting and action - observation states ( average across virtual and actual action - observation ) formed the measure of putative mna ( a ) error bars representing differences in means ( 95% confidence interval ) of mirror neuron activity ( y axis ) assessed using motor threshold-1 , ( b ) 120% resting motor threshold , ( c ) short interval intra - cortical inhibition and ( d ) long interval intra - cortical inhibition stimulus paradigms , across patients with and without symptoms of ego boundary disturbances ( x axis ) . as shown in table 1 , the two groups did not significantly differ from each other in terms of age , gender and education , illness duration , medication status ( drug - nave v / s on medication ) and symptom scores . independent samples t - test on the two groups ( patients with and without ego - boundary disturbances ) revealed significantly greater mna in patients without ego - boundary disturbances than in patients with ego - boundary disturbances for mt1 and 120%rmt stimulus paradigms , indicating lesser mirror neuron activity in patients with symptoms of ego - boundary disturbances [ table 3 , figure 2 ] . there was no significant difference in mna measured using the two paired - pulse paradigms ( sici and lici ) across the 2 patient groups . putative mna in both groups : the mep difference between resting and action - observation states ( average across virtual and actual action - observation ) formed the measure of putative mna ( a ) error bars representing differences in means ( 95% confidence interval ) of mirror neuron activity ( y axis ) assessed using motor threshold-1 , ( b ) 120% resting motor threshold , ( c ) short interval intra - cortical inhibition and ( d ) long interval intra - cortical inhibition stimulus paradigms , across patients with and without symptoms of ego boundary disturbances ( x axis ) . this study aimed to explore mna of a subset of schizophrenia patients with symptoms of ebds like thought alienation phenomena , made phenomena and somatic passivity . a common link across these symptoms is a breach in the barrier separating the self from one 's environment . a dysfunctional mirror neuron system has been proposed to underlie deficits in self - awareness and agency attribution , which may lead to symptoms of ebd . we found greater enhancement of mep during action observation relative to rest states in schizophrenia patients without ebd than those with ebd . that is to say , schizophrenia patients with symptoms of ebd had significant deficits in mna when compared to patients without symptoms of ebd . the two patient groups did not differ in terms of their age , education , and illness duration or symptom scores assessed using panss . results were consistent when mna was measured using single - pulse tms paradigms ( mt1 and 120% rmt ) . however , when mna was measured using the paired pulse paradigms ( sici and lici ) , there was no significant difference between the two groups , though the findings were in the expected direction ( i.e. , patients with ebd had poorer mna than those with ebd ) . to the best of our knowledge this is the first study to demonstrate specific associations of premotor mna deficits with specific symptom dimensions ( ebd ) of schizophrenia . aberrant hyperactivity , deficient cortical thickness and reduced volume of the inferior parietal lobule have been demonstrated in schizophrenia patients with frss . our study showing deficient putative mna in schizophrenia patients with ebd indirectly supports these findings . whenever an action is intended an efference copy of the intended action diminishes responses to sensations of limb movements because of which the action is anticipated , the response does not come as a surprise to the person and his self - monitoring is , therefore , intact . in schizophrenia patients with ebd , deficient mna may result in the absence of this attenuation of the responses to sensations of limb movements , thus resulting in a sense of alien control . functional neuroimaging studies are better suited to produce an indirect measure of mna in other parts of the core mirror system ( inferior parietal lobule ) and the extended mirror system ( insular cortex , superior temporal sulcus , primary , and secondary motor and somatosensory cortices ) . the results indicate possible mirror neuron dysfunction in schizophrenia patients with symptoms of ebd , and are not definitive . assessing the relationship between mirror neuron mechanisms , social cognition and ebds schizophrenia patients with ebd symptoms showed lower mep enhancement during action observation relative to rest states , than patients without ebd . this reflects a possible deficit in mirror neuron activation in schizophrenia patients who experience symptoms characterized by a breach in their ego - boundary . given the emerging evidence for the role of mirror neurons in psychiatric disorders in general , these findings contribute to the already existing understanding of the neurophysiology of frs in schizophrenia patients , thus providing novel treatment targets to be explored in future research .

various studies have shown a shift in focus from condom programming and provision of clinical services to initiatives that address the contextual factors underlying human immunodeficiency virus ( hiv ) risk and vulnerability1 among high - risk groups . the aastha project , funded by the bill and melinda gates foundation , a scaled - up hiv - prevention intervention since 2004 to date across mumbai and thane with a heterogeneous population of sex workers in an urban setting , is a case in point.2,3 even though maharashtra is a high - prevalence state , the number of new infections in mumbai has been reduced as a result of this intervention.4 one of the aastha strategies has been to focus on the vulnerabilities of female sex workers ( fsws ) , and identifying what makes them vulnerable and redress mechanisms . power dynamics play a crucial role in the sex industry and in the lives of fsws , right from the scope of negotiation with clients and partners on condom use and sexual acts and managing the control and influence of stakeholders , such as brothel owners , bar owners , brothel managers , and local thugs . however , what makes some fsws more vulnerable than others ? and what implications do their sociocultural vulnerabilities have for an hiv / sexually transmitted infection ( sti)-intervention program , whose prime objective is to encourage health - seeking behavior among fsws ? this paper postulates that it is the continuous interaction of various socioeconomic factors and aspects of sex work itself that together contribute toward making fsws vulnerable , and some fsws more than others.3 the paper also argues that it is important for targeted interventions to define the vulnerability of their target group prior to project design and implementation , given that factors shaping vulnerability are context - specific and project strategies need to respond accordingly . recently , through various syndemics , it has become increasingly clear that diseases do not occur in isolation , and social conditions of those who are suffering from these diseases are important factors to consider in order to understand the health impact on these individuals.5 prior research highlights that some groups in society are more vulnerable to diseases than others . a set of social and economic factors appear to be associated with ideas , attitudes , and behaviors.5 in an attempt to clarify the concept of vulnerability , delor and hubert describe it as relational , contextual , and the process aspect of risk . it thus has three coordinates : the risk of being exposed to crisis situations ( exposure ) , the risk of not having the necessary resources to cope with these situations ( capacity ) , and the risk of being subjected to serious consequences as a result of the crises ( potentiality).6 however , it is important to make a distinction between hiv risk behaviors or situations that directly increase the probability of hiv transmission and hiv vulnerability , the social context that governs the individual s ability to avoid hiv transmission.1 qualitative approaches to understanding the vulnerability of fsws have looked upon integrated structural intervention strategies to reduce vulnerability among fsws.79 for instance , a previous qualitative research study on the sociocultural context of fsws in nigeria suggested that five areas determine the degree of vulnerability , namely flawed knowledge , the psychosocial and economic context of sex work , stigma and risk - taking behavior , barriers to hiv testing , and legal constraints.8 however , there is a lack of similar quantitative analyses where the concept of vulnerability has been used as an independent measure . this paper contributes to the existing body of knowledge in this aspect by defining vulnerability and considering specific aspects . recent studies have explored the role of various factors separately , such as poverty , illiteracy , duration in sex work , and mobility for work among fsws , contributing toward hiv vulnerability . in a study in the four high - prevalence indian states , including maharashtra , a higher degree of mobility was associated with higher rates of inconsistent condom use with clients and regular partners.10,11 lack of social support in destination areas , changing environments that influence the sex worker s daily life , such as shifts in laws , changing police personnel , and high economic needs , make mobile fsws especially vulnerable.7 prior research that focused on the influence of socioeconomic factors on hiv risk and vulnerability suggests that women in india with less than 5 years of education had higher hiv prevalence compared to their counterparts , and that education has protective tendencies on hiv prevalence , as literacy is positively associated with comprehensive knowledge about hiv / acquired immunodeficiency syndrome ( aids).12 in prior research that focused on the role of debt on hiv risk in a high - prevalence state in india , fsws who reported debt were more likely to report recent violence , unprotected sex with occasional clients in the past week , and at least one sti symptom in the past 6 months.13 previous research has also focused on preexisting sociocultural vulnerabilities that encourage fsws to enter sex work in the first place.14 a microlevel study of the life circumstances of fsws in dimapur , nagaland indicated the factors of vulnerability : 1 ) time of entry into sex work , ie , early age of sexual debut , lack of other sources of employment , low levels of education , and 2 ) factors of vulnerability in their current work situation , namely high levels of mobility , the need to financially support dependants , and the need to give money to others.15 in another study among mobile fsws across andhra pradesh , tamil nadu , karnataka , and maharashtra , a significant association was seen between reasons for entry into sex work , such as poor economic conditions , and inconsistent condom use . poor economic conditions were the primary reason for entry into sex work.14 these vulnerabilities increase fsws likelihood of experiencing violence , lead to poor negotiation skills with their clients , and act as a barrier to service utilization.16,17 it is evident from the aforementioned examples that frequently one or two vulnerability factors have been considered in isolation,815 but as explained in the literature about various syndemics,5,18 vulnerability factors do not occur in isolation . figure 1 illustrates the interaction and interface of various factors of vulnerability , which lead to differing degrees and types of vulnerability . there is a need to bring together individual factors to construct an index that considers all the important vulnerability factors therefore , this paper attempts : 1 ) to define vulnerability as an independent measure , after consolidating various independent factors ; 2 ) to describe the aastha hiv / sti prevention program , considering the vulnerabilities of fsws prior to initiating the intervention ; and 3 ) to understand the association of duration of exposure to the aastha program on the relationship between vulnerability and behavioral outcomes . the aastha hiv / sti prevention program was initiated in 2004 to reach out to fsws who come from a diverse sociocultural background , predominantly from andhra pradesh , karnataka , west bengal , and nepal , operating through various locations scattered across mumbai and thane . the project witnessed a high churn rate , as the fsws that the project worked with were a highly mobile group . they also functioned from a wide range of places of solicitation : homes , streets , bars , brothels , and phone networks . the objective of the project was to empower fsws to access and manage hiv - prevention services . a key focal area of the program was 100% screening for stis , and the program achieved a 35%40% monthly clinical uptake and up to 93% on a quarterly basis . table 1 gives a profile of the fsws , in terms of the unique characteristics of each typology , the challenges faced while working with them , and the specially designed aastha response . through the program , interactive sessions are organized with fsws to understand their vulnerabilities . during these sessions , various situations faced by fsws that increased their vulnerability to hiv risk are discussed . these vulnerabilities affect their ability to seek health services , use condoms consistently , and address issues related to negotiation for safer sex behaviors , which in turn have an effect on the overall health of the fsws , including increased chances of acquiring hiv infection . during program implementation some of these included poor economic conditions , high degree of control and influence from partners and clients , lack of identity , low self - esteem and self - confidence , and lack of basic opportunities , such as schooling for fsws children and government identity cards , among others . the root cause for such common issues lay in the fact that each fsw was socially functioning alone in her given individual context . these vulnerabilities further manifested in increased cases of violence and harassment from stakeholders and regular partners , police arrests based on false charges , inconsistent condom use and a high transaction - frequency rate . the aastha project introduced strategies to empower fsws and increase their self confidence in order to improve their health - seeking behavior and condom - negotiation skills with clients . the project created a self - help group called aastha gats at the local level . the aastha gats members promoted unity among fsws to address issues related to the community . details of this process are available elsewhere.2 in addition , the program initiated a community - led rapid - response system or aastha tatkal seva ( ats ) to tackle the regular violence faced by the fsws . the ats functions through site - level , sex worker - led groups that respond to crisis situations within 30 minutes . further , the project also ensured that all fsw leaders in each hot - spot area were educated on the legal issues related to sex work and their rights as indian citizens . the data for this study are drawn from the behavioural tracking survey ( bts),2 a cross - sectional behavioral survey conducted in 2010 with 2,431 fsws from 411 sites in mumbai and thane . survey respondents included women who were 18 years who had had sex in exchange of cash / kind in the month prior to the survey . the overall survey design , including sample - size calculation and participant recruitment , has been described in detail elsewhere.2 interviews were conducted through a structured questionnaire by trained researchers in the local language ( hindi ) . the survey instrument was developed in english and translated into hindi . the translated forms were reviewed by the study investigators , who were fluent in both english and hindi . field supervisors reviewed the completed questionnaires on the same day , and these were sent every week to the data - management team for data entry . a user - written computer program in cspro ( version 4.0 ; us census bureau , suitland , md , usa ) was used for double data entry by trained data - entry officers . data were regularly processed to ensure consistency and accuracy . the bts was reviewed and approved by fhi 360 s protection of human subjects committee , based in the us . a local community advisory board , functioning as the local institutional review board , including fsws , fsw community based organizations ( cbos ) , and prominent researchers , was constituted in mumbai , which worked closely with the local research agency and fhi 360 to ensure that the study participants rights were protected . to protect confidentiality and respect privacy , oral consent was obtained from the respondents before they were interviewed to ensure they knew about the study and information sought from them . to maintain privacy , participants were not given any monetary compensation for their time given to the study , but were provided information on hiv programs in the area . the factors of vulnerability used in the current analysis are based on program experience , anecdotal sharing from fsws at the beginning of the project , and evidence from the literature.816 figure 2 presents the factors that make fsws vulnerable to hiv risk , the program strategies of aastha that focused on the vulnerabilities of fsws , and the outcome measures that were considered in the paper . the vulnerability index and duration in the aastha program were the two key independent measures considered in this paper . the factors used to construct the vulnerability index were based on several items in the questionnaire , which included literacy ( literate , can read and/or write ; illiterate , can not read or write ) , duration in sex work ( 3 + years , 2 years ) , mobility for sex work ( nonmobile , did not travel for sex work ; mobile , traveled for sex work ) , additional source of income other than sex work ( yes / no ) , currently being in debt ( yes / no ) , and having children ( yes / no ) . using these six factors , a vulnerability index was constructed , with scale values ranging from 0 to 6 . those who were new to sex work ( 2 years ) , were in debt , were illiterate , had no alternative source of income , were mobile for sex work and had children were considered vulnerable to hiv infection . using a median split , the index score was further divided into two categories to construct degree of vulnerability , with three or more factors constituting high vulnerability and fewer than three factors constituting low vulnerability . duration in the aastha project was measured by asking the participants how long they had been associated / registered with the aastha program . the responses included : less than 6 months , 7 months to 1 year , and more than a year ( a trichotomous variable ) . the responses were further grouped into two categories ( 1 year and > 1year ) . program clinic data showed that sex workers exposed to various project services for at least a year had reduced sti symptoms and improved health - seeking behavior . as a result , duration of participation the program - exposure and service - utilization indicators considered in this paper were 1 ) received counseling / referral services from aastha nongovernmental organizations in the last 6 months ( yes / no ) , 2 ) aware of ats ( yes / no ) , 3 ) aware and accessed ats services in the past 6 months ( yes / no ) , and 4 ) visits to a clinic for sti symptoms in the past year . visits to a clinic to check for sti symptoms was initially measured as a continuous variable by asking fsws the number of times they visited the aastha clinic , and was further dichotomized as visited aastha clinic to check for sti symptoms in the past year / did not visit aastha clinic to check for sti symptoms . self - confidence was measured by asking each fsw 1 ) whether or not she was confident in supporting a fellow sex worker in crisis situation , and 2 ) whether or not she was confident in going to a service provider alone , where she may have to disclose her identity as a sex worker . responses to these questions included : not at all , somewhat , very confident , and completely confident . fsws who responded very confident or completely confident were considered confident . the term individual agency was defined as each fsw s individual ability to make decisions herself . to measure fsws individual agency , a question was asked in the survey : how often do you take clients when you are tired ? the responses included : never , sometimes , usually , and always . those who responded sometimes , usually , and always were considered to be taking clients when they do not want to . the fsws ability to claim self - identity was measured by asking how much they agreed with the following two questions : whether they were not ashamed to say that they were a sex worker 1 ) in a meeting with other sex workers , and 2 ) when she met a social or health worker who worked in her community . the aforementioned two indicators for claiming identity were considered separately in this paper . in all our analyses , we controlled for age ( < 25 , 2529 , 3034 , 35 + years ) , marital status ( ever married , never married ) , and typology of sex work ( brothel - based , bar - based , street-/home-/lodge - based ) . simple descriptive analyses were run on characteristics that were used to construct the vulnerability index . the relationship between selected sample characteristics and degree of vulnerability was assessed using contingency tables . a series of multivariate logistic regression models were constructed to examine the association between vulnerability , duration of exposure to the aastha program , and fsws self - confidence , self - identity , individual agency , and service utilization , adjusting for age , marital status , and typology of sex work . all the analyses were performed using stata 12 ( statacorp , college station , tx , usa ) . the bts was reviewed and approved by fhi 360 s protection of human subjects committee , based in the us . a local community advisory board , functioning as the local institutional review board , including fsws , fsw community based organizations ( cbos ) , and prominent researchers , was constituted in mumbai , which worked closely with the local research agency and fhi 360 to ensure that the study participants rights were protected . to protect confidentiality and respect privacy , oral consent was obtained from the respondents before they were interviewed to ensure they knew about the study and information sought from them . to maintain privacy , interviews were conducted at a location dependent on the preference of the respondent . participants were not given any monetary compensation for their time given to the study , but were provided information on hiv programs in the area . the factors of vulnerability used in the current analysis are based on program experience , anecdotal sharing from fsws at the beginning of the project , and evidence from the literature.816 figure 2 presents the factors that make fsws vulnerable to hiv risk , the program strategies of aastha that focused on the vulnerabilities of fsws , and the outcome measures that were considered in the paper . the vulnerability index and duration in the aastha program were the two key independent measures considered in this paper . the factors used to construct the vulnerability index were based on several items in the questionnaire , which included literacy ( literate , can read and/or write ; illiterate , can not read or write ) , duration in sex work ( 3 + years , 2 years ) , mobility for sex work ( nonmobile , did not travel for sex work ; mobile , traveled for sex work ) , additional source of income other than sex work ( yes / no ) , currently being in debt ( yes / no ) , and having children ( yes / no ) . using these six factors , a vulnerability index was constructed , with scale values ranging from 0 to 6 . those who were new to sex work ( 2 years ) , were in debt , were illiterate , had no alternative source of income , were mobile for sex work and had children were considered vulnerable to hiv infection . using a median split , the index score was further divided into two categories to construct degree of vulnerability , with three or more factors constituting high vulnerability and fewer than three factors constituting low vulnerability . duration in the aastha project was measured by asking the participants how long they had been associated / registered with the aastha program . the responses included : less than 6 months , 7 months to 1 year , and more than a year ( a trichotomous variable ) . the responses were further grouped into two categories ( 1 year and > 1year ) . program clinic data showed that sex workers exposed to various project services for at least a year had reduced sti symptoms and improved health - seeking behavior . as a result , duration of participation the program - exposure and service - utilization indicators considered in this paper were 1 ) received counseling / referral services from aastha nongovernmental organizations in the last 6 months ( yes / no ) , 2 ) aware of ats ( yes / no ) , 3 ) aware and accessed ats services in the past 6 months ( yes / no ) , and 4 ) visits to a clinic for sti symptoms in the past year . visits to a clinic to check for sti symptoms was initially measured as a continuous variable by asking fsws the number of times they visited the aastha clinic , and was further dichotomized as visited aastha clinic to check for sti symptoms in the past year / did not visit aastha clinic to check for sti symptoms . self - confidence was measured by asking each fsw 1 ) whether or not she was confident in supporting a fellow sex worker in crisis situation , and 2 ) whether or not she was confident in going to a service provider alone , where she may have to disclose her identity as a sex worker . responses to these questions included : not at all , somewhat , very confident , and completely confident . fsws who responded very confident or completely confident were considered confident . the term individual agency was defined as each fsw s individual ability to make decisions herself . to measure fsws individual agency , a question was asked in the survey : how often do you take clients when you are tired ? the responses included : never , sometimes , usually , and always . those who responded always were considered to be taking clients when they do not want to . the fsws ability to claim self - identity was measured by asking how much they agreed with the following two questions : whether they were not ashamed to say that they were a sex worker 1 ) in a meeting with other sex workers , and 2 ) when she met a social or health worker who worked in her community . the aforementioned two indicators for claiming identity were considered separately in this paper . in all our analyses , we controlled for age ( < 25 , 2529 , 3034 , 35 + years ) , marital status ( ever married , never married ) , and typology of sex work ( brothel - based , bar - based , street-/home-/lodge - based ) . the vulnerability index and duration in the aastha program were the two key independent measures considered in this paper . the factors used to construct the vulnerability index were based on several items in the questionnaire , which included literacy ( literate , can read and/or write ; illiterate , can not read or write ) , duration in sex work ( 3 + years , 2 years ) , mobility for sex work ( nonmobile , did not travel for sex work ; mobile , traveled for sex work ) , additional source of income other than sex work ( yes / no ) , currently being in debt ( yes / no ) , and having children ( yes / no ) . using these six factors , a vulnerability index was constructed , with scale values ranging from 0 to 6 . those who were new to sex work ( 2 years ) , were in debt , were illiterate , had no alternative source of income , were mobile for sex work and had children were considered vulnerable to hiv infection . using a median split , the index score was further divided into two categories to construct degree of vulnerability , with three or more factors constituting high vulnerability and fewer than three factors constituting low vulnerability . duration in the aastha project was measured by asking the participants how long they had been associated / registered with the aastha program . the responses included : less than 6 months , 7 months to 1 year , and more than a year ( a trichotomous variable ) . the responses were further grouped into two categories ( 1 year and > 1year ) . program clinic data showed that sex workers exposed to various project services for at least a year had reduced sti symptoms and improved health - seeking behavior . as a result , duration of participation in the survey instrument , information was elicited on several dimensions of exposure to the aastha program . the program - exposure and service - utilization indicators considered in this paper were 1 ) received counseling / referral services from aastha nongovernmental organizations in the last 6 months ( yes / no ) , 2 ) aware of ats ( yes / no ) , 3 ) aware and accessed ats services in the past 6 months ( yes / no ) , and 4 ) visits to a clinic for sti symptoms in the past year . visits to a clinic to check for sti symptoms was initially measured as a continuous variable by asking fsws the number of times they visited the aastha clinic , and was further dichotomized as visited aastha clinic to check for sti symptoms in the past year / did not visit aastha clinic to check for sti symptoms . self - confidence was measured by asking each fsw 1 ) whether or not she was confident in supporting a fellow sex worker in crisis situation , and 2 ) whether or not she was confident in going to a service provider alone , where she may have to disclose her identity as a sex worker . responses to these questions included : not at all , somewhat , very confident , and completely confident . fsws who responded very confident or completely confident were considered confident . the term individual agency was defined as each fsw s individual ability to make decisions herself . to measure fsws individual agency , a question was asked in the survey : how often do you take clients when you are tired ? the responses included : never , sometimes , usually , and always . those who responded always were considered to be taking clients when they do not want to . the fsws ability to claim self - identity was measured by asking how much they agreed with the following two questions : whether they were not ashamed to say that they were a sex worker 1 ) in a meeting with other sex workers , and 2 ) when she met a social or health worker who worked in her community . in all our analyses , we controlled for age ( < 25 , 2529 , 3034 , 35 + years ) , marital status ( ever married , never married ) , and typology of sex work ( brothel - based , bar - based , street-/home-/lodge - based ) . simple descriptive analyses were run on characteristics that were used to construct the vulnerability index . the relationship between selected sample characteristics and degree of vulnerability was assessed using contingency tables . a series of multivariate logistic regression models were constructed to examine the association between vulnerability , duration of exposure to the aastha program , and fsws self - confidence , self - identity , individual agency , and service utilization , adjusting for age , marital status , and typology of sex work . all the analyses were performed using stata 12 ( statacorp , college station , tx , usa ) . of the total analytical sample of 2,431 fsws , 1,295 ( 53.3% ) were categorized as highly vulnerable . the literacy level was very low , with only a third ( 29% ) reporting that they could read or write , and nearly one in three reporting that they had children ( 29% ) . a majority of the participants were new to sex work ( 81% ) , with most ( 79% ) reporting it to be their main source of income . more than a third reported traveling for sex work ( 40% ) , and nearly a quarter ( 25% ) were currently in debt . as seen in table 3 , a higher proportion of fsws who were in the age - group 1825 years , who were never married ( 75.7% versus 48.6% , p<0.001 ) , or who were bar - based sex workers were categorized as highly vulnerable to hiv risk compared to sex workers without these characteristics . fsws who had spent less than 1 year in the aastha program were more likely to be highly vulnerable compared to those who had spent more than 1 year in the program ( 63.9% versus 51.3% , p<0.001 ) . as seen in table 4 , highly vulnerable fsws who were in the aastha program for more than 1 year were more likely to be aware of ats ( adjusted odds ratio [ or ] 2.4 , 95% confidence interval [ ci ] 1.44.0 ; p<0.001 ) , to have accessed ats services in the past 6 months ( adjusted or 2.2 , 95% ci 1.43.6 ; p<0.001 ) , and to have visited an aastha clinic to get a checkup for sti symptoms ( adjusted or 2.4 , 95% ci 1.24.8 , p<0.015 ) compared to those less vulnerable with similar exposure to the aastha program . similarly , table 5 indicates that a significantly higher proportion of highly vulnerable fsws who were associated with or registered with the aastha program for more than a year were not ashamed to disclose their identity as sex workers in a meeting with other sex workers ( adjusted or 2.3 , 95% ci 1.23.6 ; p<0.013 ) and were not ashamed to disclose their identity as sex workers while meeting a health or social worker who worked in their community ( adjusted or 2.1 , 95% ci 1.23.5 ; p<0.008 ) . additionally , highly vulnerable fsws with a longer association with the aastha program were confident in supporting a fellow sex worker in crisis ( adjusted or 1.7 , 95% ci 1.02.8 ; p<0.033 ) compared to less vulnerable fsws with similar exposure in aastha program . the paper explores how fsw intervention programs can identify and assess vulnerabilities , and correspondingly can be used to design strategies . the findings show that those reporting higher vulnerability and who are exposed to project services for a longer period of time in aastha are more likely to report a higher uptake of services and a higher degree of overall empowerment than those who are less vulnerable . this further illustrates the fact that project strategies of increasing awareness about services and risk over a period of time can lead to improved results . creating mechanisms to address cases of harassment , violence , and other contingencies , and organizing fsws in support groups has been pivotal in creating an enabling environment and challenging existing structures and power dynamics through empowered actions,19 such as disclosing their profession , effectively dealing with violence from stakeholders , and participating in friendly social interactions with peers outside of work contexts . these findings corroborate similar studies that show that empowerment strategies are a critical pathway to risk reduction , in contrast to the standard targeted intervention approach.20 while the described behavioral outcomes and individual empowerment are desirable changes in the context of hiv intervention , structural intervention approaches typically create a collective identity among fsws.7 community - led structural intervention approaches , such as the ats , are critical to the process of risk reduction . while the current paper focuses more on certain initiatives of the aastha project , a more detailed analysis of community collectivization strategies has been described elsewhere.2 the results of the paper can be viewed in parallel with the findings of a study in karnataka , which describes the use of similar strategies , among others , in influencing the context - specific social , economic , political , and environmental factors determining risk for hiv.9 it is critical to place the vulnerabilities of an individual fsw in context and to understand the complex interaction of various factors at different levels , including economic , social , and politicolegal . for instance , an ethnographic case study of the sonagachi hiv / std intervention project describes some of the factors contributing to hiv risk in the context of the brothel areas of sonagachi , including the ambiguity of the legal status of the sex - work industry , lack of education , social discrimination , and occupational structures within the industry.7 given the difficulty of measuring vulnerability , there is considerable lack of quantitative research , where a holistic definition of vulnerability has been used for the purpose of analysis . hence , similar to the ethnographic study described earlier , the aastha project also provides a useful construct by adapting a specific vulnerability index . however , though the association of vulnerability index and behavior outcomes has important implications for hiv interventions , the results need to be seen in light of several limitations . first , this paper focuses on limited project services and desired behavior outcomes due to lack of relevant data , as it is drawn from the bts , which had different objective and goals . similar analyses in future should also include more comprehensive variables for their analyses , including the impact of high mobility , increased closure of bars due to police raids , consumption of alcohol amongst fsws , and fsws economic status . second , given that identifying sociocultural vulnerabilities is context - specific , the factors of vulnerability used for the current analysis are specific to the districts of mumbai and thane , but could be replicated with suitable adaptation in other parts of india and south asian countries . third , the responses of the fsws were part of a larger study to assess the behavioral outcomes of fsws across mumbai and thane , and not to attempt to define vulnerability and assess its association with project services or desired behavior outcomes . finally , a lack of cohort data makes it difficult for the paper to establish causality between degree of vulnerability and the desired behavior outcomes or between the observed behavior changes and the community mobilization efforts . , it is critical for hiv / sti interventions to identify and define factors of vulnerability at project inception , prior to developing risk - reduction strategies . the factors should be thoroughly and strategically addressed in program design and periodically reviewed to take into account changes in economic , social , and legal scenarios . moreover , it is important to look at a holistic interaction of different sociocultural vulnerabilities , such as lack of education , poor economic status , financial debts , presence of dependants such as children , lack of other livelihood options , mobility due to sex work , and duration in sex work , as well as changes in the social and legal environment , which contribute towards an environment of increased violence and lack of individual agency and control , rather than just one or two factors . though addressing structural barriers is a complicated and long - term process , it plays a critical role in reducing hiv risk amongst high - risk groups like fsws . in summary , we would like to say that defining vulnerability and constructing a specific vulnerability index and assessing it in the context of behavioral outcomes and project inputs would create a better understanding of the effectiveness of hiv - prevention interventions with fsws .

backgroundit is important for targeted interventions to consider vulnerabilities of female sex workers ( fsws ) such as poverty , work - related mobility , and literacy , for effective human immunodeficiency virus ( hiv ) prevention . this paper describes and examines the association of the aastha hiv / sexually transmitted infection ( sti ) prevention project in mumbai and thane , india , on the relationship between vulnerability and behavioral outcomes.materials and methodsdata were drawn from the behavioural tracking survey , a cross - sectional behavioral study conducted in 2010 with 2,431 fsws recruited in mumbai and thane . the key independent measures used were program exposure and vulnerability index , a composite index of literacy , factors of dependence ( alternative livelihood options , current debt , and children ) , and aspects of sex work ( mobility and duration in sex work ) . dependent measures included service uptake , self - confidence , self - identity , and individual agency . logistic regression analysis was used to examine the study objectives.resultsof the analytical sample of 2,431 fsws , 1,295 ( 53.3% ) were categorized as highly vulnerable . highly vulnerable fsws who were associated with the aastha program for more than a year were more likely to have accessed crisis - response services in the past 6 months ( adjusted odds ratio [ aor ] 2.2 , 95% confidence interval [ ci ] 1.43.6 ; p<0.001 ) , to have visited a clinic to get a checkup for sti symptoms ( aor 2.4 , 95% ci 1.24.8 ; p<0.015 ) , not to be ashamed to disclose identity as an fsw to health workers ( aor 2.1 , 95% ci 1.23.5 ; p<0.008 ) , and to be confident in supporting a fellow fsw in crisis ( aor 1.7 , 95% ci 1.02.8 , p<0.033 ) compared to those less vulnerable with similar exposure to the aastha program.conclusionit is critical for hiv / sti interventions to consider vulnerabilities of fsws at project inception and address them with focused strategies , including a segmented service - delivery model and community involvement , in order to strengthen the structural response to hiv prevention .

Background Defining vulnerability The Aastha project Materials and methods Ethical considerations Measures Vulnerability index and duration in Aastha program Program exposure and service utilization Self-confidence, self-identity, and individual agency Control variables Statistical analyses Results Discussion

the aastha project , funded by the bill and melinda gates foundation , a scaled - up hiv - prevention intervention since 2004 to date across mumbai and thane with a heterogeneous population of sex workers in an urban setting , is a case in point.2,3 even though maharashtra is a high - prevalence state , the number of new infections in mumbai has been reduced as a result of this intervention.4 one of the aastha strategies has been to focus on the vulnerabilities of female sex workers ( fsws ) , and identifying what makes them vulnerable and redress mechanisms . power dynamics play a crucial role in the sex industry and in the lives of fsws , right from the scope of negotiation with clients and partners on condom use and sexual acts and managing the control and influence of stakeholders , such as brothel owners , bar owners , brothel managers , and local thugs . this paper postulates that it is the continuous interaction of various socioeconomic factors and aspects of sex work itself that together contribute toward making fsws vulnerable , and some fsws more than others.3 the paper also argues that it is important for targeted interventions to define the vulnerability of their target group prior to project design and implementation , given that factors shaping vulnerability are context - specific and project strategies need to respond accordingly . it thus has three coordinates : the risk of being exposed to crisis situations ( exposure ) , the risk of not having the necessary resources to cope with these situations ( capacity ) , and the risk of being subjected to serious consequences as a result of the crises ( potentiality).6 however , it is important to make a distinction between hiv risk behaviors or situations that directly increase the probability of hiv transmission and hiv vulnerability , the social context that governs the individual s ability to avoid hiv transmission.1 qualitative approaches to understanding the vulnerability of fsws have looked upon integrated structural intervention strategies to reduce vulnerability among fsws.79 for instance , a previous qualitative research study on the sociocultural context of fsws in nigeria suggested that five areas determine the degree of vulnerability , namely flawed knowledge , the psychosocial and economic context of sex work , stigma and risk - taking behavior , barriers to hiv testing , and legal constraints.8 however , there is a lack of similar quantitative analyses where the concept of vulnerability has been used as an independent measure . recent studies have explored the role of various factors separately , such as poverty , illiteracy , duration in sex work , and mobility for work among fsws , contributing toward hiv vulnerability . in a study in the four high - prevalence indian states , including maharashtra , a higher degree of mobility was associated with higher rates of inconsistent condom use with clients and regular partners.10,11 lack of social support in destination areas , changing environments that influence the sex worker s daily life , such as shifts in laws , changing police personnel , and high economic needs , make mobile fsws especially vulnerable.7 prior research that focused on the influence of socioeconomic factors on hiv risk and vulnerability suggests that women in india with less than 5 years of education had higher hiv prevalence compared to their counterparts , and that education has protective tendencies on hiv prevalence , as literacy is positively associated with comprehensive knowledge about hiv / acquired immunodeficiency syndrome ( aids).12 in prior research that focused on the role of debt on hiv risk in a high - prevalence state in india , fsws who reported debt were more likely to report recent violence , unprotected sex with occasional clients in the past week , and at least one sti symptom in the past 6 months.13 previous research has also focused on preexisting sociocultural vulnerabilities that encourage fsws to enter sex work in the first place.14 a microlevel study of the life circumstances of fsws in dimapur , nagaland indicated the factors of vulnerability : 1 ) time of entry into sex work , ie , early age of sexual debut , lack of other sources of employment , low levels of education , and 2 ) factors of vulnerability in their current work situation , namely high levels of mobility , the need to financially support dependants , and the need to give money to others.15 in another study among mobile fsws across andhra pradesh , tamil nadu , karnataka , and maharashtra , a significant association was seen between reasons for entry into sex work , such as poor economic conditions , and inconsistent condom use . there is a need to bring together individual factors to construct an index that considers all the important vulnerability factors therefore , this paper attempts : 1 ) to define vulnerability as an independent measure , after consolidating various independent factors ; 2 ) to describe the aastha hiv / sti prevention program , considering the vulnerabilities of fsws prior to initiating the intervention ; and 3 ) to understand the association of duration of exposure to the aastha program on the relationship between vulnerability and behavioral outcomes . the aastha hiv / sti prevention program was initiated in 2004 to reach out to fsws who come from a diverse sociocultural background , predominantly from andhra pradesh , karnataka , west bengal , and nepal , operating through various locations scattered across mumbai and thane . these vulnerabilities affect their ability to seek health services , use condoms consistently , and address issues related to negotiation for safer sex behaviors , which in turn have an effect on the overall health of the fsws , including increased chances of acquiring hiv infection . during program implementation some of these included poor economic conditions , high degree of control and influence from partners and clients , lack of identity , low self - esteem and self - confidence , and lack of basic opportunities , such as schooling for fsws children and government identity cards , among others . the data for this study are drawn from the behavioural tracking survey ( bts),2 a cross - sectional behavioral survey conducted in 2010 with 2,431 fsws from 411 sites in mumbai and thane . a local community advisory board , functioning as the local institutional review board , including fsws , fsw community based organizations ( cbos ) , and prominent researchers , was constituted in mumbai , which worked closely with the local research agency and fhi 360 to ensure that the study participants rights were protected . the factors of vulnerability used in the current analysis are based on program experience , anecdotal sharing from fsws at the beginning of the project , and evidence from the literature.816 figure 2 presents the factors that make fsws vulnerable to hiv risk , the program strategies of aastha that focused on the vulnerabilities of fsws , and the outcome measures that were considered in the paper . the vulnerability index and duration in the aastha program were the two key independent measures considered in this paper . the factors used to construct the vulnerability index were based on several items in the questionnaire , which included literacy ( literate , can read and/or write ; illiterate , can not read or write ) , duration in sex work ( 3 + years , 2 years ) , mobility for sex work ( nonmobile , did not travel for sex work ; mobile , traveled for sex work ) , additional source of income other than sex work ( yes / no ) , currently being in debt ( yes / no ) , and having children ( yes / no ) . those who were new to sex work ( 2 years ) , were in debt , were illiterate , had no alternative source of income , were mobile for sex work and had children were considered vulnerable to hiv infection . as a result , duration of participation the program - exposure and service - utilization indicators considered in this paper were 1 ) received counseling / referral services from aastha nongovernmental organizations in the last 6 months ( yes / no ) , 2 ) aware of ats ( yes / no ) , 3 ) aware and accessed ats services in the past 6 months ( yes / no ) , and 4 ) visits to a clinic for sti symptoms in the past year . visits to a clinic to check for sti symptoms was initially measured as a continuous variable by asking fsws the number of times they visited the aastha clinic , and was further dichotomized as visited aastha clinic to check for sti symptoms in the past year / did not visit aastha clinic to check for sti symptoms . self - confidence was measured by asking each fsw 1 ) whether or not she was confident in supporting a fellow sex worker in crisis situation , and 2 ) whether or not she was confident in going to a service provider alone , where she may have to disclose her identity as a sex worker . the fsws ability to claim self - identity was measured by asking how much they agreed with the following two questions : whether they were not ashamed to say that they were a sex worker 1 ) in a meeting with other sex workers , and 2 ) when she met a social or health worker who worked in her community . in all our analyses , we controlled for age ( < 25 , 2529 , 3034 , 35 + years ) , marital status ( ever married , never married ) , and typology of sex work ( brothel - based , bar - based , street-/home-/lodge - based ) . a series of multivariate logistic regression models were constructed to examine the association between vulnerability , duration of exposure to the aastha program , and fsws self - confidence , self - identity , individual agency , and service utilization , adjusting for age , marital status , and typology of sex work . a local community advisory board , functioning as the local institutional review board , including fsws , fsw community based organizations ( cbos ) , and prominent researchers , was constituted in mumbai , which worked closely with the local research agency and fhi 360 to ensure that the study participants rights were protected . the factors of vulnerability used in the current analysis are based on program experience , anecdotal sharing from fsws at the beginning of the project , and evidence from the literature.816 figure 2 presents the factors that make fsws vulnerable to hiv risk , the program strategies of aastha that focused on the vulnerabilities of fsws , and the outcome measures that were considered in the paper . the vulnerability index and duration in the aastha program were the two key independent measures considered in this paper . the factors used to construct the vulnerability index were based on several items in the questionnaire , which included literacy ( literate , can read and/or write ; illiterate , can not read or write ) , duration in sex work ( 3 + years , 2 years ) , mobility for sex work ( nonmobile , did not travel for sex work ; mobile , traveled for sex work ) , additional source of income other than sex work ( yes / no ) , currently being in debt ( yes / no ) , and having children ( yes / no ) . those who were new to sex work ( 2 years ) , were in debt , were illiterate , had no alternative source of income , were mobile for sex work and had children were considered vulnerable to hiv infection . as a result , duration of participation the program - exposure and service - utilization indicators considered in this paper were 1 ) received counseling / referral services from aastha nongovernmental organizations in the last 6 months ( yes / no ) , 2 ) aware of ats ( yes / no ) , 3 ) aware and accessed ats services in the past 6 months ( yes / no ) , and 4 ) visits to a clinic for sti symptoms in the past year . visits to a clinic to check for sti symptoms was initially measured as a continuous variable by asking fsws the number of times they visited the aastha clinic , and was further dichotomized as visited aastha clinic to check for sti symptoms in the past year / did not visit aastha clinic to check for sti symptoms . self - confidence was measured by asking each fsw 1 ) whether or not she was confident in supporting a fellow sex worker in crisis situation , and 2 ) whether or not she was confident in going to a service provider alone , where she may have to disclose her identity as a sex worker . the fsws ability to claim self - identity was measured by asking how much they agreed with the following two questions : whether they were not ashamed to say that they were a sex worker 1 ) in a meeting with other sex workers , and 2 ) when she met a social or health worker who worked in her community . the vulnerability index and duration in the aastha program were the two key independent measures considered in this paper . the factors used to construct the vulnerability index were based on several items in the questionnaire , which included literacy ( literate , can read and/or write ; illiterate , can not read or write ) , duration in sex work ( 3 + years , 2 years ) , mobility for sex work ( nonmobile , did not travel for sex work ; mobile , traveled for sex work ) , additional source of income other than sex work ( yes / no ) , currently being in debt ( yes / no ) , and having children ( yes / no ) . those who were new to sex work ( 2 years ) , were in debt , were illiterate , had no alternative source of income , were mobile for sex work and had children were considered vulnerable to hiv infection . the program - exposure and service - utilization indicators considered in this paper were 1 ) received counseling / referral services from aastha nongovernmental organizations in the last 6 months ( yes / no ) , 2 ) aware of ats ( yes / no ) , 3 ) aware and accessed ats services in the past 6 months ( yes / no ) , and 4 ) visits to a clinic for sti symptoms in the past year . visits to a clinic to check for sti symptoms was initially measured as a continuous variable by asking fsws the number of times they visited the aastha clinic , and was further dichotomized as visited aastha clinic to check for sti symptoms in the past year / did not visit aastha clinic to check for sti symptoms . self - confidence was measured by asking each fsw 1 ) whether or not she was confident in supporting a fellow sex worker in crisis situation , and 2 ) whether or not she was confident in going to a service provider alone , where she may have to disclose her identity as a sex worker . the fsws ability to claim self - identity was measured by asking how much they agreed with the following two questions : whether they were not ashamed to say that they were a sex worker 1 ) in a meeting with other sex workers , and 2 ) when she met a social or health worker who worked in her community . a series of multivariate logistic regression models were constructed to examine the association between vulnerability , duration of exposure to the aastha program , and fsws self - confidence , self - identity , individual agency , and service utilization , adjusting for age , marital status , and typology of sex work . of the total analytical sample of 2,431 fsws , 1,295 ( 53.3% ) were categorized as highly vulnerable . more than a third reported traveling for sex work ( 40% ) , and nearly a quarter ( 25% ) were currently in debt . as seen in table 3 , a higher proportion of fsws who were in the age - group 1825 years , who were never married ( 75.7% versus 48.6% , p<0.001 ) , or who were bar - based sex workers were categorized as highly vulnerable to hiv risk compared to sex workers without these characteristics . fsws who had spent less than 1 year in the aastha program were more likely to be highly vulnerable compared to those who had spent more than 1 year in the program ( 63.9% versus 51.3% , p<0.001 ) . as seen in table 4 , highly vulnerable fsws who were in the aastha program for more than 1 year were more likely to be aware of ats ( adjusted odds ratio [ or ] 2.4 , 95% confidence interval [ ci ] 1.44.0 ; p<0.001 ) , to have accessed ats services in the past 6 months ( adjusted or 2.2 , 95% ci 1.43.6 ; p<0.001 ) , and to have visited an aastha clinic to get a checkup for sti symptoms ( adjusted or 2.4 , 95% ci 1.24.8 , p<0.015 ) compared to those less vulnerable with similar exposure to the aastha program . similarly , table 5 indicates that a significantly higher proportion of highly vulnerable fsws who were associated with or registered with the aastha program for more than a year were not ashamed to disclose their identity as sex workers in a meeting with other sex workers ( adjusted or 2.3 , 95% ci 1.23.6 ; p<0.013 ) and were not ashamed to disclose their identity as sex workers while meeting a health or social worker who worked in their community ( adjusted or 2.1 , 95% ci 1.23.5 ; p<0.008 ) . additionally , highly vulnerable fsws with a longer association with the aastha program were confident in supporting a fellow sex worker in crisis ( adjusted or 1.7 , 95% ci 1.02.8 ; p<0.033 ) compared to less vulnerable fsws with similar exposure in aastha program . these findings corroborate similar studies that show that empowerment strategies are a critical pathway to risk reduction , in contrast to the standard targeted intervention approach.20 while the described behavioral outcomes and individual empowerment are desirable changes in the context of hiv intervention , structural intervention approaches typically create a collective identity among fsws.7 community - led structural intervention approaches , such as the ats , are critical to the process of risk reduction . while the current paper focuses more on certain initiatives of the aastha project , a more detailed analysis of community collectivization strategies has been described elsewhere.2 the results of the paper can be viewed in parallel with the findings of a study in karnataka , which describes the use of similar strategies , among others , in influencing the context - specific social , economic , political , and environmental factors determining risk for hiv.9 it is critical to place the vulnerabilities of an individual fsw in context and to understand the complex interaction of various factors at different levels , including economic , social , and politicolegal . for instance , an ethnographic case study of the sonagachi hiv / std intervention project describes some of the factors contributing to hiv risk in the context of the brothel areas of sonagachi , including the ambiguity of the legal status of the sex - work industry , lack of education , social discrimination , and occupational structures within the industry.7 given the difficulty of measuring vulnerability , there is considerable lack of quantitative research , where a holistic definition of vulnerability has been used for the purpose of analysis . third , the responses of the fsws were part of a larger study to assess the behavioral outcomes of fsws across mumbai and thane , and not to attempt to define vulnerability and assess its association with project services or desired behavior outcomes . , it is critical for hiv / sti interventions to identify and define factors of vulnerability at project inception , prior to developing risk - reduction strategies . moreover , it is important to look at a holistic interaction of different sociocultural vulnerabilities , such as lack of education , poor economic status , financial debts , presence of dependants such as children , lack of other livelihood options , mobility due to sex work , and duration in sex work , as well as changes in the social and legal environment , which contribute towards an environment of increased violence and lack of individual agency and control , rather than just one or two factors .

it is well known that transformers are regarded as indispensable and crucial components in power systems . due to market globalization , and in some cases to accommodate particular specification requests this competitive environment mandates the adoption of design strategies yielding better performance at lower costs . in the past , adly and abd - el - hafiz demonstrated that feed - forward neural networks may be utilized to predict design details of power transformers after being trained using dimensional and winding details of a set of actual transformers . alternatively , finite element analysis ( fea ) coupled to an educated trial and error approach was introduced . furthermore , a computer - aided trial search looping algorithm aiming at minimizing transformer design cost has been demonstrated . other approaches coupling fea to a knowledge - based design optimization strategy and genetic algorithms were presented [ 57 ] . hernndez and arjona proposed another approach that couples classical design equations to an intelligent design search algorithm . a quick review of these methodologies reveals that a wide span of design strategies could be utilized to achieve an optimum power transformer design . for instance , analytical formulations may be utilized for the quick estimation of transformer dimensions and design details . methodologies based upon more accurate fea computations offer precise estimation of transformer performance measures , provided that design specifics are suggested a priori . other methodologies , on the other hand , may utilize a hybrid strategy or even non - traditional heuristic and/or evolutionary computation approaches . several techniques have addressed transformer design problems using single - objective particle swarm optimization ( pso ) . demonstrated that the two objectives of minimizing power loss and leakage inductance were combined into one objective function using weighted aggregation . single - objective evolutionary optimization was , then applied using a hybrid algorithm of pso and differential evolution . jalilvand and bagheri also utilized single - objective pso in the optimal design of protective current transformers . the objectives of making current measurements more accurate and designing more efficient current transformers in terms of both size and cost were formulated as an optimization problem to be solved by pso . on the other hand , du et al . focused on improving the standard single - objective pso algorithm and utilizing the improved version in the optimal design of rectifier transformers . the reduction of a multi - objective optimization problem to a single - objective problem is usually performed by constructing a weighted sum of the original objective functions . while such methods are easy to implement and use , it is difficult to determine the appropriate weight coefficients when enough information about the problem is not available . another drawback of such approaches is that several runs of the algorithm are needed in order to obtain a set of optimal compromise solutions to choose from . furthermore , some optimal solutions can not be obtained , in some cases , regardless of the weight combinations used . hence , multi - objective pso becomes useful as it enables finding several optimal compromise solutions in a single run of the algorithm instead of having to perform a series of separate runs as in the case of classical optimization methods . the purpose of this paper is to present a power transformer design methodology using multi - objective evolutionary optimization . using this methodology , which is tailored to be target performance design - oriented estimated design parameters and details using the proposed methodology may also be considered for further refinement by other fea approaches . it should be stated that while the proposed methodology is analytical in nature , some parameter range settings have utilized previously reported power transformer field computation results . details of the proposed methodology as well as sample design results are reported in the following sections . in addition to the mandated primary line voltage vl1 , secondary line voltage vl2 and supply frequency f , a three - phase power transformer design is usually optimized to meet volt - ampere rating s , total copper losses pcu , no - load losses pnl and equivalent reactance x requirements . in other words , a performance - oriented design problem reduces to the proper selection of windings and dimensional details that would lead to a set of targeted performance figures . expressions linking the above - mentioned performance figures to the windings and dimensional details of a three - phase power transformer may be deduced in a systematic way as given below ( please refer , for instance , to [ 1517]).(1)vl13=4.44fbkfkc4d2n1,where b is the core maximum flux density ( magnetic loading ) , kf is the laminations stack factor , kc is the gross area to maximum circular area ratio , d is the core bounding diameter and n1 is the primary winding number of turns . it is also known that the window space factor of a three - phase transformer sw may be expressed as:(2)sw=2n1ac1 + 2n2ac2hwww , where n2 is the secondary winding number of turns , hw is the window height , ww is the window width , while ac1 and ac2 represent the primary and secondary winding cross sectional areas , respectively . denoting the window height to width ratio by kw and assuming a common current density ( electric loading ) j in both windings while n1iph1 = n2iph2 ( where iph1 and iph2 are the primary and secondary phase currents ) , expression ( 2 ) may be rewritten in the form:(3)sw=4n1ac1kwhw2 . it should be pointed out here that , usually , current densities in low and high voltage windings are not identical due to standard wire size availability and/or other design factor constraints . nevertheless , the assumed current density j may be regarded as an average figure for both windings . from expressions ( 1 ) and ( 3 ) , the volt - ampere rating of a three - phase transformer may thus be expressed as:(4)s=34.44fbkfkc4d2n1jswhw24kwn1=3.33kfkcswf4kwjbd2hw2 . namely , these three components are the ohmic winding losses pcu - ohmic , the eddy current losses in the windings pcu - eddy and the copper terminals connection losses pcu - con . while designing a transformer to meet pre - mandated specification , maintaining the total copper losses below the threshold values becomes a must . in order to achieve this goal alternatively , appropriate computational safety factors may be applied to fast analytical design methodologies . while pcu - con 0.05pcu - ohmic , eddy current losses in transformer windings are dependent on the window height to width ratio kw . as previously reported by saleh et al . , electromagnetic field computation results suggest that , taking 2 kw 2.5 , winding eddy current losses may be estimated as pcu - eddy 0.15pcu - ohmic . from the previous common electric loading assumptions and for the aforementioned kw range , total copper losses may thus be given by the expression:(5)pcu1.23iph12cun1lmt1ac1 + 3iph22cun2lmt2ac2=7.2j2cun1ac1lmt=1.2j2cuvolcu , where cu is the specific resistivity of copper , volcu is the overall copper volume , while lmt1 , lmt2 and lmt represent the average turn length of the primary , secondary and both windings , respectively . no - load losses pnl , on the other hand , may also be regarded as a superposition of two components . more specifically , these two components are the core losses pfe and stray losses pstray . once more , it should be stated that accurate estimation of the stray losses requires massive computational resources that involve complex models of coupled magnetic , thermal and mechanical variables ( please refer , for instance , to [ 1921 ] ) . by adopting standard fabrication methodologies [ 1517 ] , stray losses may be estimated in accordance with the inequality pstray 0.3pfe . consequently , an upper limit for the no - load losses may be expressed in the form:(6)pnl1.3pfe=1.3wfe(b , f)fevolfe1.3wfe(b , f)fekfkc4d2(3hw+4ww+6d)1.3wfe(b , f)fekfkc4d23kw+4kwhw+6d , where fe is the steel lamination density and wfe(b , f ) is the specific core losses as a function of flux density and frequency that may be deduced by referring to the core lamination specifications data sheet . please note that explicit function formulation for wfe(b , f ) is either given in manufacturers specification sheets or simply inferred by fitting reported curves . referring to , the equivalent transformer reactance may be computed from:(7)x=2fon12lmtlwha+b1+b23,where o is the permeability of free space , lwh is the windings height , a is the spacing between the low and high voltage windings , while b1 and b2 represent the gross primary and secondary winding thicknesses , respectively . following the assumption of identical current densities for both windings and assuming similar winding heights , winding thicknesses may be assumed equal such that b1 = b2 = b ( please refer to fig . 1 ) . usually , the spacing between high voltage ( outer ) windings is double the distance between a low voltage ( inner ) winding and its corresponding high voltage winding . in other words , 4(a + b ) . from practical industrial considerations a b/4 . in this case , the winding thickness may be correlated to the window dimensions according to:(8)bww5hw5kw . denoting the winding height lwh to the window height hw ratio by kh and following the previously stated practical assumptions as well as ( 8) , expression ( 7 ) may be rewritten in the form:(9)x=2fon12(d+2b+a)khhwa+2b3=112fon1230khkwd+9hw20kw . from ( 3 ) , n1 may be expressed in the form:(10)n1=swhw24kwac1=swhw2j4kwiph1 . substituting ( 10 ) into ( 9 ) , we obtain:(11)x=112fosw2480khkw3iph12j2hw4d+9hw20kw . following the same window configuration assumptions , expression ( 5 ) ( 4 ) , ( 6 ) , ( 11 ) and ( 12 ) , it is clear that the target performance oriented three - phase transformer design problem may be reduced to the proper selection of four unknowns . namely , those unknowns are the current density j , the maximum core magnetic flux density b , the transformer core diameter d , and the window height hw . dividing ( 11 ) by ( 12 ) , we get:(13)xpcu=11fosw864cukhkw2iph12hw2 . consequently , hw may be deduced from the expression:(14)hw=864cukhkw2iph1211foswxpcu.after obtaining hw value , remaining unknowns may be deduced by solving ( 4 ) , ( 6 ) and ( 12 ) . given the highly nonlinear nature of the equations under consideration , multi - objective optimization is needed to achieve a minimum cost design subject to the range restrictions for unknowns j , b and d. details of the multi - objective problem formulation are given in the following section . in engineering design problems , computational models are often used to describe the complex behaviors of physical systems and optimal solutions are sought with respect to some performance criteria . hence , multi - objective optimization becomes useful in obtaining a set of optimal compromise solutions ( pareto - optimal front ) so that the designer can select the best choice . the basic concepts of multi - objective optimization are introduced using a d - dimensional search space , srd , and k objective functions defined over s as given by bui and alam : ( 15)f(x)=[f1(x),f2(x), ,fk(x)],subject to m inequality constraints:(16)gi(x)0,i=1, ,m . the objective functions fi(x ) may be conflicting with each other , thereby preventing the detection of a single global minimum at the same point in . consequently , optimality of a solution in multi - objective problems is defined differently . a vector v = ( v1 , v2 , , vk ) is said to dominate a vector u = ( u1 , u2 , , uk ) for a multi - objective minimization problem if and only if vi ui for all i = 1 , 2 , ... , k and vi < ui for at least one component , where k is the dimension of the objective space . a solution u u , where u is the universe , is said to be pareto optimal if and only if there exists no other solution v u , such that u is dominated by v. such solutions , u , are called non - dominated solutions . the set of all such non - dominated solutions constitutes the pareto - optimal front . for the transformer design approach under consideration , s , pcu , pnl and x multi - objective optimization is then utilized to determine the other leading design parameters ; j , b and d. hence , x = ( j , b , d ) . within the current implementation , expressions ( 15 ) and ( 16 ) are formulated as:(17)f(x)=scmp(x)-s24,(pcucmp(x)-pcu)4,pnlcmp(x)-pnl44,subject to the following x inequality constraints:(18)jlbldljbdjubudu , where s , pcucmp and pnlcmp are the computed volt - ampere rating , the computed total copper losses and the computed no - load losses , respectively . it should be pointed out that the inequality ranges given in ( 18 ) should be in accordance with the typical lower and upper limits of j , b and d for power transformers in the range under consideration . inspired by the behavior of bird flocks or insect swarms , kennedy and eberhart first proposed pso in 1995 . pso is a population based heuristic , where the population of the potential solutions is called a swarm and each individual solution within the swarm is called a particle . considering a d - dimensional search space , an ith particle is associated with a position in the search space xi = ( xi,1 , , xi , d ) , a velocity vi = ( vi,1 , , vi , d ) and an individual experience vector pbi = ( pbi,1 , , pbi , d ) storing the position corresponding to the particle s personal best performance . experience of the whole swarm is captured in the vector gb = ( gb1 , , gbd ) , which corresponds to the position of the global best performance in the swarm . the movement of a particle toward the optimum solution is governed by updating its velocity and position according to eqs . 2 , respectively . while the parameter w is the inertia weight , parameters c1 and c2 are acceleration coefficients . parameters r1 and r2 are random numbers , generated uniformly in the interval [ 0 , 1 ] and are responsible for providing randomness to the flight of the swarm . the second term in eq . 2 is the cognition term , which takes into account only the particle s individual experience . the third term in eq . the values of c1 and c2 allow the particle to tune the cognition and social terms , respectively . a larger value of c1 allows exploration , while a larger value of c2 encourages exploitation . single objective pso has been successfully utilized in many engineering applications such as the optimization of devices and systems and field computation in nonlinear magnetic media . in order to handle multi - objective optimization , several approaches adapt single objective pso using the pareto dominance concept to determine the best positions that will guide the swarm during search . additional criteria are also imposed to take into consideration further issues such as swarm diversity and pareto front spread . in this paper , the time variant multi - objective particle swarm optimization ( tv - mopso ) algorithm is utilized . to achieve good balance between exploration and exploitation of the search space , tv - mopso is adaptive in nature with respect to its inertia weight and acceleration coefficients . a mutation operator is incorporated to resolve the problem of premature convergence to the local pareto - optimal front that is often observed in multi - objective pso . an archive is also maintained to store the non - dominated solutions found during execution . the first step generates an initial swarm swrmo of size ms with zero velocities and random values for the coordinates from the respective domains of each dimension . an archive of maximum size ma is initialized to contain the non - dominated solutions from swrmo . the second step represents the main iteration cycle in which the swarm is updated , the archive is updated and the swarm is mutated at each iteration t. the swarm swrmt is updated , in step ( 2.1 ) of the algorithm , by updating the velocity and coordinates of each particle using eqs . 2 , respectively . to update the velocity , the global best solution is obtained from the archive using a diversity consideration . the method for computing diversity of the solutions is based on a nearest neighbor concept . the present solution is compared with the personal best solution , and replaces the latter only if it dominates that solution . moreover , these parameters include an inertia coefficient , w , a local acceleration coefficient , c1 , and a global acceleration coefficient , c2 . the inertia coefficient w is decreased linearly with each iteration from an initial value wi to a final value wf . the value of w at iteration number t is calculated as:(19)w=(wi - wf)(m - t)m+wf , where m is the maximum number of iterations . to compromise between exploration and exploitation of the search space , the cognitive acceleration coefficient c1 and the social acceleration coefficient c2 are varied linearly with each iteration as given by ( 20 ) and ( 21 ) , respectively . while c1 decreases from the initial value c1i to the final value c1f , c2 increases from c2i to c2f.(20)c1=(c1f - c1i)tm+c1i,(21)c2=(c2f - c2i)tm+c2i . the archive at is updated , in step ( 2.2 ) of the algorithm , by including the non - dominated solutions from the combined population of the swarm and the archive . if the size of the archive exceeds the maximum limit ( ma ) , it is truncated using the diversity consideration . to explore the search space to a greater extent , while obtaining better diversity , a mutation operator is used in step ( 2.3 ) of the algorithm shown in fig . mutation is performed with probability inversely proportional to the chromosome length d. given a particle p , a randomly chosen coordinate ( i.e. , variable ) of the particle , pk , is mutated as follows:(22)pk=pk+(t , pku - pk)if flip=0pk-(t , pk - pkl)if flip=1,where flip , pkl and pku denote the random event of returning 0 or 1 , the lower and the upper limits of pk . the function is defined by:(23)(t , x)=x1-r(1-tm)q , where r is a random number in the range [ 0 , 1 ] , m is the maximum number of iterations and t is the iteration number . the parameter q determines the mutation s dependence level on the iteration number . after executing the specified number of iterations this archive contains the final non - dominated front ( i.e. , pareto optimal front ) . to serve the testing and estimation purposes , the proposed design approach has been implemented in digital form . the methodology has been particularly utilized to design 2550 mva , 66 kv/11 kv , dyn11 , 50 hz power transformers subject to a variety of design performance constraints . as per practical transformer stacking and assembly measures for the mva range under consideration , it was decided to set throughout the computations sw = 0.2 , kh = 0.9 , kf = 0.95 , and consider 11-step cores ( leading to kc = 0.958 ) [ 1517 ] . common values for o , cu and fe were taken as 4 10 h / m , 2.1 10 m and 7.65 10 kg / m , respectively . using armco steel tran - cor - h0 carlite-3 core laminations , an expression for wfe(b , 50 hz ) was inferred from data offered by the manufacturer ( please refer to ) . the typical values of jl , ju , bl , bu , dl and du for power transformers in the range under consideration are set as 1.1 10 , 3.2 10 , 1.0 , 1.8 , 0.1 , 0.7 , respectively . in order to test the proposed performance - oriented design methodology , two transformers ( rated 25 mva and 40 mva ) of known manufacturer design details and measured performances are considered . it should be stated here that a considerable number of units of these particular transformer designs , which obviously passed all standard routine tests , has been acquired and installed in several national and regional grid sub - stations . as previously discussed , measured performance figures of actual transformers are taken as the target design requirements for the design methodology . the tv - mopso algorithm is executed using a swarm of ms = 50 particles , a maximum archive size of ma = 200 and for m = 1000 iterations . the parameters used in the reported results are wi = 0.7 , wf = 0.4 , c1i = 2.5 , c1f = 0.5 , c2i = 0.5 , c2f = 2.5 and q = 5 . 3 . out of a set of design parameters inferred by the tv - mopso implementation , the design corresponding to a minimum iron core volume is taken as the optimum choice . comparisons between design parameters of the actual transformers and those proposed by the design methodology under consideration are given in tables 1 and 2 . variations between actual and computed performance ( as well as cost ) figures are also given in the same tables . with the exception of the suggested current density j , it is clear that the proposed approach leads to good qualitative and quantitative performance - oriented design results . moreover , the proposed higher j value by the suggested methodology may be regarded as a possible cost minimization option as indicated in tables 1 and 2 by the possible reduction in the transformers copper volume . using the proposed approach , computations are also carried out to investigate the design parameters deviation from those of the 40 mva test transformer as a result of changing volt ampere rating and power loss requirements . in the first computation set , design parameters corresponding to 30 mva and 50 mva transformer ratings having the same specifics , per - unit reactances and percentage total copper and no - load losses ( i.e. , efficiencies ) were computed . 4 , almost all design parameter values increase as the transformer rating is increased while maintaining the same percentage total copper and no - load losses . it should be mentioned here that hw variation is minimal in these cases since transformer voltages are assumed unchanged . this suggests that any rating variation will similarly affect the phase currents squared and total copper loss values , thus minimally affecting hw as indicated by expression ( 14 ) . in the second computation set , design parameters corresponding to 40 mva transformer ratings having the same specifics , per - unit reactances but while varying the percentage total copper and no - load losses ( i.e. , efficiencies ) are computed . as shown in fig . 5 , a smaller loss restriction is achieved by a larger size transformer with reduced current and flux density values . this is a particularly encountered design trade - off between capital and running costs for a power transformer . in this paper , a performance - oriented power transformer design methodology using multi - objective evolutionary optimization has been introduced in detail . experimental testing as well as other presented computational results clearly demonstrates the qualitative and quantitative accuracy of the methodology . one advantage of using multi - objective evolutionary optimization is that it deals simultaneously with a set of possible solutions ( i.e. , a population ) . this enables finding several members of the pareto front in a single run of the algorithm instead of having to perform a series of separate runs as in the case of classical optimization methods . these options can be extremely useful to minimize overall production costs in view of the changing global prices of different transformer components , especially copper and steel laminations . the proposed methodology may be easily utilized to obtain a quick first guess design details for more sophisticated design approaches such as those utilizing fea packages . moreover , in the presence of detailed design strategies , the proposed methodology may be easily improved to relax some assumptions by including those strategies . future work is planned to enhance the accuracy of the proposed methodology as well as to extend its applicability to cover more detailed transformer design aspects .

transformers are regarded as crucial components in power systems . due to market globalization , power transformer manufacturers are facing an increasingly competitive environment that mandates the adoption of design strategies yielding better performance at lower costs . in this paper , a power transformer design methodology using multi - objective evolutionary optimization is proposed . using this methodology , which is tailored to be target performance design - oriented , quick rough estimation of transformer design specifics may be inferred . testing of the suggested approach revealed significant qualitative and quantitative match with measured design and performance values . details of the proposed methodology as well as sample design results are reported in the paper .

Introduction Performance-oriented power transformer design approach Formulation as a multi-objective optimization problem Multi-objective particle swarm optimization Implementation and design examples Conclusions Conflict of interest Compliance with Ethics Requirements

it is well known that transformers are regarded as indispensable and crucial components in power systems . due to market globalization , and in some cases to accommodate particular specification requests this competitive environment mandates the adoption of design strategies yielding better performance at lower costs . in the past , adly and abd - el - hafiz demonstrated that feed - forward neural networks may be utilized to predict design details of power transformers after being trained using dimensional and winding details of a set of actual transformers . furthermore , a computer - aided trial search looping algorithm aiming at minimizing transformer design cost has been demonstrated . a quick review of these methodologies reveals that a wide span of design strategies could be utilized to achieve an optimum power transformer design . for instance , analytical formulations may be utilized for the quick estimation of transformer dimensions and design details . methodologies based upon more accurate fea computations offer precise estimation of transformer performance measures , provided that design specifics are suggested a priori . several techniques have addressed transformer design problems using single - objective particle swarm optimization ( pso ) . single - objective evolutionary optimization was , then applied using a hybrid algorithm of pso and differential evolution . jalilvand and bagheri also utilized single - objective pso in the optimal design of protective current transformers . the objectives of making current measurements more accurate and designing more efficient current transformers in terms of both size and cost were formulated as an optimization problem to be solved by pso . focused on improving the standard single - objective pso algorithm and utilizing the improved version in the optimal design of rectifier transformers . the reduction of a multi - objective optimization problem to a single - objective problem is usually performed by constructing a weighted sum of the original objective functions . another drawback of such approaches is that several runs of the algorithm are needed in order to obtain a set of optimal compromise solutions to choose from . furthermore , some optimal solutions can not be obtained , in some cases , regardless of the weight combinations used . hence , multi - objective pso becomes useful as it enables finding several optimal compromise solutions in a single run of the algorithm instead of having to perform a series of separate runs as in the case of classical optimization methods . the purpose of this paper is to present a power transformer design methodology using multi - objective evolutionary optimization . using this methodology , which is tailored to be target performance design - oriented estimated design parameters and details using the proposed methodology may also be considered for further refinement by other fea approaches . it should be stated that while the proposed methodology is analytical in nature , some parameter range settings have utilized previously reported power transformer field computation results . details of the proposed methodology as well as sample design results are reported in the following sections . in addition to the mandated primary line voltage vl1 , secondary line voltage vl2 and supply frequency f , a three - phase power transformer design is usually optimized to meet volt - ampere rating s , total copper losses pcu , no - load losses pnl and equivalent reactance x requirements . in other words , a performance - oriented design problem reduces to the proper selection of windings and dimensional details that would lead to a set of targeted performance figures . expressions linking the above - mentioned performance figures to the windings and dimensional details of a three - phase power transformer may be deduced in a systematic way as given below ( please refer , for instance , to [ 1517]). (1)vl13=4.44fbkfkc4d2n1,where b is the core maximum flux density ( magnetic loading ) , kf is the laminations stack factor , kc is the gross area to maximum circular area ratio , d is the core bounding diameter and n1 is the primary winding number of turns . it is also known that the window space factor of a three - phase transformer sw may be expressed as:(2)sw=2n1ac1 + 2n2ac2hwww , where n2 is the secondary winding number of turns , hw is the window height , ww is the window width , while ac1 and ac2 represent the primary and secondary winding cross sectional areas , respectively . denoting the window height to width ratio by kw and assuming a common current density ( electric loading ) j in both windings while n1iph1 = n2iph2 ( where iph1 and iph2 are the primary and secondary phase currents ) , expression ( 2 ) may be rewritten in the form:(3)sw=4n1ac1kwhw2 . it should be pointed out here that , usually , current densities in low and high voltage windings are not identical due to standard wire size availability and/or other design factor constraints . nevertheless , the assumed current density j may be regarded as an average figure for both windings . namely , these three components are the ohmic winding losses pcu - ohmic , the eddy current losses in the windings pcu - eddy and the copper terminals connection losses pcu - con . in order to achieve this goal alternatively , appropriate computational safety factors may be applied to fast analytical design methodologies . while pcu - con 0.05pcu - ohmic , eddy current losses in transformer windings are dependent on the window height to width ratio kw . as previously reported by saleh et al . , electromagnetic field computation results suggest that , taking 2 kw 2.5 , winding eddy current losses may be estimated as pcu - eddy 0.15pcu - ohmic . from the previous common electric loading assumptions and for the aforementioned kw range , total copper losses may thus be given by the expression:(5)pcu1.23iph12cun1lmt1ac1 + 3iph22cun2lmt2ac2=7.2j2cun1ac1lmt=1.2j2cuvolcu , where cu is the specific resistivity of copper , volcu is the overall copper volume , while lmt1 , lmt2 and lmt represent the average turn length of the primary , secondary and both windings , respectively . no - load losses pnl , on the other hand , may also be regarded as a superposition of two components . once more , it should be stated that accurate estimation of the stray losses requires massive computational resources that involve complex models of coupled magnetic , thermal and mechanical variables ( please refer , for instance , to [ 1921 ] ) . by adopting standard fabrication methodologies [ 1517 ] , stray losses may be estimated in accordance with the inequality pstray 0.3pfe . consequently , an upper limit for the no - load losses may be expressed in the form:(6)pnl1.3pfe=1.3wfe(b , f)fevolfe1.3wfe(b , f)fekfkc4d2(3hw+4ww+6d)1.3wfe(b , f)fekfkc4d23kw+4kwhw+6d , where fe is the steel lamination density and wfe(b , f ) is the specific core losses as a function of flux density and frequency that may be deduced by referring to the core lamination specifications data sheet . referring to , the equivalent transformer reactance may be computed from:(7)x=2fon12lmtlwha+b1+b23,where o is the permeability of free space , lwh is the windings height , a is the spacing between the low and high voltage windings , while b1 and b2 represent the gross primary and secondary winding thicknesses , respectively . following the assumption of identical current densities for both windings and assuming similar winding heights , winding thicknesses may be assumed equal such that b1 = b2 = b ( please refer to fig . in this case , the winding thickness may be correlated to the window dimensions according to:(8)bww5hw5kw . denoting the winding height lwh to the window height hw ratio by kh and following the previously stated practical assumptions as well as ( 8) , expression ( 7 ) may be rewritten in the form:(9)x=2fon12(d+2b+a)khhwa+2b3=112fon1230khkwd+9hw20kw . from ( 3 ) , n1 may be expressed in the form:(10)n1=swhw24kwac1=swhw2j4kwiph1 . following the same window configuration assumptions , expression ( 5 ) ( 4 ) , ( 6 ) , ( 11 ) and ( 12 ) , it is clear that the target performance oriented three - phase transformer design problem may be reduced to the proper selection of four unknowns . namely , those unknowns are the current density j , the maximum core magnetic flux density b , the transformer core diameter d , and the window height hw . consequently , hw may be deduced from the expression:(14)hw=864cukhkw2iph1211foswxpcu.after obtaining hw value , remaining unknowns may be deduced by solving ( 4 ) , ( 6 ) and ( 12 ) . given the highly nonlinear nature of the equations under consideration , multi - objective optimization is needed to achieve a minimum cost design subject to the range restrictions for unknowns j , b and d. details of the multi - objective problem formulation are given in the following section . hence , multi - objective optimization becomes useful in obtaining a set of optimal compromise solutions ( pareto - optimal front ) so that the designer can select the best choice . the basic concepts of multi - objective optimization are introduced using a d - dimensional search space , srd , and k objective functions defined over s as given by bui and alam : ( 15)f(x)=[f1(x),f2(x), ,fk(x)],subject to m inequality constraints:(16)gi(x)0,i=1, ,m . the objective functions fi(x ) may be conflicting with each other , thereby preventing the detection of a single global minimum at the same point in . consequently , optimality of a solution in multi - objective problems is defined differently . a vector v = ( v1 , v2 , , vk ) is said to dominate a vector u = ( u1 , u2 , , uk ) for a multi - objective minimization problem if and only if vi ui for all i = 1 , 2 , ... , k and vi < ui for at least one component , where k is the dimension of the objective space . a solution u u , where u is the universe , is said to be pareto optimal if and only if there exists no other solution v u , such that u is dominated by v. such solutions , u , are called non - dominated solutions . for the transformer design approach under consideration , s , pcu , pnl and x multi - objective optimization is then utilized to determine the other leading design parameters ; j , b and d. hence , x = ( j , b , d ) . it should be pointed out that the inequality ranges given in ( 18 ) should be in accordance with the typical lower and upper limits of j , b and d for power transformers in the range under consideration . pso is a population based heuristic , where the population of the potential solutions is called a swarm and each individual solution within the swarm is called a particle . considering a d - dimensional search space , an ith particle is associated with a position in the search space xi = ( xi,1 , , xi , d ) , a velocity vi = ( vi,1 , , vi , d ) and an individual experience vector pbi = ( pbi,1 , , pbi , d ) storing the position corresponding to the particle s personal best performance . experience of the whole swarm is captured in the vector gb = ( gb1 , , gbd ) , which corresponds to the position of the global best performance in the swarm . parameters r1 and r2 are random numbers , generated uniformly in the interval [ 0 , 1 ] and are responsible for providing randomness to the flight of the swarm . 2 is the cognition term , which takes into account only the particle s individual experience . in order to handle multi - objective optimization , several approaches adapt single objective pso using the pareto dominance concept to determine the best positions that will guide the swarm during search . in this paper , the time variant multi - objective particle swarm optimization ( tv - mopso ) algorithm is utilized . to achieve good balance between exploration and exploitation of the search space , tv - mopso is adaptive in nature with respect to its inertia weight and acceleration coefficients . a mutation operator is incorporated to resolve the problem of premature convergence to the local pareto - optimal front that is often observed in multi - objective pso . the second step represents the main iteration cycle in which the swarm is updated , the archive is updated and the swarm is mutated at each iteration t. the swarm swrmt is updated , in step ( 2.1 ) of the algorithm , by updating the velocity and coordinates of each particle using eqs . to update the velocity , the global best solution is obtained from the archive using a diversity consideration . the method for computing diversity of the solutions is based on a nearest neighbor concept . moreover , these parameters include an inertia coefficient , w , a local acceleration coefficient , c1 , and a global acceleration coefficient , c2 . to compromise between exploration and exploitation of the search space , the cognitive acceleration coefficient c1 and the social acceleration coefficient c2 are varied linearly with each iteration as given by ( 20 ) and ( 21 ) , respectively . the archive at is updated , in step ( 2.2 ) of the algorithm , by including the non - dominated solutions from the combined population of the swarm and the archive . if the size of the archive exceeds the maximum limit ( ma ) , it is truncated using the diversity consideration . to explore the search space to a greater extent , while obtaining better diversity , a mutation operator is used in step ( 2.3 ) of the algorithm shown in fig . mutation is performed with probability inversely proportional to the chromosome length d. given a particle p , a randomly chosen coordinate ( i.e. , variable ) of the particle , pk , is mutated as follows:(22)pk=pk+(t , pku - pk)if flip=0pk-(t , pk - pkl)if flip=1,where flip , pkl and pku denote the random event of returning 0 or 1 , the lower and the upper limits of pk . the function is defined by:(23)(t , x)=x1-r(1-tm)q , where r is a random number in the range [ 0 , 1 ] , m is the maximum number of iterations and t is the iteration number . to serve the testing and estimation purposes , the proposed design approach has been implemented in digital form . the methodology has been particularly utilized to design 2550 mva , 66 kv/11 kv , dyn11 , 50 hz power transformers subject to a variety of design performance constraints . the typical values of jl , ju , bl , bu , dl and du for power transformers in the range under consideration are set as 1.1 10 , 3.2 10 , 1.0 , 1.8 , 0.1 , 0.7 , respectively . in order to test the proposed performance - oriented design methodology , two transformers ( rated 25 mva and 40 mva ) of known manufacturer design details and measured performances are considered . it should be stated here that a considerable number of units of these particular transformer designs , which obviously passed all standard routine tests , has been acquired and installed in several national and regional grid sub - stations . as previously discussed , measured performance figures of actual transformers are taken as the target design requirements for the design methodology . the tv - mopso algorithm is executed using a swarm of ms = 50 particles , a maximum archive size of ma = 200 and for m = 1000 iterations . the parameters used in the reported results are wi = 0.7 , wf = 0.4 , c1i = 2.5 , c1f = 0.5 , c2i = 0.5 , c2f = 2.5 and q = 5 . out of a set of design parameters inferred by the tv - mopso implementation , the design corresponding to a minimum iron core volume is taken as the optimum choice . comparisons between design parameters of the actual transformers and those proposed by the design methodology under consideration are given in tables 1 and 2 . variations between actual and computed performance ( as well as cost ) figures are also given in the same tables . with the exception of the suggested current density j , it is clear that the proposed approach leads to good qualitative and quantitative performance - oriented design results . moreover , the proposed higher j value by the suggested methodology may be regarded as a possible cost minimization option as indicated in tables 1 and 2 by the possible reduction in the transformers copper volume . using the proposed approach , computations are also carried out to investigate the design parameters deviation from those of the 40 mva test transformer as a result of changing volt ampere rating and power loss requirements . in the first computation set , design parameters corresponding to 30 mva and 50 mva transformer ratings having the same specifics , per - unit reactances and percentage total copper and no - load losses ( i.e. in the second computation set , design parameters corresponding to 40 mva transformer ratings having the same specifics , per - unit reactances but while varying the percentage total copper and no - load losses ( i.e. as shown in fig . 5 , a smaller loss restriction is achieved by a larger size transformer with reduced current and flux density values . this is a particularly encountered design trade - off between capital and running costs for a power transformer . in this paper , a performance - oriented power transformer design methodology using multi - objective evolutionary optimization has been introduced in detail . experimental testing as well as other presented computational results clearly demonstrates the qualitative and quantitative accuracy of the methodology . one advantage of using multi - objective evolutionary optimization is that it deals simultaneously with a set of possible solutions ( i.e. , a population ) . this enables finding several members of the pareto front in a single run of the algorithm instead of having to perform a series of separate runs as in the case of classical optimization methods . these options can be extremely useful to minimize overall production costs in view of the changing global prices of different transformer components , especially copper and steel laminations . the proposed methodology may be easily utilized to obtain a quick first guess design details for more sophisticated design approaches such as those utilizing fea packages . moreover , in the presence of detailed design strategies , the proposed methodology may be easily improved to relax some assumptions by including those strategies . future work is planned to enhance the accuracy of the proposed methodology as well as to extend its applicability to cover more detailed transformer design aspects .

premature coronary artery disease ( cad ) is the occurrence of cardiac events at a younger age ( before the age 45 years in men and before the age of 55 years in women ) . in its severe form premature cad the incidence and prevalence of cad in india is very high and currently india is having a maximum number of cad patients in the world putting india in the state of a cad epidemic . high degree of mortality , morbidity , and prematurity of cad has put a great burden on the society and health care expenditure , and it is the most common cause of death worldwide . the higher degree of prematurity and the severity of cad in the indian population as compared to any other ethnic group in the world can only be partially explained on the basis of conventional lipid parameters . partial explanation is based on the fact that the indians generally tend to have a higher level of low density lipoprotein ( ldl)-c and lower level of high - density lipoprotein ( hdl)-c as compared to their western counterparts . dyslipidemia is one of the most common risk factors for the development of atherosclerosis , and the increased values of the conventional lipid parameters are associated with increased risk of cad . although many studies have shown a strong correlation between dyslipidemia and cad , but it does not explain the high prevalence and severity of cad completely in all the cases . it has been found in many studies that cad occurs even in the patients who have normal levels of ldl and hdl . therefore , researchers have defined many indices such as ai , lipid tetrad index ( lti ) , and lipid pentad index ( lpi ) on the basis of the values of the conventional lipid parameters for assessing consolidated lipid risk factor in cad . although these existing indices are better than the individual lipid parameters , the values of these indices are many times high even in normal persons free from cad . moreover , the occurrence of cad in normolipidemic persons gives the insight to look beyond the conventional lipid profile and researchers have been trying to elucidate the possibility of other parameters that must be statistically better predictors in the pathogenesis of atherosclerosis which is the underlying pathology of cad . hence , in our study , we have estimated nonconventional lipid parameters such as oxidized ( ox ldl ) , small dense ( sd ldl ) , apolipoprotein a1 ( apo a1 ) , lipoprotein ( a ) , and apolipoprotein b ( apo b ) and used these parameters for the calculation of advanced atherogenic index ( aai ) . then we compared the values of aai with that of the existing lipid indices that have already defined as an assessment of consolidated lipid risk factor in cad . lti was given by enas , and it explains the risk of dyslipidemia in cad in the indian population . lti includes triglyceride ( tc ) , total cholesterol ( tg ) , lipoprotein ( a ) and hdl - c . for assessment of risk factors in the children of premature cad patients includes tc , tg , apo b , lipoprotein ( a ) , and apo a1 . although lpi have shown better discrimination between cases and controls but this index was actually calculated in the children of cad patients rather than the patients themselves , and limited data are available in india , which has evaluated this index in cad patients . most importantly , none of the above indices included the more important lipid parameters such as ox ldl and sd ldl which are better markers of cad as proved by many studies . the significance to include sd ldl instead of ldl lies in the fact that ldl is not homogenous in size and shape and on differential centrifugation it is divided into two fractions . one fraction is small and dense , and the other one is large and buoyant . studies have shown that higher proportion of sd ldl in an individual is associated with higher risk of occurrence of cad because it has a higher tendency to get ox and ox ldl , in turn , is actually deposited inside the macrophages to form the foam cells . normal ldl deposited inside macrophages can be taken back by reverse transport cholesterol ( rct ) by hdl , but once it is converted to ox ldl , then it down - regulates the scavenger receptors on the macrophages and decreases the extent of rct because apo a1 of hdl acts on these receptors . hence , higher amount of sd ldl will lead to the formation of increased amount of ox ldl . studies have shown a strong correlation between the values of sd ldl and severity of cad . therefore , the levels of sd ldl and ox ldl assumes greater importance as the lipid risk factors for the development of atherosclerosis . the increased values of sd ldl and ox ldl can explain the occurrence of cad in patients with normal levels of ldl as many studies have shown the occurrence of cad even in normolipidemic patients having a normal or subnormal level of ldl . therefore , lipid index which will include these parameters this led us to calculate a new aai from the values of the nonconventional lipid risk factors such as sd ldl , ox ldl , apo a1 , apo b , and lipoprotein ( a ) . another importance of this index is that all the parameters used in its calculation are genetically determined and can explain the prematurity and severity of cad in indian population . then we used various statistical methods to compare the aai the existing indices such as ai , lti , and lpi to check whether it offers any advantage in predicting the consolidated lipid risk in cad . after taking prior ethical clearance from the ethical committee of the institute , this study was conducted in a tertiary care multispecialty hospital in new delhi . our study included two hundred and forty subjects divided into two equal groups ( cases and controls ) . one hundred and twenty patients suffering from premature cad ( males of the age < 45 years and females of the age < 55 years ) admitted to the coronary care unit of a tertiary care hospital , were taken as the cases for this study . the diagnosis was based on detailed history , clinical examination , electrocardiography changes , angiography , increased cardiac enzymes ( troponin t , ck - mb fraction ) , and echocardiography . one hundred and twenty age and gender matched ( males below the age of 45 years and females below the age of 55 years ) healthy individuals from the general population who were free from cad based on clinical history or detailed examination constituted the control group . patients having a history of smoking , chronic alcoholism , concomitant liver , or kidney disease and acute or chronic infection were excluded . patients taking hypolipidemic drugs , oral contraceptives , or hormone replacement therapy were also excluded from this study . informed and written consent was taken from all the subjects ( cases and controls ) after explaining the details and the utility of this study . a detailed history of all the subjects admitted with premature cad was taken , and clinical examination was performed . details of laboratory investigations were noted for all the subjects . because fasting samples are needed for the estimation of various lipid parameters , hence after taking advice from the attending cardio physician , the willing patients were asked for an overnight fasting for 1014 h a day before taking samples . the fasting samples were taken the next morning taking all aseptic precautions from an antecubital vein . the blood was centrifuged for 10 min and separated serum was collected in different aliquots for the estimation of routine lipid profile , ox ldl , apo a1 , lipoprotein ( a ) , sd ldl , and apo b ( apo b-100 ) . olympus - au 400 tc was estimated by enzymatic end - point cholesterol esterase - peroxidase method ( accurex biomedical pvt . tcs were estimated by enzymatic end - point glycerol phosphate oxidase - peroxidase method ( accurex biomedical pvt limited , mumbai ) . hdl - c was estimated by direct assay based on precipitation method where selective precipitation of ldl , very ldl ( vldl ) and tg occurs , and only hdl is measured enzymatically by cholesterol oxidase and cholesterol esterase followed by trinder reaction . ldl - c was estimated by direct assay based on precipitation method where ldl , vldl , and chylomicrons react with precipitating reagents and selected detergents which prevent these to react with cholesterol oxidase and cholesterol esterase , whereas hdl reacts . further addition of specific detergent releases ldl from the precipitated complex which is now measured enzymatically followed by trinder reactionon . ox ldl was estimated by elisa kit ( mercodia , sweden ) which is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the ox apo b molecule . lipoprotein ( a ) was estimated by elisa kit ( immunozym , progen biotechnik gmbh , germany ) which is based on a one - step sandwich elisa using specific monovalent anti - apo ( a ) antibodies . apo a1 and apo b were measured by using immunoassay based immunoturbidimetric kit ( diasys ) on fully automated ai was defined by dobiov and frohlich by using the formula : ai = ( log [ tg / hdl - c ] ) . lti , which was given by enas et al . , was calculated by the formula : lti = tc tg lipoprotein ( a ) hdl - c . lpi was calculated by the formula : lpi = tc tg lipoprotein ( a ) apo b apo a-1 . we defined a new aai for the estimation of consolidated lipid risk in premature cad patients in india . the formula for the calculation of this index is based on the earlier indices in which the parameters which correlate directly with cad is taken as the numerator and those which correlate indirectly are taken as the denominator . but this index includes sd ldl and ox ldl in addition to apo b , apo a1 , and lipoprotein ( a ) . the formula used for calculating aai is as follows : aai = { ox ldl lipoprotein ( a ) apo b } ( apo a1 sd ldl ) the statistical analysis was performed by using spss version 17.0 by spss inc . , chicago , usa . continuous variables were expressed as mean standard deviation and discrete variables were expressed as a percentage . a t - test was applied to lognormal values of lipoprotein ( a ) , tg and apo b as they followed lognormal distribution as proved by probability plots while t - test was applied directly to the rest of the lipid values because they followed normal distribution . f - test was applied to paired data in the cases and controls to determine the significance of variance . spearman 's rank correlation ( which theoretically lies between 1 and + 1 ) was used to calculate the correlation between different parameters . cumulative distribution function ( cdf ) sminrov d statistics was applied to various lipid indices to check their ability to discriminate cases from controls . histogram was used to assess the frequency of patients in a different range of the aai . because fasting samples are needed for the estimation of various lipid parameters , hence after taking advice from the attending cardio physician , the willing patients were asked for an overnight fasting for 1014 h a day before taking samples . the fasting samples were taken the next morning taking all aseptic precautions from an antecubital vein . the blood was centrifuged for 10 min and separated serum was collected in different aliquots for the estimation of routine lipid profile , ox ldl , apo a1 , lipoprotein ( a ) , sd ldl , and apo b ( apo b-100 ) . olympus - au 400 tc was estimated by enzymatic end - point cholesterol esterase - peroxidase method ( accurex biomedical pvt . tcs were estimated by enzymatic end - point glycerol phosphate oxidase - peroxidase method ( accurex biomedical pvt limited , mumbai ) . hdl - c was estimated by direct assay based on precipitation method where selective precipitation of ldl , very ldl ( vldl ) and tg occurs , and only hdl is measured enzymatically by cholesterol oxidase and cholesterol esterase followed by trinder reaction . ldl - c was estimated by direct assay based on precipitation method where ldl , vldl , and chylomicrons react with precipitating reagents and selected detergents which prevent these to react with cholesterol oxidase and cholesterol esterase , whereas hdl reacts . further addition of specific detergent releases ldl from the precipitated complex which is now measured enzymatically followed by trinder reactionon . ox ldl was estimated by elisa kit ( mercodia , sweden ) which is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the ox apo b molecule . lipoprotein ( a ) was estimated by elisa kit ( immunozym , progen biotechnik gmbh , germany ) which is based on a one - step sandwich elisa using specific monovalent anti - apo ( a ) antibodies . apo a1 and apo b were measured by using immunoassay based immunoturbidimetric kit ( diasys ) on fully automated ai was defined by dobiov and frohlich by using the formula : ai = ( log [ tg / hdl - c ] ) . lti , which was given by enas et al . , was calculated by the formula : lti = tc tg lipoprotein ( a ) hdl - c . lpi was calculated by the formula : lpi = tc tg lipoprotein ( a ) apo b apo a-1 . we defined a new aai for the estimation of consolidated lipid risk in premature cad patients in india . the formula for the calculation of this index is based on the earlier indices in which the parameters which correlate directly with cad is taken as the numerator and those which correlate indirectly are taken as the denominator . but this index includes sd ldl and ox ldl in addition to apo b , apo a1 , and lipoprotein ( a ) . the formula used for calculating aai is as follows : aai = { ox ldl lipoprotein ( a ) apo b } ( apo a1 sd ldl ) the statistical analysis was performed by using spss version 17.0 by spss inc . , chicago , usa . continuous variables were expressed as mean standard deviation and discrete variables were expressed as a percentage . a t - test was applied to lognormal values of lipoprotein ( a ) , tg and apo b as they followed lognormal distribution as proved by probability plots while t - test was applied directly to the rest of the lipid values because they followed normal distribution . f - test was applied to paired data in the cases and controls to determine the significance of variance . spearman 's rank correlation ( which theoretically lies between 1 and + 1 ) was used to calculate the correlation between different parameters . cumulative distribution function ( cdf ) sminrov d statistics was applied to various lipid indices to check their ability to discriminate cases from controls . histogram was used to assess the frequency of patients in a different range of the aai . the number of males was more than that of females ( male : female ratio 2.15 ) suggesting a higher prevalence of premature cad in the males as compared to the females . as shown in table 2 , a maximum number of patients in our study were in the age of 3645 years ( 80 out of 120 , 66.67% ) . figures 1 and 2 show that 24 out of 38 female patients were in the age group of 4655 years , which explains that with increasing age , chances of ischemic heart disease also increase in the females . age and gender distribution in the subjects lipid parameters in cases and controls age distribution in cases and controls gender distribution in cases according to the table 2 , the values of tc ( p < 0.01 ) , tg ( p < 0.032 ) , ldl ( p < 0.001 ) , ox ldl ( p < 0.0001 ) , lipoprotein ( a ) ( p < 0.0001 ) , and apo b ( p < 0.001 ) were significantly higher while hdl ( p < 0.01 ) , sd ldl ( p < 0.0001 ) , and apo a1 ( p < as shown in table 3 , the values of ai , lti , lpi , and aai were significantly high in cases as compared to controls . out of the all indices newly defined aai showed maximum correlation comparisons of lipid tetrad index , lipid pentad index and advanced atherogenic index in cases and controls ( p < 0.0001 , r = 0.737 ) with the disease as compared to ai ( p < 0.01 , r = 0.520 ) , lti ( p < 0.001 , r = 0.677 ) and lpi ( p < 0.001 , r = 0.622 ) . according to cdf plots , the chances of wrongly classifying the cases as controls were 78% , 38% , and 23% in ai , lti , and lpi , respectively [ figures 35 ] . the percentage of cases which can be wrongly classified as controls by aai was only 17% . therefore , it is evident from cdf plots , that if aai is used as a consolidated lipid risk marker in cad than chances of wrongly classifying patients as normal is minimum . then we applied kolmogorov statistic to find the d value which a measure of the extent to which cases can be discriminated from controls . kolmogorov statistic was highly significant in aai and lpi ( < 0.00001 ) and significant in lti ( < 0.001 ) and ai ( p < 0.01 ) . d value for aai was higher ( 0.84 ) as compared to d values of lpi ( 0.72 ) , lti ( 0.64 ) and ai ( 0.51 ) . higher d value of aai implies that it can discriminate between cases and controls better than other indices . as shown by dotted lines in the cdf plots of various indices , aai shows the maximum vertical deviation between the curves of cases and controls [ figure 6 ] . the more the vertical separation between the curves higher is power to discriminate between the cases and the controls . hence , the order of discriminating power of various indices is aai > lpi > lti > ai . cumulative distribution function plots of atherogenic index in cases and controls ( staircase curves show empirical cumulative distribution function from the observed data and smooth curves are lognormal cumulative distribution function fit of the observed data ) cumulative distribution function plots of lipid tetrad index in cases and controls ( staircase curves show empirical cumulative distribution function from the observed data and smooth curves are lognormal cumulative distribution function fit of the observed data ) cumulative distribution function plots of lipid pentad index in cases and controls ( staircase curves show empirical cumulative distribution function from the observed data and smooth curves are lognormal cumulative distribution function fit of the observed data ) cumulative distribution function plots of advanced atherogenic index in cases and controls ( staircase curves show empirical cumulative distribution function from the observed data and smooth curves are lognormal cumulative distribution function fit of the observed data ) histogram of aai with the corresponding lognormal fit curve shows that the number of patients in a different range of values of this index [ figures 7 and 8 ] . the value of aai is > 7500 in 74/120 patients and only 23/120 controls which further support the importance of this index for better discrimination between cases and controls groups [ figure 9 ] . histogram of advanced atherogenic index with lognormal fitcurve in controls histogram of advanced atherogenic index with lognormal fit curve in cases histogram of advanced atherogenic index in cases and controls abnormal lipid parameters have been used for many years as the risk factors for cad . many studies have been done and proved the importance of the conventional lipid parameters , i.e. , tc , ldl , hdl , and tg in the pathogenesis of atherosclerosis which is a basic pathology in cad . but in many cases , the conventional lipid parameters fail to explain the higher occurrence or severity of cad in indian population which emphasize that other lipid parameters are involved in the pathogenesis of cad . recent studies have shown that the advanced lipid parameters such as ox ldl , sd ldl apo a1 , and apo b , a lipoprotein ( a ) , are better discriminator of cad and can explain higher incidence and prevalence of cad in the indian population and has shown better correlation with the severity of cad as compared to conventional lipid parameters an existing lipid indices . for determination of consolidated lipid risk factor , ai was given by dobisov et al . and calculated by taking the logarithm of the ratio of tg to hdl - c . the values of ai were higher in patients with cad as compared to the controls . this index does not include ldl , which is a more important risk factor for cad . lti is calculated by multiplying atherogenic particles , i.e. , tc , tg , and lipoprotein ( a ) and dividing it by hdl which is antiatherogenic . in our study , lti values were significantly higher in patients of cad as compared to controls as compared to the healthy persons ( p < 0.001 ) . a study by morais et al . showed a positive correlation between lti and ldl which is important risk factor for the pathogenesis of cad but ldl is not included in the calculation of this index . lpi was given by das et al . and is calculated by the product of tc , tg , lipoprotein ( a ) , and apo b divided by apo a1 . however , lpi was given in the children of the premature cad patients in india . only a few studies are available to the best of our knowledge , which have evaluated lpi and lti in healthy individuals . although lpi values were higher in children of premature cad patients as compared to the children of normal healthy individuals but to the best of our knowledge , lpi is not tested in the premature cad patients for the assessment of lipid risk factors . in this study , we have compared this index in cases and of premature cad with the healthy individuals . although lti and lpi have shown higher values in cad patients , these indices do not include ox ldl and sd ldl . ox ldl and sd ldl are metabolically better indicators of dyslipidemia and have emerged out to be the recent putative markers implicated in cad . any index which will incorporate these all important lipid parameters may prove to be a better marker in the assessment of consolidated lipid risk in the cad . therefore , in this study we have defined aai which is calculated by multiplying ox ldl , lipoprotein ( a ) and apo b and dividing it by apo a1 and sd ldl . surprisingly , the value of aai was significantly high ( p < 0.0001 ) in patients of premature cad patients as compared to the normal individuals . moreover , aai has shown strong correlation with premature cad and its correlation coefficient ( r = 0.737 ) was higher as compared to ai ( r = 0.520 ) , lti ( r = 0.677 ) and lpi ( r = 0.622 ) . thus according to our study aai seems to have better discriminating power ( d value 0.84 ) between cases of premature cad from controls . moreover , ox ldl , sd ldl , lipoprotein ( a ) , apo b , and apo a1 are genetically determined and are included in the aai . hence , this index can explain higher incidence , severity , and prematurity of cad in the indian population . aai may prove to be an important screening test for early detection and intervention in cad patients in the indian population . however , its importance as marker of dyslipidemia in premature cad patients has to be further explored in larger groups before it can actually be used clinically . despite the limitation of sample size , our study does prove that this new aai is a statistically better index as compared to previously known indices , i.e. , ai , lti , and lpi . the values of aai which is calculated from various lipid parameters including the important sd ldl and ox ldl were significantly higher in premature cad patients in indian population as compared to the age and gender matched controls ( p < 0.001 ) . aai showed maximum correlation with the disease ( r = 0.737 ) as compared to ai ( 0.520 ) , lti ( r = 0.677 ) and lpi ( r = 0.622 ) . statistically , it appears that aai has better discriminating value in cad patients as compared to earlier indices . because parameters included in this index are genetically determined , it may also explain the higher degree of prevalence and prematurity of cad in indian population . however , this index has to further explore , by taking bigger sample size before it can be used clinically .

introduction : the high prevalence , severity , and prematurity of coronary artery disease ( cad ) in the indian population can not be completely explained by the conventional lipid parameters and the existing lipid indices.aims and objectives : to calculate newly defined advanced atherogenic index ( aai ) in premature cad patients and compare it between cases and controls and correlate its values with the existing indices.material and methods : one hundred and twenty premature cad patients and an equal number of age and sex matched healthy individuals were included in this study . lipid profile and nonconventional lipid parameters like oxidized low density lipoprotein ( ox ldl ) , small dense ldl ( sd ldl ) , lipoprotein ( a ) apolipoprotein b ( apo b ) , and apolipoprotein a1 ( apo a1 ) were estimated and their values were used to define aai and existing lipid indices like ai , lipid tetrad index ( lti ) and lipid pentad index ( lpi).results : the mean age of cases and controls was 37.29 + 4.50 and 36.13 + 3.53 years , respectively . the value of aai was highly significant in cases ( 3461.22 45.20 ) as compared to controls ( 305.84 21.80 ) . aai has shown better statistical significance and correlation ( p < 0.0001 , r = 0.737 ) as compared to the earlier indices such as ai ( p < 0.01 , r = 0.52 ) , lti ( p < 0.001 , r = 0.677 ) and lpi ( p < 0.001 , r = 0.622 ) in premature cad . kolmogorov d statistic and cumulative distribution function plot has shown that aai can discriminate cases and controls more accurately as compared to the earlier indices.conclusion:statistically aai appears to be a better marker of consolidated lipid risk in premature cad patients as compared to the earlier indices .

INTRODUCTION MATERIALS AND METHODS Collection of blood samples Statistical analysis RESULTS DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

premature coronary artery disease ( cad ) is the occurrence of cardiac events at a younger age ( before the age 45 years in men and before the age of 55 years in women ) . in its severe form premature cad the incidence and prevalence of cad in india is very high and currently india is having a maximum number of cad patients in the world putting india in the state of a cad epidemic . high degree of mortality , morbidity , and prematurity of cad has put a great burden on the society and health care expenditure , and it is the most common cause of death worldwide . the higher degree of prematurity and the severity of cad in the indian population as compared to any other ethnic group in the world can only be partially explained on the basis of conventional lipid parameters . partial explanation is based on the fact that the indians generally tend to have a higher level of low density lipoprotein ( ldl)-c and lower level of high - density lipoprotein ( hdl)-c as compared to their western counterparts . dyslipidemia is one of the most common risk factors for the development of atherosclerosis , and the increased values of the conventional lipid parameters are associated with increased risk of cad . although many studies have shown a strong correlation between dyslipidemia and cad , but it does not explain the high prevalence and severity of cad completely in all the cases . therefore , researchers have defined many indices such as ai , lipid tetrad index ( lti ) , and lipid pentad index ( lpi ) on the basis of the values of the conventional lipid parameters for assessing consolidated lipid risk factor in cad . moreover , the occurrence of cad in normolipidemic persons gives the insight to look beyond the conventional lipid profile and researchers have been trying to elucidate the possibility of other parameters that must be statistically better predictors in the pathogenesis of atherosclerosis which is the underlying pathology of cad . hence , in our study , we have estimated nonconventional lipid parameters such as oxidized ( ox ldl ) , small dense ( sd ldl ) , apolipoprotein a1 ( apo a1 ) , lipoprotein ( a ) , and apolipoprotein b ( apo b ) and used these parameters for the calculation of advanced atherogenic index ( aai ) . then we compared the values of aai with that of the existing lipid indices that have already defined as an assessment of consolidated lipid risk factor in cad . lti was given by enas , and it explains the risk of dyslipidemia in cad in the indian population . lti includes triglyceride ( tc ) , total cholesterol ( tg ) , lipoprotein ( a ) and hdl - c . for assessment of risk factors in the children of premature cad patients includes tc , tg , apo b , lipoprotein ( a ) , and apo a1 . although lpi have shown better discrimination between cases and controls but this index was actually calculated in the children of cad patients rather than the patients themselves , and limited data are available in india , which has evaluated this index in cad patients . most importantly , none of the above indices included the more important lipid parameters such as ox ldl and sd ldl which are better markers of cad as proved by many studies . the significance to include sd ldl instead of ldl lies in the fact that ldl is not homogenous in size and shape and on differential centrifugation it is divided into two fractions . one fraction is small and dense , and the other one is large and buoyant . studies have shown that higher proportion of sd ldl in an individual is associated with higher risk of occurrence of cad because it has a higher tendency to get ox and ox ldl , in turn , is actually deposited inside the macrophages to form the foam cells . normal ldl deposited inside macrophages can be taken back by reverse transport cholesterol ( rct ) by hdl , but once it is converted to ox ldl , then it down - regulates the scavenger receptors on the macrophages and decreases the extent of rct because apo a1 of hdl acts on these receptors . hence , higher amount of sd ldl will lead to the formation of increased amount of ox ldl . therefore , the levels of sd ldl and ox ldl assumes greater importance as the lipid risk factors for the development of atherosclerosis . therefore , lipid index which will include these parameters this led us to calculate a new aai from the values of the nonconventional lipid risk factors such as sd ldl , ox ldl , apo a1 , apo b , and lipoprotein ( a ) . then we used various statistical methods to compare the aai the existing indices such as ai , lti , and lpi to check whether it offers any advantage in predicting the consolidated lipid risk in cad . our study included two hundred and forty subjects divided into two equal groups ( cases and controls ) . one hundred and twenty patients suffering from premature cad ( males of the age < 45 years and females of the age < 55 years ) admitted to the coronary care unit of a tertiary care hospital , were taken as the cases for this study . the diagnosis was based on detailed history , clinical examination , electrocardiography changes , angiography , increased cardiac enzymes ( troponin t , ck - mb fraction ) , and echocardiography . one hundred and twenty age and gender matched ( males below the age of 45 years and females below the age of 55 years ) healthy individuals from the general population who were free from cad based on clinical history or detailed examination constituted the control group . informed and written consent was taken from all the subjects ( cases and controls ) after explaining the details and the utility of this study . a detailed history of all the subjects admitted with premature cad was taken , and clinical examination was performed . the blood was centrifuged for 10 min and separated serum was collected in different aliquots for the estimation of routine lipid profile , ox ldl , apo a1 , lipoprotein ( a ) , sd ldl , and apo b ( apo b-100 ) . hdl - c was estimated by direct assay based on precipitation method where selective precipitation of ldl , very ldl ( vldl ) and tg occurs , and only hdl is measured enzymatically by cholesterol oxidase and cholesterol esterase followed by trinder reaction . ox ldl was estimated by elisa kit ( mercodia , sweden ) which is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the ox apo b molecule . lipoprotein ( a ) was estimated by elisa kit ( immunozym , progen biotechnik gmbh , germany ) which is based on a one - step sandwich elisa using specific monovalent anti - apo ( a ) antibodies . apo a1 and apo b were measured by using immunoassay based immunoturbidimetric kit ( diasys ) on fully automated ai was defined by dobiov and frohlich by using the formula : ai = ( log [ tg / hdl - c ] ) . , was calculated by the formula : lti = tc tg lipoprotein ( a ) hdl - c . lpi was calculated by the formula : lpi = tc tg lipoprotein ( a ) apo b apo a-1 . we defined a new aai for the estimation of consolidated lipid risk in premature cad patients in india . but this index includes sd ldl and ox ldl in addition to apo b , apo a1 , and lipoprotein ( a ) . the formula used for calculating aai is as follows : aai = { ox ldl lipoprotein ( a ) apo b } ( apo a1 sd ldl ) the statistical analysis was performed by using spss version 17.0 by spss inc . a t - test was applied to lognormal values of lipoprotein ( a ) , tg and apo b as they followed lognormal distribution as proved by probability plots while t - test was applied directly to the rest of the lipid values because they followed normal distribution . f - test was applied to paired data in the cases and controls to determine the significance of variance . spearman 's rank correlation ( which theoretically lies between 1 and + 1 ) was used to calculate the correlation between different parameters . cumulative distribution function ( cdf ) sminrov d statistics was applied to various lipid indices to check their ability to discriminate cases from controls . the blood was centrifuged for 10 min and separated serum was collected in different aliquots for the estimation of routine lipid profile , ox ldl , apo a1 , lipoprotein ( a ) , sd ldl , and apo b ( apo b-100 ) . hdl - c was estimated by direct assay based on precipitation method where selective precipitation of ldl , very ldl ( vldl ) and tg occurs , and only hdl is measured enzymatically by cholesterol oxidase and cholesterol esterase followed by trinder reaction . lipoprotein ( a ) was estimated by elisa kit ( immunozym , progen biotechnik gmbh , germany ) which is based on a one - step sandwich elisa using specific monovalent anti - apo ( a ) antibodies . , was calculated by the formula : lti = tc tg lipoprotein ( a ) hdl - c . lpi was calculated by the formula : lpi = tc tg lipoprotein ( a ) apo b apo a-1 . we defined a new aai for the estimation of consolidated lipid risk in premature cad patients in india . but this index includes sd ldl and ox ldl in addition to apo b , apo a1 , and lipoprotein ( a ) . the formula used for calculating aai is as follows : aai = { ox ldl lipoprotein ( a ) apo b } ( apo a1 sd ldl ) the statistical analysis was performed by using spss version 17.0 by spss inc . a t - test was applied to lognormal values of lipoprotein ( a ) , tg and apo b as they followed lognormal distribution as proved by probability plots while t - test was applied directly to the rest of the lipid values because they followed normal distribution . f - test was applied to paired data in the cases and controls to determine the significance of variance . spearman 's rank correlation ( which theoretically lies between 1 and + 1 ) was used to calculate the correlation between different parameters . cumulative distribution function ( cdf ) sminrov d statistics was applied to various lipid indices to check their ability to discriminate cases from controls . the number of males was more than that of females ( male : female ratio 2.15 ) suggesting a higher prevalence of premature cad in the males as compared to the females . as shown in table 2 , a maximum number of patients in our study were in the age of 3645 years ( 80 out of 120 , 66.67% ) . age and gender distribution in the subjects lipid parameters in cases and controls age distribution in cases and controls gender distribution in cases according to the table 2 , the values of tc ( p < 0.01 ) , tg ( p < 0.032 ) , ldl ( p < 0.001 ) , ox ldl ( p < 0.0001 ) , lipoprotein ( a ) ( p < 0.0001 ) , and apo b ( p < 0.001 ) were significantly higher while hdl ( p < 0.01 ) , sd ldl ( p < 0.0001 ) , and apo a1 ( p < as shown in table 3 , the values of ai , lti , lpi , and aai were significantly high in cases as compared to controls . out of the all indices newly defined aai showed maximum correlation comparisons of lipid tetrad index , lipid pentad index and advanced atherogenic index in cases and controls ( p < 0.0001 , r = 0.737 ) with the disease as compared to ai ( p < 0.01 , r = 0.520 ) , lti ( p < 0.001 , r = 0.677 ) and lpi ( p < 0.001 , r = 0.622 ) . according to cdf plots , the chances of wrongly classifying the cases as controls were 78% , 38% , and 23% in ai , lti , and lpi , respectively [ figures 35 ] . the percentage of cases which can be wrongly classified as controls by aai was only 17% . therefore , it is evident from cdf plots , that if aai is used as a consolidated lipid risk marker in cad than chances of wrongly classifying patients as normal is minimum . kolmogorov statistic was highly significant in aai and lpi ( < 0.00001 ) and significant in lti ( < 0.001 ) and ai ( p < 0.01 ) . d value for aai was higher ( 0.84 ) as compared to d values of lpi ( 0.72 ) , lti ( 0.64 ) and ai ( 0.51 ) . higher d value of aai implies that it can discriminate between cases and controls better than other indices . as shown by dotted lines in the cdf plots of various indices , aai shows the maximum vertical deviation between the curves of cases and controls [ figure 6 ] . cumulative distribution function plots of atherogenic index in cases and controls ( staircase curves show empirical cumulative distribution function from the observed data and smooth curves are lognormal cumulative distribution function fit of the observed data ) cumulative distribution function plots of lipid tetrad index in cases and controls ( staircase curves show empirical cumulative distribution function from the observed data and smooth curves are lognormal cumulative distribution function fit of the observed data ) cumulative distribution function plots of lipid pentad index in cases and controls ( staircase curves show empirical cumulative distribution function from the observed data and smooth curves are lognormal cumulative distribution function fit of the observed data ) cumulative distribution function plots of advanced atherogenic index in cases and controls ( staircase curves show empirical cumulative distribution function from the observed data and smooth curves are lognormal cumulative distribution function fit of the observed data ) histogram of aai with the corresponding lognormal fit curve shows that the number of patients in a different range of values of this index [ figures 7 and 8 ] . the value of aai is > 7500 in 74/120 patients and only 23/120 controls which further support the importance of this index for better discrimination between cases and controls groups [ figure 9 ] . histogram of advanced atherogenic index with lognormal fitcurve in controls histogram of advanced atherogenic index with lognormal fit curve in cases histogram of advanced atherogenic index in cases and controls abnormal lipid parameters have been used for many years as the risk factors for cad . many studies have been done and proved the importance of the conventional lipid parameters , i.e. , tc , ldl , hdl , and tg in the pathogenesis of atherosclerosis which is a basic pathology in cad . but in many cases , the conventional lipid parameters fail to explain the higher occurrence or severity of cad in indian population which emphasize that other lipid parameters are involved in the pathogenesis of cad . recent studies have shown that the advanced lipid parameters such as ox ldl , sd ldl apo a1 , and apo b , a lipoprotein ( a ) , are better discriminator of cad and can explain higher incidence and prevalence of cad in the indian population and has shown better correlation with the severity of cad as compared to conventional lipid parameters an existing lipid indices . for determination of consolidated lipid risk factor , ai was given by dobisov et al . the values of ai were higher in patients with cad as compared to the controls . , tc , tg , and lipoprotein ( a ) and dividing it by hdl which is antiatherogenic . in our study , lti values were significantly higher in patients of cad as compared to controls as compared to the healthy persons ( p < 0.001 ) . showed a positive correlation between lti and ldl which is important risk factor for the pathogenesis of cad but ldl is not included in the calculation of this index . and is calculated by the product of tc , tg , lipoprotein ( a ) , and apo b divided by apo a1 . however , lpi was given in the children of the premature cad patients in india . only a few studies are available to the best of our knowledge , which have evaluated lpi and lti in healthy individuals . although lpi values were higher in children of premature cad patients as compared to the children of normal healthy individuals but to the best of our knowledge , lpi is not tested in the premature cad patients for the assessment of lipid risk factors . in this study , we have compared this index in cases and of premature cad with the healthy individuals . although lti and lpi have shown higher values in cad patients , these indices do not include ox ldl and sd ldl . ox ldl and sd ldl are metabolically better indicators of dyslipidemia and have emerged out to be the recent putative markers implicated in cad . any index which will incorporate these all important lipid parameters may prove to be a better marker in the assessment of consolidated lipid risk in the cad . therefore , in this study we have defined aai which is calculated by multiplying ox ldl , lipoprotein ( a ) and apo b and dividing it by apo a1 and sd ldl . surprisingly , the value of aai was significantly high ( p < 0.0001 ) in patients of premature cad patients as compared to the normal individuals . moreover , aai has shown strong correlation with premature cad and its correlation coefficient ( r = 0.737 ) was higher as compared to ai ( r = 0.520 ) , lti ( r = 0.677 ) and lpi ( r = 0.622 ) . thus according to our study aai seems to have better discriminating power ( d value 0.84 ) between cases of premature cad from controls . moreover , ox ldl , sd ldl , lipoprotein ( a ) , apo b , and apo a1 are genetically determined and are included in the aai . hence , this index can explain higher incidence , severity , and prematurity of cad in the indian population . aai may prove to be an important screening test for early detection and intervention in cad patients in the indian population . however , its importance as marker of dyslipidemia in premature cad patients has to be further explored in larger groups before it can actually be used clinically . , ai , lti , and lpi . the values of aai which is calculated from various lipid parameters including the important sd ldl and ox ldl were significantly higher in premature cad patients in indian population as compared to the age and gender matched controls ( p < 0.001 ) . aai showed maximum correlation with the disease ( r = 0.737 ) as compared to ai ( 0.520 ) , lti ( r = 0.677 ) and lpi ( r = 0.622 ) . statistically , it appears that aai has better discriminating value in cad patients as compared to earlier indices . because parameters included in this index are genetically determined , it may also explain the higher degree of prevalence and prematurity of cad in indian population .

leukoaraiosis is associated with an increased risk of stroke , and stroke outcome in patients with leukoaraiosis is poor . however , the pathophysiology of leukoaraiosis remains incompletely understood , and the mechanism of the increased stroke risk is uncertain . atheromatous disease of the carotid arteries is associated with a high risk of stroke , and it may also have a role in the development of leukoaraiosis , although this remains controversial . first , the presence of carotid atheroma may indicate more widespread atheromatous changes and increased arterial stiffness , both potentially affecting the small penetrating arteries and arterioles of the brain . chronic hypoperfusion is thought to be important in the aetiology of leukoaraiosis and , by reducing vascular reserve , may also increase the risk of stroke . our aim was to determine the role of carotid disease and of other clinical variables in the development of leukoaraiosis and to define their contribution to the associated increased risk of stroke . we prospectively studied a large cohort of consecutive patients with tia and minor stroke , who had all undergone magnetic resonance ( mr ) imaging of the brain and related the presence and severity of leukoaraiosis to multiple clinical factors , in particular the presence of carotid stenosis . we prospectively studied consecutive patients referred to a hospitalbased neurovascular clinic between 2002 and 2009 . all patients were assessed by a trained neurologist and diagnosed with a tia or stroke defined according to world health organization criteria . all patients included in the study had a minor , nondisabling ischemic event and could be managed as outpatients . detailed clinical data for vascular risk factors , previous cerebrovascular events , and the nature of the presenting event were collected on a standardized proforma . all patients underwent brain mr imaging on the same day using a 1.5t siemens symphony system with quantum gradients . the study protocol included a t2weighted turbo gradient spin echo axial sequence and a diffusionweighted sequence ( dwi ) . where appropriate , patients also underwent timeofflight mr angiography of the brainsupplying arteries . in patients in whom the mr angiogram suggested a vascular stenosis , confirmatory imaging with doppler ultrasound or contrastenhanced mr or ct angiography two trained independent observers , both neurologists , assessed the presence of leukoaraiosis with use of the arwmc ( age related white matter changes ) score . this wellvalidated score grades the presence of white matter changes in 10 regions of the brain from 0 ( no lesions ) to 3 ( diffuse involvement of an entire region ) , with a maximum score of 30 . in cases of disagreement , scans were also assessed for the presence of acute ischemic lesions ( high signal on dwi and low signal on the apparent diffusion coefficient [ adc ] maps ) and of established infarction ( high signal on t2 images , not visible or low signal on dwi ) . carotid stenosis was assessed by the nascet ( north american symptomatic carotid endarterectomy trial ) method . with this method , the degree of stenosis is calculated as [ ( ba)/b]*100 , with a being the vessel lumen at the site of the stenosis , and b being the diameter of the internal carotid artery distal to the stenosis , measured at a point where the walls are first parallel . patients were regarded as having no significant carotid disease if they had < 50% stenosis , as this is the cutoff beyond which surgical intervention for symptomatic carotid disease is considered , and a stenosis of 70% was regarded as severe , potentially flow limiting and leading to hypoperfusion . we analyzed the arwmc score as a categorical variable by dividing the scores into approximate quartiles , allowing for the fact that > 25% of patients had no leukoaraiosis and that all patients with the same score needed to be grouped into the same category . this led to the following categories : 0 no disease ; 1 mild disease , scores 1 to 4 ; 2 moderate disease , scores 5 to 7 ; 3 severe disease , score > 7 . we tested categorical variables with the pearson 2 test for heterogeneity across groups of different degrees of disease severity and with the 2 for trend ( p trend ) to detect a potential association with increasing disease severity . for continuous variables , we performed an anova to test for heterogeneity and trend , respectively . for variables associated at the p<0.05 significance level in the univariate analysis across categories , we performed a stepwise backward multiple logistic regression analysis comparing patients with no disease ( lowest quartile of the arwmc score ) versus patients with at least some leukoaraiosis ( ie , the other 3 categories combined ) . to study the association between leukoaraiosis and flowlimiting carotid disease , we compared arwmc scores between patients with and without carotid stenosis > 70% or carotid occlusion with the independentsamples median test . we assessed interobserver agreement with the statistic for the category assignments for the arwmc score by the 2 observers . two trained independent observers , both neurologists , assessed the presence of leukoaraiosis with use of the arwmc ( age related white matter changes ) score . this wellvalidated score grades the presence of white matter changes in 10 regions of the brain from 0 ( no lesions ) to 3 ( diffuse involvement of an entire region ) , with a maximum score of 30 . in cases of disagreement , scans were also assessed for the presence of acute ischemic lesions ( high signal on dwi and low signal on the apparent diffusion coefficient [ adc ] maps ) and of established infarction ( high signal on t2 images , not visible or low signal on dwi ) . carotid stenosis was assessed by the nascet ( north american symptomatic carotid endarterectomy trial ) method . with this method , the degree of stenosis is calculated as [ ( ba)/b]*100 , with a being the vessel lumen at the site of the stenosis , and b being the diameter of the internal carotid artery distal to the stenosis , measured at a point where the walls are first parallel . patients were regarded as having no significant carotid disease if they had < 50% stenosis , as this is the cutoff beyond which surgical intervention for symptomatic carotid disease is considered , and a stenosis of 70% was regarded as severe , potentially flow limiting and leading to hypoperfusion . we analyzed the arwmc score as a categorical variable by dividing the scores into approximate quartiles , allowing for the fact that > 25% of patients had no leukoaraiosis and that all patients with the same score needed to be grouped into the same category . this led to the following categories : 0 no disease ; 1 mild disease , scores 1 to 4 ; 2 moderate disease , scores 5 to 7 ; 3 severe disease , score > 7 . we tested categorical variables with the pearson 2 test for heterogeneity across groups of different degrees of disease severity and with the 2 for trend ( p trend ) to detect a potential association with increasing disease severity . for continuous variables , we performed an anova to test for heterogeneity and trend , respectively . for variables associated at the p<0.05 significance level in the univariate analysis across categories , we performed a stepwise backward multiple logistic regression analysis comparing patients with no disease ( lowest quartile of the arwmc score ) versus patients with at least some leukoaraiosis ( ie , the other 3 categories combined ) . to study the association between leukoaraiosis and flowlimiting carotid disease , we compared arwmc scores between patients with and without carotid stenosis > 70% or carotid occlusion with the independentsamples median test . we assessed interobserver agreement with the statistic for the category assignments for the arwmc score by the 2 observers . of those , 469 were excluded because they were considered not to have had a cerebrovascular event , and 51 were excluded because of refusal , or because of contraindications to mr imaging . of the 671 remaining patients ( 374 [ 56% ] men ; mean [ sd ] age 71 years ) , 301 ( 45% ) were diagnosed with a transient ischemic attack ( tia ) and 370 ( 55% ) with a stroke . leukoaraiosis of at least a mild degree was present in 415 ( 62% ) patients . interobserver agreement for assessing the severity of leukoaraiosis was very good with a value of 0.82 ( 95% ci 0.78 to 0.85 , p<0.0001 ) . eightyfive ( 12.6% ) patients did not undergo carotid imaging ( possible vascular intervention not felt to be appropriate or refused ) . mean age ( 71 versus 73 years , p=0.08 ) and the presence of leukoaraiosis ( 61% versus 68% , p=0.2 ) did not differ significantly between patients who had or had not undergone carotid imaging , respectively . otherwise , data collection was near complete , with < 1% missing data for vascular risk factors , and complete clinical and brain imaging data . table 1 shows the prevalence of clinical baseline variables , vascular risk factors , and their association with leukoaraiosis in the univariate analysis . we confirmed the wellknown association of leukoaraiosis with increasing age ( p<0.0001 ) and with a history of hypertension ( p=0.003 ) . women were more likely to have severe leukoaraiosis than were men , but this may have been due to confounding by age , as women in this cohort were older than men ( mean [ sd ] age=72.5 [ 11.3 ] versus 70.1 [ 11.5 ] years , p=0.008 ) . furthermore , increasing severity of leukoaraiosis was associated with a current diagnosis of stroke versus tia ( p=0.008 ) , a history of previous stroke ( p=0.03 ) but not previous tia ( p=0.561 ) , and imaging markers of focal ischemic damage , such as acute infarction on dwi ( p<0.0001 ) and chronic infarction on t2weighted imaging ( p<0.0001 ) . in the multivariate analysis , only age , hypertension , acute and chronic infarction , and the presence of lacunar infarction on mr imaging remained associated with the presence of leukoaraiosis ( table 2 ) . while the association with stroke versus tia was no longer significant in the multivariate analysis , the clinical presentation was strongly correlated with infarct presence on imaging . presence of acute infarction on dwi was much more common in patients with stroke versus tia ( or 5.43 , 95% ci 3.90 to 7.58 , p<0.0001 ) and presence of chronic infarction was associated with a history of previous stroke ( or 5.92 , 95% ci 2.67 to 13.16 , p<0.0001 ) but not previous tia ( or 1.20 , 95% ci 0.72 to 2.01 , p=0.49 ) . hypertension was associated with a diagnosis of previous ( p<0.0001 ) and current stroke ( p=0.02 ) and with chronic ( p=0.04 ) infarction on mr imaging , as well as showing a borderline association with lesion presence on dwi ( p=0.07 ) . there was no significant age difference between patients with and without a history of hypertension ( mean [ sd ] age=71.7 [ 10.9 ] versus 70.3 [ 12.2 ] years , p=0.12 ) , with and without a past history of stroke ( mean [ sd ] age=71.6 [ 11.8 ] versus 71.1 [ 11.4 ] years , p=0.76 ) or a current diagnosis of stroke versus tia ( mean [ sd ] age=71.6 [ 11.4 ] versus 70.6 [ 11.6 ] years , p=0.28 ) . however , patients with acute and chronic infarction on mr imaging were significantly older that those without any infarcts ( mean [ sd ] age=73.0 [ 10.8 ] versus 69.3 [ 11.8 ] years , p<0.0001 ) and ( mean [ sd ] age=72.6 [ 10.7 ] versus 68.8 [ 12.1 ] years , p<0.0001 ) , respectively . risk factor associations for leukoaraiosis , assessed by arwmc score ( arwmc = age related white matter changes ) . patient cohort divided into approximate quartiles first quartile : arwmc ( age related white matter changes ) score=0 ; [ q2 ] : scores=1 to 4 ; [ q3 ] : scores 5 to 7 ; [ q4 ] : score 8 . phet indicates p for heterogeneity ; p trend , p for trend ; dwi , diffusionweighted sequence ; tia , transient ischemic attack . multivariate logistic regression analysis of variables associated with the arwmc score , using a backward stepwise procedure variables included were those showing an association at the 0.05 significance level in the univariate analyses : age , sex , hypertension , diagnosis stroke , previous stroke , acute infarct on dwi , chronic infarct on t2 , lacunar infarct on dwi and peripheral vascular disease . table 3 shows the risk factor associations for carotid artery disease , defined as the presence of at least one carotid artery stenosis of 50% and compares them with the risk factor associations for leukoaraiosis . whereas smoking and ischemic heart disease were associated with carotid disease , they showed no association with leukoaraiosis , and the risk factor associations for leukoaraiosis ( female sex , increasing age , hypertension , and current stroke ) showed no association with carotid atheroma . risk factors for carotid disease ( presence of carotid stenosis 50% ) and for leukoaraiosis for categorical variables , the unadjusted ors ( 95% cis ) for a risk factor being present vs absent in patients with 50% stenosis vs < 50% stenosis are shown . mean ( sd ) age did not differ significantly between patients with and without carotid disease . for leukoaraiosis , the same analyses as shown in table 1 were performed , but only including the patients in whom carotid imaging was available . numbers in italics show associations significant at the p<0.05 level . to determine if leukoaraiosis was more severe distal to a flowlimiting stenosis , we compared arwmc scores in both hemispheres in patients who had unilateral 70% stenosis or occlusion and no significant contralateral disease ( stenosis < 50% ) , and we also compared patients with bilateral carotid stenosis 70% with patients with no significant stenosis ( table 4 ) . arwmc scores did not differ significantly distal to a flowlimiting stenosis compared with patients with no or mild disease only . lack of association between flowlimiting carotid stenosis and severity of leukoaraiosis the withinpatient comparison shows the median ( interquartile range [ iqr ] ) arwmc ( age related white matter changes ) score for each hemisphere in patients with unilateral 70% carotid stenosis or occlusion , and no significant disease ( < 50% ) in the contralateral artery . betweenpatient comparison shows total arwmc scores for patients with bilateral 70% carotid stenosis vs patients with no carotid atheroma . phet indicates p for heterogeneity , the comparison was performed with the independent samples median test ; ica , internal carotid artery . in this study , the association with past and present stroke rather than tia suggests that patients with leukoaraiosis have more severe cerebrovascular events , and the association with old and acute infarction on mr imaging shows that brain tissue may have a greater tendency to develop ischemic tissue damage in the presence of increasingly severe leukoaraiosis . while the association with stroke versus tia was no longer significant in the multivariate analysis , this was explained by the strong association between a clinical diagnosis of stroke with infarct presence on imaging . the fact that imaging findings rather than the clinical presentation remained significant is most likely explained by imaging being a better marker of tissue damage . our findings are in keeping with those of two recent studies , which found higher white matter lesion volumes in patients with stroke compared with patients with tia or transient symptoms with infarction . all three studies show that in the presence of leukoaraiosis , the brain is more vulnerable to acute ischemic damage and suggest that leukoaraiosis is an indicator for increased cerebral susceptibility to ischemia . previous studies have suggested that leukoaraiosis reflects damage from chronic hypoperfusion and that increased vulnerability to ischemia may be due to reduced vascular reserve . however , if chronic hypoperfusion was the sole contributing factor , we would expect leukoaraiosis to be more severe distal to flowlimiting arterial occlusive disease . in our cohort , while these data are limited by lack of information about the presence of collateral flow , several other recent studies also found no association of leukoaraiosis with carotid disease and with collateral supply . hypoperfusion alone , at least when caused by large vessel occlusion , does not appear to contribute to leukoaraiosis . alternatively , any damaging effect of hypoperfusion might be offset by an opposite effect of the stenosis protecting the small vessels from the damaging effects of hypertension , with no overall change . in this study , leukoaraiosis was unrelated to atheromatous disease in the carotid and other circulations , and the risk factor profiles for leukoaraiosis and carotid disease differed . while it has been suggested that leukoaraiosis may at least partly be caused by atheroma of the small penetrating arteries , the complete lack of association between leukoaraiosis and the presence of atheroma and its risk factors in our study and others suggest that atheroma plays little role in the pathogenesis of leukoaraiosis . in contrast , two big populationbased studies found an association between leukoaraiosis , carotid intimamedia thickness , and carotid plaque , although the findings of the studies differed to some extent , in that in the rotterdam scan study , carotid plaque was associated with periventricular white matter lesions and not with subcortical white matter lesions , whereas in the cardiovascular health study ( chs ) , these associations were reversed . as these studies were population based , the prevalence of carotid stenosis > 50% was low ( 5.4% in the chs and 1.4% in the rotterdam study ) , and the studies concentrated much more on the early stages of atheroma , whereas our cohort consisted of patients with already established cerebrovascular disease . some risk factors , such as increasing age or hypertension , will contribute to large and to small vessel damage and may explain the association between early atheroma and leukoaraiosis described in these two studies . additional risk factors , such as smoking or genetic factors , may be required to lead to more severe atheromatous disease , explaining the differences in risk factor profiles between patients with 50% carotid stenosis and leukoaraiosis in our study . in this study and others , there was a strong association of leukoaraiosis with lacunar infarction , which suggests that these are two manifestations of a shared underlying aetiology . most commonly , this is likely to be due to small vessel changes associated with hypertension and increasing age , as further discussed later . however , there are also other disease processes that cause leukoaraiosis and lacunar infarcts , such as cerebral amyloid angiopathy and cadasil ( cerebral autosomal dominant arteriopathy with subcortical infarcts and leukencephalopathy ) . there are several explanations why a small vessel vasculopathy may cause both leukoaraiosis and focal infarction . they may be different degrees of severity of the same process ; leukoaraiosis may increase the susceptibility to developing infarction ; or infarcts may contribute to the appearance of leukoaraiosis , with the distinction between leukoaraiosis and infarction being less clear than previously thought . hypertension is the most consistently reported modifiable risk factor for leukoaraiosis and showed a strong association in our cohort . similar to leukoaraiosis , it was associated with a past and current diagnosis of stroke versus tia and with infarction on mr imaging . indeed , adjusting for hypertension in the multivariate analysis reduced the strength of the association of leukoaraiosis with past and current stroke . our findings indicate that hypertension plays an important role in increasing cerebral susceptibility to ischemia . this is further supported by animal studies , in which the same ischemic stimulus led to bigger infarcts in hypertensive versus normotensive rats , and human studies , in which acute infarct growth was bigger in hypertensive patients . possible mechanisms by which hypertension may increase susceptibility to ischemia include the release of vasoactive substances , which lead to changes in vascular tone , reduced cerebral autoregulation , and subsequently increased vulnerability to reductions in perfusion pressure . furthermore , cytokine release may render the endothelium more procoagulant , and hypertension may cause longerterm changes in arteriolar wall thickness and reduction in lumen , with subsequently reduced blood flow . of course , the latter mechanism is also thought to cause leukoaraiosis . increasing age is widely accepted as the most important risk factor for leukoaraiosis , and was strongly associated in our cohort . while it showed no association with past and current stroke , patients with infarction on imaging were significantly older than those without , perhaps indicating that increasing age also increases cerebral susceptibility to ischemia . this has been suggested previously by a study that found greater conversion of tissue at risk to infarction in older patients with acute stroke . older age leads to a number of changes that could increase cerebral susceptibility to ischemia , such as reduced capillary lumen diameter , increased vascular tortuosity , thickening of vessel walls , and impairment in cerebral autoregulation . while we believe our findings to be valid , this study has several potential limitations . first , we used the arwmc score rather than volumetric analysis to grade the severity of leukoaraiosis . visual rating scales can make comparison between studies difficult if different scales are used ; they depend on the expertise of the rater , and they may not detect small changes in disease severity in followup studies . in contrast , volumetric analysis may be more sensitive to detecting small changes in lesion load . by using the now wellestablished arwmc score we aimed to make our study comparable to others . interrater agreement was high , supporting the validity of the readings , and it is uncertain if a volumetric analysis would have been more accurate or meaningful , in particular as it is also subject to rater experience . we believe that the visual semiquantitative arwmc score helped to identify relevant differences between scans and offered the most robust analysis . second , our imaging sequence did not include any t1weighted imaging , which can help to differentiate focal infarction from the lesions of leukoaraiosis . however , most studies of leukoaraiosis are based on t2weighted imaging alone , and differentiating a focal infarct from leukoaraiosis may not always be possible even with t1weighted imaging . third , not all patients had carotid imaging , as this was only done when thought to be of therapeutic consequence . however , risk factors and severity of leukoaraiosis did not differ significantly between patients with and without carotid imaging , and we do not think that this introduced any bias to the results . fourth , the proportion of patients with severe carotid disease in this cohort was low , perhaps making it difficult to detect any association between leukoaraiosis and carotid stenosis . while this does not eliminate the low patient numbers , we believe that the consistent results , and previous similar reports in other studies , support the validity of our findings . in this large cohort of patients with established cerebrovascular disease , neither the presence or severity of atheromatous disease , nor the presence of risk factors for atheroma were associated with leukoaraiosis , suggesting that these are two unrelated disease processes . in contrast , increasing age and hypertension were strongly associated , as were past and current stroke , and infarct presence on imaging . our findings suggest that leukoaraiosis represents the visible structural damage caused by age , hypertension , and potentially other factors and that these changes indicate that the brain is more vulnerable to ischemic damage . however , it is a marker of increased cerebral susceptibility to ischemia , and this makes it more likely that , if the brain is exposed an acute ischemic challenge , infarction will develop and clinically the patient will have a stroke .

backgroundleukoaraiosis is associated with an increased risk of stroke , but the underlying mechanism remains uncertain , as do the associations with other risk factors , such as carotid disease . we aimed to determine the role of carotid disease and of other clinical variables in the development of leukoaraiosis and to define their contributions to the associated increased risk of stroke.methods and resultswe prospectively studied a large cohort of consecutive patients with transient ischemic attack ( tia ) and minor stroke who attended a tia clinic between 2002 and 2009 . detailed clinical data were obtained , and patients underwent magnetic resonance brain and vascular imaging . we assessed the severity of leukoaraiosis with use of the arwmc ( age related white matter changes ) score : 671 patients ( 374 [ 56% ] men ; mean [ sd ] age 71 [ 11 ] years ) were studied , of whom 415 ( 62% ) had leukoaraiosis . in a multivariate analysis , leukoaraiosis was associated with increasing age ( p<0.0001 ) and hypertension ( p=0.01 ) , as well as the presence of acute ( p<0.0001 ) and chronic ( p=0.014 ) infarction on magnetic resonance imaging . in the univariate analysis , a current and past diagnosis of stroke versus tia also showed a strong association . carotid disease was not associated with leukoaraiosis , even in the presence of a flowlimiting ( > 70% ) stenosis or occlusion , and the risk factor profiles for leukoaraiosis and carotid disease differed.conclusionsthe association with more severe ischemic events ( stroke versus tia ) and infarction on imaging is consistent with leukoaraiosis being a marker of increased cerebral susceptibility to ischemia . in contrast , the presence , severity of , and risk factors for atheromatous disease showed no association with leukoaraiosis , suggesting that these are two unrelated disease processes .

Introduction Methods Assessment of White Matter Disease/MR Imaging Scans Statistical Analysis Results Discussion Conclusion

leukoaraiosis is associated with an increased risk of stroke , and stroke outcome in patients with leukoaraiosis is poor . however , the pathophysiology of leukoaraiosis remains incompletely understood , and the mechanism of the increased stroke risk is uncertain . atheromatous disease of the carotid arteries is associated with a high risk of stroke , and it may also have a role in the development of leukoaraiosis , although this remains controversial . first , the presence of carotid atheroma may indicate more widespread atheromatous changes and increased arterial stiffness , both potentially affecting the small penetrating arteries and arterioles of the brain . chronic hypoperfusion is thought to be important in the aetiology of leukoaraiosis and , by reducing vascular reserve , may also increase the risk of stroke . our aim was to determine the role of carotid disease and of other clinical variables in the development of leukoaraiosis and to define their contribution to the associated increased risk of stroke . we prospectively studied a large cohort of consecutive patients with tia and minor stroke , who had all undergone magnetic resonance ( mr ) imaging of the brain and related the presence and severity of leukoaraiosis to multiple clinical factors , in particular the presence of carotid stenosis . we prospectively studied consecutive patients referred to a hospitalbased neurovascular clinic between 2002 and 2009 . detailed clinical data for vascular risk factors , previous cerebrovascular events , and the nature of the presenting event were collected on a standardized proforma . in patients in whom the mr angiogram suggested a vascular stenosis , confirmatory imaging with doppler ultrasound or contrastenhanced mr or ct angiography two trained independent observers , both neurologists , assessed the presence of leukoaraiosis with use of the arwmc ( age related white matter changes ) score . this wellvalidated score grades the presence of white matter changes in 10 regions of the brain from 0 ( no lesions ) to 3 ( diffuse involvement of an entire region ) , with a maximum score of 30 . in cases of disagreement , scans were also assessed for the presence of acute ischemic lesions ( high signal on dwi and low signal on the apparent diffusion coefficient [ adc ] maps ) and of established infarction ( high signal on t2 images , not visible or low signal on dwi ) . with this method , the degree of stenosis is calculated as [ ( ba)/b]*100 , with a being the vessel lumen at the site of the stenosis , and b being the diameter of the internal carotid artery distal to the stenosis , measured at a point where the walls are first parallel . patients were regarded as having no significant carotid disease if they had < 50% stenosis , as this is the cutoff beyond which surgical intervention for symptomatic carotid disease is considered , and a stenosis of 70% was regarded as severe , potentially flow limiting and leading to hypoperfusion . we analyzed the arwmc score as a categorical variable by dividing the scores into approximate quartiles , allowing for the fact that > 25% of patients had no leukoaraiosis and that all patients with the same score needed to be grouped into the same category . we tested categorical variables with the pearson 2 test for heterogeneity across groups of different degrees of disease severity and with the 2 for trend ( p trend ) to detect a potential association with increasing disease severity . for variables associated at the p<0.05 significance level in the univariate analysis across categories , we performed a stepwise backward multiple logistic regression analysis comparing patients with no disease ( lowest quartile of the arwmc score ) versus patients with at least some leukoaraiosis ( ie , the other 3 categories combined ) . to study the association between leukoaraiosis and flowlimiting carotid disease , we compared arwmc scores between patients with and without carotid stenosis > 70% or carotid occlusion with the independentsamples median test . two trained independent observers , both neurologists , assessed the presence of leukoaraiosis with use of the arwmc ( age related white matter changes ) score . this wellvalidated score grades the presence of white matter changes in 10 regions of the brain from 0 ( no lesions ) to 3 ( diffuse involvement of an entire region ) , with a maximum score of 30 . in cases of disagreement , scans were also assessed for the presence of acute ischemic lesions ( high signal on dwi and low signal on the apparent diffusion coefficient [ adc ] maps ) and of established infarction ( high signal on t2 images , not visible or low signal on dwi ) . with this method , the degree of stenosis is calculated as [ ( ba)/b]*100 , with a being the vessel lumen at the site of the stenosis , and b being the diameter of the internal carotid artery distal to the stenosis , measured at a point where the walls are first parallel . patients were regarded as having no significant carotid disease if they had < 50% stenosis , as this is the cutoff beyond which surgical intervention for symptomatic carotid disease is considered , and a stenosis of 70% was regarded as severe , potentially flow limiting and leading to hypoperfusion . we analyzed the arwmc score as a categorical variable by dividing the scores into approximate quartiles , allowing for the fact that > 25% of patients had no leukoaraiosis and that all patients with the same score needed to be grouped into the same category . for variables associated at the p<0.05 significance level in the univariate analysis across categories , we performed a stepwise backward multiple logistic regression analysis comparing patients with no disease ( lowest quartile of the arwmc score ) versus patients with at least some leukoaraiosis ( ie , the other 3 categories combined ) . to study the association between leukoaraiosis and flowlimiting carotid disease , we compared arwmc scores between patients with and without carotid stenosis > 70% or carotid occlusion with the independentsamples median test . we assessed interobserver agreement with the statistic for the category assignments for the arwmc score by the 2 observers . of the 671 remaining patients ( 374 [ 56% ] men ; mean [ sd ] age 71 years ) , 301 ( 45% ) were diagnosed with a transient ischemic attack ( tia ) and 370 ( 55% ) with a stroke . leukoaraiosis of at least a mild degree was present in 415 ( 62% ) patients . interobserver agreement for assessing the severity of leukoaraiosis was very good with a value of 0.82 ( 95% ci 0.78 to 0.85 , p<0.0001 ) . mean age ( 71 versus 73 years , p=0.08 ) and the presence of leukoaraiosis ( 61% versus 68% , p=0.2 ) did not differ significantly between patients who had or had not undergone carotid imaging , respectively . otherwise , data collection was near complete , with < 1% missing data for vascular risk factors , and complete clinical and brain imaging data . table 1 shows the prevalence of clinical baseline variables , vascular risk factors , and their association with leukoaraiosis in the univariate analysis . we confirmed the wellknown association of leukoaraiosis with increasing age ( p<0.0001 ) and with a history of hypertension ( p=0.003 ) . women were more likely to have severe leukoaraiosis than were men , but this may have been due to confounding by age , as women in this cohort were older than men ( mean [ sd ] age=72.5 [ 11.3 ] versus 70.1 [ 11.5 ] years , p=0.008 ) . furthermore , increasing severity of leukoaraiosis was associated with a current diagnosis of stroke versus tia ( p=0.008 ) , a history of previous stroke ( p=0.03 ) but not previous tia ( p=0.561 ) , and imaging markers of focal ischemic damage , such as acute infarction on dwi ( p<0.0001 ) and chronic infarction on t2weighted imaging ( p<0.0001 ) . in the multivariate analysis , only age , hypertension , acute and chronic infarction , and the presence of lacunar infarction on mr imaging remained associated with the presence of leukoaraiosis ( table 2 ) . while the association with stroke versus tia was no longer significant in the multivariate analysis , the clinical presentation was strongly correlated with infarct presence on imaging . presence of acute infarction on dwi was much more common in patients with stroke versus tia ( or 5.43 , 95% ci 3.90 to 7.58 , p<0.0001 ) and presence of chronic infarction was associated with a history of previous stroke ( or 5.92 , 95% ci 2.67 to 13.16 , p<0.0001 ) but not previous tia ( or 1.20 , 95% ci 0.72 to 2.01 , p=0.49 ) . hypertension was associated with a diagnosis of previous ( p<0.0001 ) and current stroke ( p=0.02 ) and with chronic ( p=0.04 ) infarction on mr imaging , as well as showing a borderline association with lesion presence on dwi ( p=0.07 ) . there was no significant age difference between patients with and without a history of hypertension ( mean [ sd ] age=71.7 [ 10.9 ] versus 70.3 [ 12.2 ] years , p=0.12 ) , with and without a past history of stroke ( mean [ sd ] age=71.6 [ 11.8 ] versus 71.1 [ 11.4 ] years , p=0.76 ) or a current diagnosis of stroke versus tia ( mean [ sd ] age=71.6 [ 11.4 ] versus 70.6 [ 11.6 ] years , p=0.28 ) . however , patients with acute and chronic infarction on mr imaging were significantly older that those without any infarcts ( mean [ sd ] age=73.0 [ 10.8 ] versus 69.3 [ 11.8 ] years , p<0.0001 ) and ( mean [ sd ] age=72.6 [ 10.7 ] versus 68.8 [ 12.1 ] years , p<0.0001 ) , respectively . risk factor associations for leukoaraiosis , assessed by arwmc score ( arwmc = age related white matter changes ) . patient cohort divided into approximate quartiles first quartile : arwmc ( age related white matter changes ) score=0 ; [ q2 ] : scores=1 to 4 ; [ q3 ] : scores 5 to 7 ; [ q4 ] : score 8 . multivariate logistic regression analysis of variables associated with the arwmc score , using a backward stepwise procedure variables included were those showing an association at the 0.05 significance level in the univariate analyses : age , sex , hypertension , diagnosis stroke , previous stroke , acute infarct on dwi , chronic infarct on t2 , lacunar infarct on dwi and peripheral vascular disease . table 3 shows the risk factor associations for carotid artery disease , defined as the presence of at least one carotid artery stenosis of 50% and compares them with the risk factor associations for leukoaraiosis . whereas smoking and ischemic heart disease were associated with carotid disease , they showed no association with leukoaraiosis , and the risk factor associations for leukoaraiosis ( female sex , increasing age , hypertension , and current stroke ) showed no association with carotid atheroma . risk factors for carotid disease ( presence of carotid stenosis 50% ) and for leukoaraiosis for categorical variables , the unadjusted ors ( 95% cis ) for a risk factor being present vs absent in patients with 50% stenosis vs < 50% stenosis are shown . for leukoaraiosis , the same analyses as shown in table 1 were performed , but only including the patients in whom carotid imaging was available . to determine if leukoaraiosis was more severe distal to a flowlimiting stenosis , we compared arwmc scores in both hemispheres in patients who had unilateral 70% stenosis or occlusion and no significant contralateral disease ( stenosis < 50% ) , and we also compared patients with bilateral carotid stenosis 70% with patients with no significant stenosis ( table 4 ) . lack of association between flowlimiting carotid stenosis and severity of leukoaraiosis the withinpatient comparison shows the median ( interquartile range [ iqr ] ) arwmc ( age related white matter changes ) score for each hemisphere in patients with unilateral 70% carotid stenosis or occlusion , and no significant disease ( < 50% ) in the contralateral artery . in this study , the association with past and present stroke rather than tia suggests that patients with leukoaraiosis have more severe cerebrovascular events , and the association with old and acute infarction on mr imaging shows that brain tissue may have a greater tendency to develop ischemic tissue damage in the presence of increasingly severe leukoaraiosis . while the association with stroke versus tia was no longer significant in the multivariate analysis , this was explained by the strong association between a clinical diagnosis of stroke with infarct presence on imaging . all three studies show that in the presence of leukoaraiosis , the brain is more vulnerable to acute ischemic damage and suggest that leukoaraiosis is an indicator for increased cerebral susceptibility to ischemia . in our cohort , while these data are limited by lack of information about the presence of collateral flow , several other recent studies also found no association of leukoaraiosis with carotid disease and with collateral supply . in this study , leukoaraiosis was unrelated to atheromatous disease in the carotid and other circulations , and the risk factor profiles for leukoaraiosis and carotid disease differed . while it has been suggested that leukoaraiosis may at least partly be caused by atheroma of the small penetrating arteries , the complete lack of association between leukoaraiosis and the presence of atheroma and its risk factors in our study and others suggest that atheroma plays little role in the pathogenesis of leukoaraiosis . in contrast , two big populationbased studies found an association between leukoaraiosis , carotid intimamedia thickness , and carotid plaque , although the findings of the studies differed to some extent , in that in the rotterdam scan study , carotid plaque was associated with periventricular white matter lesions and not with subcortical white matter lesions , whereas in the cardiovascular health study ( chs ) , these associations were reversed . as these studies were population based , the prevalence of carotid stenosis > 50% was low ( 5.4% in the chs and 1.4% in the rotterdam study ) , and the studies concentrated much more on the early stages of atheroma , whereas our cohort consisted of patients with already established cerebrovascular disease . some risk factors , such as increasing age or hypertension , will contribute to large and to small vessel damage and may explain the association between early atheroma and leukoaraiosis described in these two studies . additional risk factors , such as smoking or genetic factors , may be required to lead to more severe atheromatous disease , explaining the differences in risk factor profiles between patients with 50% carotid stenosis and leukoaraiosis in our study . in this study and others , there was a strong association of leukoaraiosis with lacunar infarction , which suggests that these are two manifestations of a shared underlying aetiology . most commonly , this is likely to be due to small vessel changes associated with hypertension and increasing age , as further discussed later . however , there are also other disease processes that cause leukoaraiosis and lacunar infarcts , such as cerebral amyloid angiopathy and cadasil ( cerebral autosomal dominant arteriopathy with subcortical infarcts and leukencephalopathy ) . they may be different degrees of severity of the same process ; leukoaraiosis may increase the susceptibility to developing infarction ; or infarcts may contribute to the appearance of leukoaraiosis , with the distinction between leukoaraiosis and infarction being less clear than previously thought . hypertension is the most consistently reported modifiable risk factor for leukoaraiosis and showed a strong association in our cohort . similar to leukoaraiosis , it was associated with a past and current diagnosis of stroke versus tia and with infarction on mr imaging . indeed , adjusting for hypertension in the multivariate analysis reduced the strength of the association of leukoaraiosis with past and current stroke . our findings indicate that hypertension plays an important role in increasing cerebral susceptibility to ischemia . possible mechanisms by which hypertension may increase susceptibility to ischemia include the release of vasoactive substances , which lead to changes in vascular tone , reduced cerebral autoregulation , and subsequently increased vulnerability to reductions in perfusion pressure . furthermore , cytokine release may render the endothelium more procoagulant , and hypertension may cause longerterm changes in arteriolar wall thickness and reduction in lumen , with subsequently reduced blood flow . increasing age is widely accepted as the most important risk factor for leukoaraiosis , and was strongly associated in our cohort . while it showed no association with past and current stroke , patients with infarction on imaging were significantly older than those without , perhaps indicating that increasing age also increases cerebral susceptibility to ischemia . older age leads to a number of changes that could increase cerebral susceptibility to ischemia , such as reduced capillary lumen diameter , increased vascular tortuosity , thickening of vessel walls , and impairment in cerebral autoregulation . first , we used the arwmc score rather than volumetric analysis to grade the severity of leukoaraiosis . however , risk factors and severity of leukoaraiosis did not differ significantly between patients with and without carotid imaging , and we do not think that this introduced any bias to the results . fourth , the proportion of patients with severe carotid disease in this cohort was low , perhaps making it difficult to detect any association between leukoaraiosis and carotid stenosis . in this large cohort of patients with established cerebrovascular disease , neither the presence or severity of atheromatous disease , nor the presence of risk factors for atheroma were associated with leukoaraiosis , suggesting that these are two unrelated disease processes . in contrast , increasing age and hypertension were strongly associated , as were past and current stroke , and infarct presence on imaging . however , it is a marker of increased cerebral susceptibility to ischemia , and this makes it more likely that , if the brain is exposed an acute ischemic challenge , infarction will develop and clinically the patient will have a stroke .

the stomatognathic system , an integral component of the upper body , may play an important role in postural control . several studies suggest that spatial relationships between the jaws may influence the distal musculature and induce body postural adaptations . however , perinetti et al . concluded that mandibular position , asymmetric occlusion , and temporomandibular disorders do not appear to correlate with body sway or muscle activity in other parts of the body , including those responsible for maintaining posture , at a clinically relevant level . several studies have examined the relationship between malocclusion and parameters of body posture in the sagittal and frontal planes ; the results identified a correlation between structural orthopaedic diseases and occlusal morphology . found that children with various spinal deformities have a high number of malocclusions , and ben - bassat et al . found that patients with idiopathic scoliosis showed more asymmetric features characteristic of malocclusion than a random control group . also , children with congenital hip dislocation are more predisposed to the development of a lateral cross - bite . however , the results from studies looking at the correlation between poor body posture and dental occlusion are conflicting . for example , lippold et al . examined 59 pre - school children and found statistically significant correlations between weak body posture and class ii malocclusion . also , lippold et al . used rasterstereography to examine the sagittal profile of the spine in 53 adults with skeletal class ii and class iii malocclusions , and found a correlation between the vertical and sagittal position of the lower jaw and thoracic , lordotic , and pelvic inclination , and between the vertical and sagittal position of the lower jaw and pelvic rotation . thus , 2 different models of back shape were devised based on of the results on these studies : 1 ) a more distal and vertical craniofacial pattern is associated with an increase in the upper thoracic , lumbar - lordotic , and pelvic angles ; and 2 ) a more mesial and horizontal craniofacial pattern is associated with smaller upper thoracic , lumbar - lordotic , and pelvic angles . compared body posture in 29 class ii and class iii patients , and found that the apex of the thoracic kyphosis was more cranial in class iii patients than in class ii patients or healthy controls . however , these studies are based on small samples . when perillo et al . examined 703 children , they found no association between body posture and clinically assessed dental occlusion . they observed that about 14% of all patients had a pathological gait ; among them , children demonstrated a higher prevalence of vertical occlusion anomalies . contradictory results of studies can arise because there was a large diversity among the studies with regard to the protocols used ; some studies assessed body posture by physical examination while other studies used body photographs and rasterstereography . enlarged tonsils and adenoids , allergic rhinitis , and chronic respiratory problems cause a mouth breathing syndrome , resulting in adaptive head and body postures , which also affects the development of the facial skeleton . it is generally accepted that anterior tilting of the head is the main postural change in such subjects , who push their heads forward and extend their neck to facilitate air flow through the mouth . the forward position of the head causes protraction and rotation of the shoulders , elevation and abduction of the scapulae , depression of the thoracic anterior region , and forward displacement of the whole body . unlike in nasal - breathing children , these postural changes in mouth - breathing children do not improve spontaneously once they are older ( > 8 years - of - age ) . demonstrated that adults who were mouth - breathers during childhood had a more anterior head posture and a larger lumbar lordosis angle than individuals in a control group . to date , no study has examined the association between malocclusion , body posture , and breathing pattern . the assessment of correlations between orthopedic , otorhinolaryngologic , and orthodontic findings derived from interdisciplinary studies appears to be of practical importance in diagnosis and prevention . therefore , the aim of the present study was to examine the relationship between the type of malocclusion , body posture , and nasopharyngeal obstruction in children aged 714 years . the tested null hypotheses were that : 1 ) sagittal craniofacial skeletal morphology depends on the nasopharyngeal obstruction and body posture , and 2 ) vertical craniofacial skeletal morphology depends on the nasopharyngeal obstruction and body posture . the study sample was obtained from consecutive patients attending for orthodontic treatment at the department of orthodontics who agreed to participate in the study from september 2013 through may 2014 . a full explanation of the study aims and procedures was provided to the parents of each patient and signed consent forms were obtained . the study was approved by the regional biomedical research ethics committee ( no . be-2 - 48 ) . the study group comprised 94 patients aged 714 years ( mean sd : 11.92.1 years ) . forty - four were male ( 46.8% ) and 50 were female ( 53.2% ) . all patients passed an examination performed by the same clinical team in a blinded manner . power analysis was used to determine the sample size . performing a power calculation , we anticipated changes in snb angle by 2 ( sd=2 ) , =0.05 . in such circumstances , this study aimed to investigate 90 patients ( n1=45 , n2=45 ) to achieve 0.99 power . after investigation we concluded the study needed a power 0.802 ( n1=49 , n2=45 ; =0.05 ; change in snb angle 2.21 ) ( sd=3.77 ) . the inclusion criteria into the study were as follows : age 714 years ; no history of maxillofacial trauma or surgery , syndromes , clefts , or orthodontic treatment ; no previous treatment for orthopedic disorders ; and no previous injury to the pelvis , spine , or long bones . the orthodontic examination consisted of the study model and cephalometric radiograph analysis . the study model examination consisted of a transverse examination in which a posterior cross - bite was confirmed ( at least 2 teeth showed a cross - relationship with the opposite teeth in the posterior segments of the dental arches . the cross - bite was categorized as unilateral or bilateral , and we performed a space analysis in which the difference between the available space and the necessary space in the dental arch was calculated . crowding was categorized as mild ( lack of space : 24 mm ) , moderate ( 59 mm ) , or severe ( > 9 mm ) . a standardized lateral cephalometric radiograph was taken for each patient ( kodak 8000c ; enlargement factor 1.15 ; exposure : 12 mas , 7680 kv ) and analyzed using dolphin software ( version 10.5 ) . the sagittal position of the maxilla ( sna ) and the mandible ( snb ) , the sagittal jaw relationship ( anb ) , the mandibular plane angle ( mp - sn ) , and the inclination of the maxillary incisors and mandibular incisors ( u1-ans / pns and l1-mp , respectively ) were used to analyze the facial skull parameters . the error margins for the study models and lateral cephalometric analysis were determined by repeatedly measuring the 6 variables on 10 randomly selected models and radiographs at 2-week intervals . parametric data were subjected to a paired - samples t - test and non - parametric wilcoxon signed ranks test , which showed that there was no significant difference between the data sets . the patient was examined in a relaxed standing posture : subjects were asked to stand in an upright position , barefoot , without moving , looking straight ahead , with relaxed shoulders and arms resting at their sides for a natural head and body position . a standard routine examination from the front , side , and back degrees of severity can not be differentiated with adequate precision by manual orthopedic diagnostics ; therefore , the findings were graded either as normal or abnormal . patients were first examined from the side and the thoracic kyphosis was evaluated . if an increased , but adjustable , asymptomatic curvature of the thoracic spine was observed , the posture was classed as kyphotic . all patients underwent tests to rule out scheuermann s disease and ankylosing spondylitis ; briefly , each patient was asked to stand upright and pull back the shoulders to induce thoracic extension . in cases of postural kyphosis , an increased curvature , which is regular and mobile , was found . next , patients were examined from the front , and the position of shoulders , the symmetry of the waist triangles , and the horizontal alignment of the upper iliac crests were noted . finally , patients were examined from the back , and the position of the shoulders , the scapular height , and the symmetry of the waist triangles , iliac crests , and thoracic rib hump were noted . briefly , patients were asked to bring their chin to their chest , relax the hands , and flex the hips with the knees extended . the examiner then looked for the emergence of a paravertebral muscle roller in the lumbar region or a rib hump in the thoracic region . if a rib prominence hump was present ( > 1 cm ) , then full - length frontal and lateral spinal radiographs were obtained to evaluate the degree of spinal deformation ( by measuring the cobb s angle ) ( figure 2 ) . the following diagnoses were made based on the findings : hypertrophy of the adenoids ( grade 23 ) was diagnosed when up to 2/3 of the choana was compromised ; hypertrophy of the palatal tonsils ( grade 24 ) was diagnosed when there was < 50% of normal space between tonsillar pillars ; nasal septum deviation was diagnosed when the nasal septum was severely shifted away from the midline ; and allergic rhinitis was diagnosed when the patient showed typical allergy symptoms ( nasal congestion , runny nose , sneezing , and watery eyes ) and skin - prick test results were positive . nasopharyngeal obstruction was determined when hypertrophy of the adenoids ( 2 degree or higher ) and/or hypertrophy of the tonsils ( 2 degree or higher ) , and/or nasal septum deviation and/or allergic rhinitis was diagnosed for the patient . the orthopedic and otorhinolaryngological examination was performed by expert investigators ( ec and rp ) . to assess the method error of clinical investigation , prior to the survey , the investigators calibrated and standardized their procedures by repeating examinations of 10 patients at 2 different times all statistical analyses were performed using the statistical software package spss 20.0 for windows . to compare the mean values , the student s t test was used if the distribution of data was normal . in case of non - normal data , hypotheses of interrelations between characteristics were verified using the criterion method and spearman correlation coefficients ( r ) . the most specific predictors of the decrease in the snb angle were assessed using logistic regression analysis and receiver operating characteristic ( roc ) curve analysis . a p - value of < 0.05 was considered statistically significant . the orthodontic examination consisted of the study model and cephalometric radiograph analysis . the study model examination consisted of a transverse examination in which a posterior cross - bite was confirmed ( at least 2 teeth showed a cross - relationship with the opposite teeth in the posterior segments of the dental arches . the cross - bite was categorized as unilateral or bilateral , and we performed a space analysis in which the difference between the available space and the necessary space in the dental arch was calculated . crowding was categorized as mild ( lack of space : 24 mm ) , moderate ( 59 mm ) , or severe ( > 9 mm ) . a standardized lateral cephalometric radiograph was taken for each patient ( kodak 8000c ; enlargement factor 1.15 ; exposure : 12 mas , 7680 kv ) and analyzed using dolphin software ( version 10.5 ) . the sagittal position of the maxilla ( sna ) and the mandible ( snb ) , the sagittal jaw relationship ( anb ) , the mandibular plane angle ( mp - sn ) , and the inclination of the maxillary incisors and mandibular incisors ( u1-ans / pns and l1-mp , respectively ) were used to analyze the facial skull parameters . the error margins for the study models and lateral cephalometric analysis were determined by repeatedly measuring the 6 variables on 10 randomly selected models and radiographs at 2-week intervals . parametric data were subjected to a paired - samples t - test and non - parametric wilcoxon signed ranks test , which showed that there was no significant difference between the data sets . the patient was examined in a relaxed standing posture : subjects were asked to stand in an upright position , barefoot , without moving , looking straight ahead , with relaxed shoulders and arms resting at their sides for a natural head and body position . a standard routine examination from the front , side , and back degrees of severity can not be differentiated with adequate precision by manual orthopedic diagnostics ; therefore , the findings were graded either as normal or abnormal . patients were first examined from the side and the thoracic kyphosis was evaluated . if an increased , but adjustable , asymptomatic curvature of the thoracic spine was observed , the posture was classed as kyphotic . all patients underwent tests to rule out scheuermann s disease and ankylosing spondylitis ; briefly , each patient was asked to stand upright and pull back the shoulders to induce thoracic extension . in cases of postural kyphosis , an increased curvature , which is regular and mobile , was found . next , patients were examined from the front , and the position of shoulders , the symmetry of the waist triangles , and the horizontal alignment of the upper iliac crests were noted . finally , patients were examined from the back , and the position of the shoulders , the scapular height , and the symmetry of the waist triangles , iliac crests , and thoracic rib hump were noted . briefly , patients were asked to bring their chin to their chest , relax the hands , and flex the hips with the knees extended . the examiner then looked for the emergence of a paravertebral muscle roller in the lumbar region or a rib hump in the thoracic region . if a rib prominence hump was present ( > 1 cm ) , then full - length frontal and lateral spinal radiographs were obtained to evaluate the degree of spinal deformation ( by measuring the cobb s angle ) ( figure 2 ) . the following diagnoses were made based on the findings : hypertrophy of the adenoids ( grade 23 ) was diagnosed when up to 2/3 of the choana was compromised ; hypertrophy of the palatal tonsils ( grade 24 ) was diagnosed when there was < 50% of normal space between tonsillar pillars ; nasal septum deviation was diagnosed when the nasal septum was severely shifted away from the midline ; and allergic rhinitis was diagnosed when the patient showed typical allergy symptoms ( nasal congestion , runny nose , sneezing , and watery eyes ) and skin - prick test results were positive . nasopharyngeal obstruction was determined when hypertrophy of the adenoids ( 2 degree or higher ) and/or hypertrophy of the tonsils ( 2 degree or higher ) , and/or nasal septum deviation and/or allergic rhinitis was diagnosed for the patient . the orthopedic and otorhinolaryngological examination was performed by expert investigators ( ec and rp ) . to assess the method error of clinical investigation , prior to the survey , the investigators calibrated and standardized their procedures by repeating examinations of 10 patients at 2 different times ( measuring agreement was calculated by kappa ; kappa values were > 0.8 ) . all statistical analyses were performed using the statistical software package spss 20.0 for windows . to compare the mean values , the student s t test was used if the distribution of data was normal . in case of non - normal data , hypotheses of interrelations between characteristics were verified using the criterion method and spearman correlation coefficients ( r ) . the most specific predictors of the decrease in the snb angle were assessed using logistic regression analysis and receiver operating characteristic ( roc ) curve analysis . a p - value of < 0.05 was considered statistically significant . the orthodontic , orthopedic , and otorhinolaryngological characteristics of the patients are described in table 1 . postural disorders were observed in 72 ( 76.6% ) patients . structural orthopedic anomaly ( scoliosis ) kyphotic posture was more common among males ( 26 ; 59.1% ) than females ( 19 ; 38% ) ( p=0.01 ) . there was no statistically significant association between sex and the occurrence of transverse orthopedic pathology . the relationships between malocclusion , sex , and sagittal orthopaedic pathology are presented in table 2 . there was a statistically significant correlation between presence of kyphotic posture and a reduction in the snb angle ( statistically significant in males but not significant in females ) . there was no significant association between the presence of transverse orthopedic pathology and orthodontic or otorhinolaryngologic pathology . also , there was no relationship between crowding , posterior cross - bite , and orthopedic or otorhinolaryngologic parameters . the findings evaluating the relationship between nasopharyngeal obstruction and sagittal orthopedic pathology indicated that kyphotic posture was significantly more common among patients with nasopharyngeal obstruction 54.1% patients with nasopharyngeal obstruction were kyphotic , compared with 25% patients with no nasopharyngeal obstruction ( spearman s correlation coefficient=0.24 ; p=0.02 ) . because we identified a significant decrease in the snb angle in patients with kyphotic posture and nasopharyngeal obstruction , we performed logistic regression analysis to evaluate the risk of a decrease in the snb angle . the critical value of the snb angle was determined using roc curve analysis ( figure 3 ) . the threshold of 77 was crucial for the snb angle ( sensitivity 71.1% ; specificity , 69.8% ; p=0,002 ) . we found that among patients with snb angle < 77 , kyphotic posture was found in 71.1% of patients and normal thoracic kyphosis was found in 38.8% . therefore , we performed binary logistic regression analysis , which revealed that kyphotic posture increases odds ratio of the snb<77 angle by 3.887 ( 95% ci ; 1.6399.218 ) . this calculation adjusted with nasopharyngeal obstruction indicated odds ratio of the snb<77 angle by 4.037 ( 95% ci ; 1.6529.861 ) . malocclusion has a multifactorial etiology ; several of these factors , including oral habits and breathing mode , play an important role in pathogenesis . an improved understanding of the mechanism underlying normal craniofacial development is needed for the accurate diagnosis and appropriate treatment of malocclusion . the present study was based on the hypothesis that body posture , breathing pattern , and the type of malocclusion are inter - dependent . the study group comprised consecutive orthodontic patients aged 714 years ( the age during which transition from primary to permanent dentition occurs ) . the rate of spinal growth is not constant there is a period of accelerated growth between 10.5 and 15.5 years of age , and peak height velocity occurs at an average of 12.2 years in girls and 13.9 years in boys . during this period we detected a high prevalence of orthopedic anomalies in the study group , the most common being kyphotic posture ( 47.9% ) and a thoracic rib hump ( 51.1% ) . for example , lippold et al . reported orthopedic pathological findings in 52% of pre - school children , and hagner et al . identified poor body posture in 65.71% of 10-year - old and 54.29% of 13-year - old non - orthodontic children . according to the literature , hypertrophy of the adenoids and tonsils , which causes mouth breathing , is common in children ( varying from 40% to 60% ) . the present study identified hypertrophy of the adenoids in 57.4% and of the tonsils in 90.3% of subjects ; these high levels may be due to the selection of the specific group of patients . overall , the results showed that sagittal body posture was related to sagittal craniofacial parameters . such a difference could occur because kyphotic posture was more common among males ( 59.1% ) than females ( 38% ) ( p=0.01 ) . this is in agreement with the results of lippold et al . , who identified correlations between the sagittal position of the lower jaw and thoracic inclination . lippold et al . also reported that the position of the maxilla does not correlate with spinal curvature , which also agrees with our results . however , we found no relationship of the vertical position of the lower jaw and thoracic inclination , which is in contrast to the results of lippold et al . . the results of our study show that the facial angle ( mp - sn ) tended to be increased in patients with a kyphotic posture ; however , this difference was not significant . reported that about 13% of children 8.52.3 years old showed a pathological gait , and among them there was a higher prevalence of patients with a deep bite and open bite . the authors also suggested that vertical malocclusions are correlated to the dominant eye . in our study we found that dental overjet and overbite were statistically significantly greater in patients with a kyphotic posture ; however , skeletal vertical parameters ( angle mp - sn ) did not differ in groups with normal or kyphotic posture . according to our results , previous studies showed that nasopharyngeal pathology causes changes in the growth of the mandible ( which rotates downward and backward ) , and an increase in the height of the lower face . we also found that a kyphotic posture was statistically significantly more common among patients with nasopharyngeal pathology ; however , neiva et al . did not find an increase in thoracic kyphosis in mouth - breathing subjects . when we evaluated body posture in the transverse plane , we identified any association between asymmetric posture and orthodontic parameters . the malocclusion most likely to be related to asymmetric orthopaedic anomalies should be a posterior cross - bite . examined 55 children referred to an orthopedic center and found that those with a unilateral cross - bite were more likely to have an oblique shoulder , oblique pelvis , functional leg length differences , and scoliosis than children with dental symmetry . mouth breathing is also associated with narrowing of the upper dental arch and a posterior cross - bite . however , we did not find any relationship between body posture , nasopharyngeal pathology , and a posterior cross - bite . michelotti et al . also failed to demonstrate a significant association between a posterior cross - bite and postural stability or transverse orthopedic pathology . here , we found no relationship between crowding of the dental arches and body posture or nasopharyngeal pathology . this is in contrast to the findings of pachi et al . and solow and sonnesen , who reported that crowding was associated with craniocervical posture . the discrepancy between the results reported herein and those of others can be explained , at least in part , by differences in study design ( e.g. , type of orthopedic evaluation ) , specific patient groups ( e.g. , orthodontic / non - orthodontic patients ) , different age groups , and different sample sizes . in summary , the results of the present study suggest that there is a significant association between a decrease in the snb angle , kyphotic posture , and nasopharyngeal pathology . the presence of kyphotic posture , especially together with a nasopharyngeal obstruction , increases the possibility of the mandibular retrusion . the null hypotheses were tested : 1 ) sagittal craniofacial skeletal morphology depended on the nasopharyngeal obstruction and body posture ; and 2 ) vertical craniofacial skeletal morphology did not depend on the nasopharyngeal obstruction and body posture . which of these morphologic changes are primary and which are consequential ? to answer this question , we looked at a few studies that evaluated changes in body posture after the correction of malocclusion . conducted a randomized clinical trial in a juvenile population with a unilateral posterior cross - bite and found that early orthodontic treatment had no effect on postural parameters . found that there was no significant difference between body posture before orthognathic surgery and at 1 year after surgery . suggest that improvements in nasopharyngeal airway adequacy after rapid maxillary expansion were only mildly associated with changes in the craniocervical angle and tipping of the head , and a review by michelotti et al . concluded that even if there is an association between occlusal factors and postural alterations , there is not enough scientific evidence to support a cause - effect relationship . therefore , although it is reasonable to suppose that the stomatognathic system can affect cervical region function , its overall relevance to body posture is still unclear . this lack of scientific evidence in the literature of a cause - effect relationship between occlusion , nasopharyngeal pathology , and postural disorders makes this question difficult to answer . the present study has 2 main findings : 1 ) there was a significant association between the sagittal position of the mandible ( snb angle ) and a kyphotic posture ; and 2 ) based on study results , kyphotic posture was significantly more common among patients with nasopharyngeal obstruction .

backgroundmalocclusion , body posture , and breathing pattern may be correlated , but this issue is still controversial . the aim of the study was to examine the relationship between the type of malocclusion , body posture , and nasopharyngeal obstruction in children aged 714 years.material/methodsthe study group comprised 94 patients aged 714 years ( meansd : 11.92.1 years ) ; 44 ( 46.8% ) males and 50 ( 53.2% ) females . all patients passed an examination performed by the same orthodontist ( study model and cephalometric radiograph analysis ) , orthopedic surgeon ( body posture examined from the front , side , and back ) , and otorhinolaryngologist ( anterior and posterior rhinoscopy and pharyngoscopy ) in a blind manner.resultspostural disorders were observed in 72 ( 76.6% ) patients . hypertrophy of the adenoids was diagnosed in 54 ( 57.4% ) patients , hypertrophy of the tonsils in 85 ( 90.3% ) , nasal septum deviation in 51 ( 54.3% ) , and allergic rhinitis in 19 ( 20.2% ) patients . there was a statistically significant correlation between presence of kyphotic posture and a reduction in the snb angle , representing sagittal position of the mandible . also , there was a statistically significant association between kyphotic posture and nasopharyngeal obstruction ( 54.1% of patients with nasopharyngeal obstruction were kyphotic , compared with 25% of patients with no nasopharyngeal obstruction ; p=0.02 ) . kyphotic posture and reduced snb angle were more common among males.conclusionswe concluded that : 1 ) there was a significant association between the sagittal position of the mandible ( snb angle ) and a kyphotic posture ; 2 ) kyphotic posture was significantly more common among patients with nasopharyngeal obstruction .

Background Material and Methods Orthodontic examination Orthopedic examination Otorhinolaryngological examination Statistical analysis Results Discussion Conclusions

concluded that mandibular position , asymmetric occlusion , and temporomandibular disorders do not appear to correlate with body sway or muscle activity in other parts of the body , including those responsible for maintaining posture , at a clinically relevant level . several studies have examined the relationship between malocclusion and parameters of body posture in the sagittal and frontal planes ; the results identified a correlation between structural orthopaedic diseases and occlusal morphology . found that patients with idiopathic scoliosis showed more asymmetric features characteristic of malocclusion than a random control group . however , the results from studies looking at the correlation between poor body posture and dental occlusion are conflicting . examined 59 pre - school children and found statistically significant correlations between weak body posture and class ii malocclusion . used rasterstereography to examine the sagittal profile of the spine in 53 adults with skeletal class ii and class iii malocclusions , and found a correlation between the vertical and sagittal position of the lower jaw and thoracic , lordotic , and pelvic inclination , and between the vertical and sagittal position of the lower jaw and pelvic rotation . thus , 2 different models of back shape were devised based on of the results on these studies : 1 ) a more distal and vertical craniofacial pattern is associated with an increase in the upper thoracic , lumbar - lordotic , and pelvic angles ; and 2 ) a more mesial and horizontal craniofacial pattern is associated with smaller upper thoracic , lumbar - lordotic , and pelvic angles . compared body posture in 29 class ii and class iii patients , and found that the apex of the thoracic kyphosis was more cranial in class iii patients than in class ii patients or healthy controls . examined 703 children , they found no association between body posture and clinically assessed dental occlusion . contradictory results of studies can arise because there was a large diversity among the studies with regard to the protocols used ; some studies assessed body posture by physical examination while other studies used body photographs and rasterstereography . enlarged tonsils and adenoids , allergic rhinitis , and chronic respiratory problems cause a mouth breathing syndrome , resulting in adaptive head and body postures , which also affects the development of the facial skeleton . the forward position of the head causes protraction and rotation of the shoulders , elevation and abduction of the scapulae , depression of the thoracic anterior region , and forward displacement of the whole body . demonstrated that adults who were mouth - breathers during childhood had a more anterior head posture and a larger lumbar lordosis angle than individuals in a control group . to date , no study has examined the association between malocclusion , body posture , and breathing pattern . therefore , the aim of the present study was to examine the relationship between the type of malocclusion , body posture , and nasopharyngeal obstruction in children aged 714 years . the tested null hypotheses were that : 1 ) sagittal craniofacial skeletal morphology depends on the nasopharyngeal obstruction and body posture , and 2 ) vertical craniofacial skeletal morphology depends on the nasopharyngeal obstruction and body posture . the study sample was obtained from consecutive patients attending for orthodontic treatment at the department of orthodontics who agreed to participate in the study from september 2013 through may 2014 . a full explanation of the study aims and procedures was provided to the parents of each patient and signed consent forms were obtained . the study was approved by the regional biomedical research ethics committee ( no . the study group comprised 94 patients aged 714 years ( mean sd : 11.92.1 years ) . forty - four were male ( 46.8% ) and 50 were female ( 53.2% ) . all patients passed an examination performed by the same clinical team in a blinded manner . performing a power calculation , we anticipated changes in snb angle by 2 ( sd=2 ) , =0.05 . after investigation we concluded the study needed a power 0.802 ( n1=49 , n2=45 ; =0.05 ; change in snb angle 2.21 ) ( sd=3.77 ) . the inclusion criteria into the study were as follows : age 714 years ; no history of maxillofacial trauma or surgery , syndromes , clefts , or orthodontic treatment ; no previous treatment for orthopedic disorders ; and no previous injury to the pelvis , spine , or long bones . the orthodontic examination consisted of the study model and cephalometric radiograph analysis . the study model examination consisted of a transverse examination in which a posterior cross - bite was confirmed ( at least 2 teeth showed a cross - relationship with the opposite teeth in the posterior segments of the dental arches . the cross - bite was categorized as unilateral or bilateral , and we performed a space analysis in which the difference between the available space and the necessary space in the dental arch was calculated . a standardized lateral cephalometric radiograph was taken for each patient ( kodak 8000c ; enlargement factor 1.15 ; exposure : 12 mas , 7680 kv ) and analyzed using dolphin software ( version 10.5 ) . the sagittal position of the maxilla ( sna ) and the mandible ( snb ) , the sagittal jaw relationship ( anb ) , the mandibular plane angle ( mp - sn ) , and the inclination of the maxillary incisors and mandibular incisors ( u1-ans / pns and l1-mp , respectively ) were used to analyze the facial skull parameters . parametric data were subjected to a paired - samples t - test and non - parametric wilcoxon signed ranks test , which showed that there was no significant difference between the data sets . the patient was examined in a relaxed standing posture : subjects were asked to stand in an upright position , barefoot , without moving , looking straight ahead , with relaxed shoulders and arms resting at their sides for a natural head and body position . a standard routine examination from the front , side , and back degrees of severity can not be differentiated with adequate precision by manual orthopedic diagnostics ; therefore , the findings were graded either as normal or abnormal . patients were first examined from the side and the thoracic kyphosis was evaluated . if an increased , but adjustable , asymptomatic curvature of the thoracic spine was observed , the posture was classed as kyphotic . next , patients were examined from the front , and the position of shoulders , the symmetry of the waist triangles , and the horizontal alignment of the upper iliac crests were noted . finally , patients were examined from the back , and the position of the shoulders , the scapular height , and the symmetry of the waist triangles , iliac crests , and thoracic rib hump were noted . if a rib prominence hump was present ( > 1 cm ) , then full - length frontal and lateral spinal radiographs were obtained to evaluate the degree of spinal deformation ( by measuring the cobb s angle ) ( figure 2 ) . the following diagnoses were made based on the findings : hypertrophy of the adenoids ( grade 23 ) was diagnosed when up to 2/3 of the choana was compromised ; hypertrophy of the palatal tonsils ( grade 24 ) was diagnosed when there was < 50% of normal space between tonsillar pillars ; nasal septum deviation was diagnosed when the nasal septum was severely shifted away from the midline ; and allergic rhinitis was diagnosed when the patient showed typical allergy symptoms ( nasal congestion , runny nose , sneezing , and watery eyes ) and skin - prick test results were positive . nasopharyngeal obstruction was determined when hypertrophy of the adenoids ( 2 degree or higher ) and/or hypertrophy of the tonsils ( 2 degree or higher ) , and/or nasal septum deviation and/or allergic rhinitis was diagnosed for the patient . the most specific predictors of the decrease in the snb angle were assessed using logistic regression analysis and receiver operating characteristic ( roc ) curve analysis . the orthodontic examination consisted of the study model and cephalometric radiograph analysis . the study model examination consisted of a transverse examination in which a posterior cross - bite was confirmed ( at least 2 teeth showed a cross - relationship with the opposite teeth in the posterior segments of the dental arches . the cross - bite was categorized as unilateral or bilateral , and we performed a space analysis in which the difference between the available space and the necessary space in the dental arch was calculated . a standardized lateral cephalometric radiograph was taken for each patient ( kodak 8000c ; enlargement factor 1.15 ; exposure : 12 mas , 7680 kv ) and analyzed using dolphin software ( version 10.5 ) . the sagittal position of the maxilla ( sna ) and the mandible ( snb ) , the sagittal jaw relationship ( anb ) , the mandibular plane angle ( mp - sn ) , and the inclination of the maxillary incisors and mandibular incisors ( u1-ans / pns and l1-mp , respectively ) were used to analyze the facial skull parameters . parametric data were subjected to a paired - samples t - test and non - parametric wilcoxon signed ranks test , which showed that there was no significant difference between the data sets . a standard routine examination from the front , side , and back degrees of severity can not be differentiated with adequate precision by manual orthopedic diagnostics ; therefore , the findings were graded either as normal or abnormal . patients were first examined from the side and the thoracic kyphosis was evaluated . if an increased , but adjustable , asymptomatic curvature of the thoracic spine was observed , the posture was classed as kyphotic . next , patients were examined from the front , and the position of shoulders , the symmetry of the waist triangles , and the horizontal alignment of the upper iliac crests were noted . finally , patients were examined from the back , and the position of the shoulders , the scapular height , and the symmetry of the waist triangles , iliac crests , and thoracic rib hump were noted . if a rib prominence hump was present ( > 1 cm ) , then full - length frontal and lateral spinal radiographs were obtained to evaluate the degree of spinal deformation ( by measuring the cobb s angle ) ( figure 2 ) . the following diagnoses were made based on the findings : hypertrophy of the adenoids ( grade 23 ) was diagnosed when up to 2/3 of the choana was compromised ; hypertrophy of the palatal tonsils ( grade 24 ) was diagnosed when there was < 50% of normal space between tonsillar pillars ; nasal septum deviation was diagnosed when the nasal septum was severely shifted away from the midline ; and allergic rhinitis was diagnosed when the patient showed typical allergy symptoms ( nasal congestion , runny nose , sneezing , and watery eyes ) and skin - prick test results were positive . nasopharyngeal obstruction was determined when hypertrophy of the adenoids ( 2 degree or higher ) and/or hypertrophy of the tonsils ( 2 degree or higher ) , and/or nasal septum deviation and/or allergic rhinitis was diagnosed for the patient . the most specific predictors of the decrease in the snb angle were assessed using logistic regression analysis and receiver operating characteristic ( roc ) curve analysis . the orthodontic , orthopedic , and otorhinolaryngological characteristics of the patients are described in table 1 . postural disorders were observed in 72 ( 76.6% ) patients . structural orthopedic anomaly ( scoliosis ) kyphotic posture was more common among males ( 26 ; 59.1% ) than females ( 19 ; 38% ) ( p=0.01 ) . there was no statistically significant association between sex and the occurrence of transverse orthopedic pathology . the relationships between malocclusion , sex , and sagittal orthopaedic pathology are presented in table 2 . there was a statistically significant correlation between presence of kyphotic posture and a reduction in the snb angle ( statistically significant in males but not significant in females ) . there was no significant association between the presence of transverse orthopedic pathology and orthodontic or otorhinolaryngologic pathology . also , there was no relationship between crowding , posterior cross - bite , and orthopedic or otorhinolaryngologic parameters . the findings evaluating the relationship between nasopharyngeal obstruction and sagittal orthopedic pathology indicated that kyphotic posture was significantly more common among patients with nasopharyngeal obstruction 54.1% patients with nasopharyngeal obstruction were kyphotic , compared with 25% patients with no nasopharyngeal obstruction ( spearman s correlation coefficient=0.24 ; p=0.02 ) . because we identified a significant decrease in the snb angle in patients with kyphotic posture and nasopharyngeal obstruction , we performed logistic regression analysis to evaluate the risk of a decrease in the snb angle . the critical value of the snb angle was determined using roc curve analysis ( figure 3 ) . the threshold of 77 was crucial for the snb angle ( sensitivity 71.1% ; specificity , 69.8% ; p=0,002 ) . we found that among patients with snb angle < 77 , kyphotic posture was found in 71.1% of patients and normal thoracic kyphosis was found in 38.8% . therefore , we performed binary logistic regression analysis , which revealed that kyphotic posture increases odds ratio of the snb<77 angle by 3.887 ( 95% ci ; 1.6399.218 ) . this calculation adjusted with nasopharyngeal obstruction indicated odds ratio of the snb<77 angle by 4.037 ( 95% ci ; 1.6529.861 ) . an improved understanding of the mechanism underlying normal craniofacial development is needed for the accurate diagnosis and appropriate treatment of malocclusion . the present study was based on the hypothesis that body posture , breathing pattern , and the type of malocclusion are inter - dependent . the study group comprised consecutive orthodontic patients aged 714 years ( the age during which transition from primary to permanent dentition occurs ) . during this period we detected a high prevalence of orthopedic anomalies in the study group , the most common being kyphotic posture ( 47.9% ) and a thoracic rib hump ( 51.1% ) . according to the literature , hypertrophy of the adenoids and tonsils , which causes mouth breathing , is common in children ( varying from 40% to 60% ) . the present study identified hypertrophy of the adenoids in 57.4% and of the tonsils in 90.3% of subjects ; these high levels may be due to the selection of the specific group of patients . overall , the results showed that sagittal body posture was related to sagittal craniofacial parameters . such a difference could occur because kyphotic posture was more common among males ( 59.1% ) than females ( 38% ) ( p=0.01 ) . , who identified correlations between the sagittal position of the lower jaw and thoracic inclination . also reported that the position of the maxilla does not correlate with spinal curvature , which also agrees with our results . however , we found no relationship of the vertical position of the lower jaw and thoracic inclination , which is in contrast to the results of lippold et al . the results of our study show that the facial angle ( mp - sn ) tended to be increased in patients with a kyphotic posture ; however , this difference was not significant . reported that about 13% of children 8.52.3 years old showed a pathological gait , and among them there was a higher prevalence of patients with a deep bite and open bite . in our study we found that dental overjet and overbite were statistically significantly greater in patients with a kyphotic posture ; however , skeletal vertical parameters ( angle mp - sn ) did not differ in groups with normal or kyphotic posture . according to our results , previous studies showed that nasopharyngeal pathology causes changes in the growth of the mandible ( which rotates downward and backward ) , and an increase in the height of the lower face . we also found that a kyphotic posture was statistically significantly more common among patients with nasopharyngeal pathology ; however , neiva et al . when we evaluated body posture in the transverse plane , we identified any association between asymmetric posture and orthodontic parameters . examined 55 children referred to an orthopedic center and found that those with a unilateral cross - bite were more likely to have an oblique shoulder , oblique pelvis , functional leg length differences , and scoliosis than children with dental symmetry . mouth breathing is also associated with narrowing of the upper dental arch and a posterior cross - bite . however , we did not find any relationship between body posture , nasopharyngeal pathology , and a posterior cross - bite . also failed to demonstrate a significant association between a posterior cross - bite and postural stability or transverse orthopedic pathology . here , we found no relationship between crowding of the dental arches and body posture or nasopharyngeal pathology . , type of orthopedic evaluation ) , specific patient groups ( e.g. , orthodontic / non - orthodontic patients ) , different age groups , and different sample sizes . in summary , the results of the present study suggest that there is a significant association between a decrease in the snb angle , kyphotic posture , and nasopharyngeal pathology . the presence of kyphotic posture , especially together with a nasopharyngeal obstruction , increases the possibility of the mandibular retrusion . the null hypotheses were tested : 1 ) sagittal craniofacial skeletal morphology depended on the nasopharyngeal obstruction and body posture ; and 2 ) vertical craniofacial skeletal morphology did not depend on the nasopharyngeal obstruction and body posture . to answer this question , we looked at a few studies that evaluated changes in body posture after the correction of malocclusion . found that there was no significant difference between body posture before orthognathic surgery and at 1 year after surgery . suggest that improvements in nasopharyngeal airway adequacy after rapid maxillary expansion were only mildly associated with changes in the craniocervical angle and tipping of the head , and a review by michelotti et al . concluded that even if there is an association between occlusal factors and postural alterations , there is not enough scientific evidence to support a cause - effect relationship . therefore , although it is reasonable to suppose that the stomatognathic system can affect cervical region function , its overall relevance to body posture is still unclear . this lack of scientific evidence in the literature of a cause - effect relationship between occlusion , nasopharyngeal pathology , and postural disorders makes this question difficult to answer . the present study has 2 main findings : 1 ) there was a significant association between the sagittal position of the mandible ( snb angle ) and a kyphotic posture ; and 2 ) based on study results , kyphotic posture was significantly more common among patients with nasopharyngeal obstruction .

yaound is the capital city of cameroon and has more than two million inhabitants and a land area of 297 km . douala is located in the coastal zone of the central province , the economic center of cameroon , and is one of the hottest cities . douala has more than three million inhabitants ( and is the most densely populated area in cameroon ) , and contains most of the urbanized and industrial areas of the country . the main diseases in douala and yaound are malaria , respiratory diseases , diarrheal diseases , hiv / aids , tuberculosis , hypertension , obesity , and cardiovascular diseases . houses are made of cement blocks , bricks , or shaped stones with a roof of cement , zinc , or tile ( 19 ) . household furnishings and belongings are characterized by cooking with gas ( 45% ) or an open fire using wood , ownership of televisions , radios ( 81% ) , mobile phones ( 10% ) , and cars ( 19 ) . about 22% of the population uses a water closet , cameroon is experiencing climate change characterized by increased temperatures during dry seasons and early rains ( with droughts and flooding ) . this problem is exacerbated in the north and southwest provinces and cities of yaound and douala as compared to other provinces and cities . the temperature trends on fig . 1 , assembled by kjellstrom and lemke for this project from the us noaa global climate website ( http://www.noaa.gov/ ) , show that both average temperature and average dew point ( absolute humidity ) increased consistently during the last 30 years at the two main cameroon city airports . 1 ) . these data show annual trends and to get a better understanding of the actual heat stress during the hottest periods , monthly and daily data would be required . in douala , the increasing dew point combined with the increasing temperature means a significant increase in heat stress . t(ave ) is mean annual temperature , dewpt is mean annual dew point ( an index of absolute humidity ) , tmax and tmin are annual averages of maximum and minimum temperature . the lines are fitted linear regressions with x = years : 19802008 and y = the level of each variable in degrees celsius . the school day is from 7:30 am to 15:30 pm with two breaks ; a morning break is from 10:15 to 10:30 am and a lunch break is from 12:30 pm to 13:30 pm . on wednesdays , schools sometimes , windows are unconventionally made by creating box - like openings in one wall section . the floors and walls are made of cement blocks and the roof is made of cement or tile . boys and girls , aged 1216 years , were recruited from three public secondary schools randomly selected in yaound and douala . two schools were randomly selected from yaound ( school 1 , school 2 ) and one was randomly selected from douala ( school 3 ) . all students from each classroom who were present at the time of data collection were included . in total , 285 schoolchildren completed the study . permission to carry out the study was obtained from the school headmasters and each of the schoolchildren prior to study conduct . ethical permission was obtained from the medical faculty of the university of yaound , cameroon . study personnel consisted of one public health expert , two nurses , and four university students . most of the study personnel were trained during a previous study about schoolchildren 's nutrition . we could not interview all of the schoolchildren each day due to time constraints and absence of those who went out for lunch , breaks , to drink water , or left for home before the interviews were conducted . moreover , the study team was not large enough to interview all schoolchildren each day / hour . however , interview speed and skills improved during data collection . all schoolchildren ( n = 285 ) in yaound ( n = 174 ) and douala ( n = 111 ) completed the questionnaire about their background ( age , sex ) , general symptoms , and hot feeling . the background questionnaire was done at the beginning of the week and the general questionnaire / hot feeling was done at the end of the week . in yaound ( school 1 , march 1519 , 2010 ; school 2 , march 2226 , 2010 ) , 50 of 174 students were individually interviewed about hourly symptoms and performance . a lascar datalogger was used to measure indoor temperature , dew point , and humidity in the school 1 classroom ( from 7:38 am to 15:38 pm ) march 1519 , 2010 and from march 2226 , 2010 in the classroom for school 2 ( from 7:34 am to 14:34 pm ) in yaound . the datalogger was fixed in one roof corner of the classroom . in school 3 , a celsius thermometer was used to measure the daily indoor temperature and relative humidity in classrooms from march 2226 , 2010 in douala ( school 3 ) . in addition , outdoor temperatures were measured using a celsius thermometer in douala and yaound . the thermometer was placed across the shadow of a window with an outside cable ( in the shadow ) to measure outdoor temperatures . the body weight of each student in light clothing and no shoes was measured to the nearest 0.1 kg using an electronic scale ( seca model 826 , bradford , uk ) in the morning in yaound . an 8 hour ( from 7:30 am to 15:30 pm ) symptoms and school performance interview was recorded for monday , tuesday , wednesday , and thursday in yaound ( school 1 and school 2 ) . the interview recall for friday should have been done on saturday but as there is no school on saturday we had to omit these . the students were asked to recall all symptoms that they felt and school performance undertaken during the previous day from 7:30 am to 15:30 pm . this interview recall was about symptoms ( i.e. fatigue , headache ) , hot feeling ( i.e. hot , very hot ) , and school performance ( i.e. writing speed , level of understanding , absentmindedness ) from 7:30 am to 15:30 pm during the previous day . we have previously used the same method for 24-h dietary recall to collect information about food consumption ( 21 ) . descriptive statistics by school and week ( yaound and douala ) were done for fatigue , hot / very hot , fever , vertigo , headache , nausea , thirst , malaria , and fever . scatter plots , linear regression , and correlation ( with pearson correlation coefficient ) were used to investigate the relationship between indoor air temperature and the percentage of schoolchildren who felt very hot , had fatigue , and had headaches in yaound . daily ( monday to friday ) and hourly ( 7:30 am to 15:30 pm ) indoor air temperature , humidity , and dew point in yaound ( week 1 and week 2 ) were computed . the percentage of schoolchildren performance in yaound was computed and included writing speed , lack of understanding , and absentmindedness . outdoor air temperature in yaound and douala as well as indoor air temperature and humidity in douala was described . yaound is the capital city of cameroon and has more than two million inhabitants and a land area of 297 km . douala is located in the coastal zone of the central province , the economic center of cameroon , and is one of the hottest cities . douala has more than three million inhabitants ( and is the most densely populated area in cameroon ) , and contains most of the urbanized and industrial areas of the country . the main diseases in douala and yaound are malaria , respiratory diseases , diarrheal diseases , hiv / aids , tuberculosis , hypertension , obesity , and cardiovascular diseases . houses are made of cement blocks , bricks , or shaped stones with a roof of cement , zinc , or tile ( 19 ) . household furnishings and belongings are characterized by cooking with gas ( 45% ) or an open fire using wood , ownership of televisions , radios ( 81% ) , mobile phones ( 10% ) , and cars ( 19 ) . about 22% of the population uses a water closet , cameroon is experiencing climate change characterized by increased temperatures during dry seasons and early rains ( with droughts and flooding ) . this problem is exacerbated in the north and southwest provinces and cities of yaound and douala as compared to other provinces and cities . the temperature trends on fig . 1 , assembled by kjellstrom and lemke for this project from the us noaa global climate website ( http://www.noaa.gov/ ) , show that both average temperature and average dew point ( absolute humidity ) increased consistently during the last 30 years at the two main cameroon city airports . 1 ) . these data show annual trends and to get a better understanding of the actual heat stress during the hottest periods , monthly and daily data would be required . in douala , the increasing dew point combined with the increasing temperature means a significant increase in heat stress . t(ave ) is mean annual temperature , dewpt is mean annual dew point ( an index of absolute humidity ) , tmax and tmin are annual averages of maximum and minimum temperature . the lines are fitted linear regressions with x = years : 19802008 and y = the level of each variable in degrees celsius . the school day is from 7:30 am to 15:30 pm with two breaks ; a morning break is from 10:15 to 10:30 am and a lunch break is from 12:30 pm to 13:30 pm . on wednesdays , schools close at 12:30 pm . sometimes , windows are unconventionally made by creating box - like openings in one wall section . the floors and walls are made of cement blocks and the roof is made of cement or tile . boys and girls , aged 1216 years , were recruited from three public secondary schools randomly selected in yaound and douala . two schools were randomly selected from yaound ( school 1 , school 2 ) and one was randomly selected from douala ( school 3 ) . all students from each classroom who were present at the time of data collection were included . in total , 285 schoolchildren completed the study . permission to carry out the study was obtained from the school headmasters and each of the schoolchildren prior to study conduct . ethical permission was obtained from the medical faculty of the university of yaound , cameroon . study personnel consisted of one public health expert , two nurses , and four university students . most of the study personnel were trained during a previous study about schoolchildren 's nutrition . we could not interview all of the schoolchildren each day due to time constraints and absence of those who went out for lunch , breaks , to drink water , or left for home before the interviews were conducted . moreover , the study team was not large enough to interview all schoolchildren each day / hour . however , interview speed and skills improved during data collection . all schoolchildren ( n = 285 ) in yaound ( n = 174 ) and douala ( n = 111 ) completed the questionnaire about their background ( age , sex ) , general symptoms , and hot feeling . the background questionnaire was done at the beginning of the week and the general questionnaire / hot feeling was done at the end of the week . in yaound ( school 1 , march 1519 , 2010 ; school 2 , march 2226 , 2010 ) , 50 of 174 students were individually interviewed about hourly symptoms and performance . a lascar datalogger was used to measure indoor temperature , dew point , and humidity in the school 1 classroom ( from 7:38 am to 15:38 pm ) march 1519 , 2010 and from march 2226 , 2010 in the classroom for school 2 ( from 7:34 am to 14:34 pm ) in yaound . the datalogger was fixed in one roof corner of the classroom . in school 3 , a celsius thermometer was used to measure the daily indoor temperature and relative humidity in classrooms from march 2226 , 2010 in douala ( school 3 ) . in addition , outdoor temperatures were measured using a celsius thermometer in douala and yaound . the thermometer was placed across the shadow of a window with an outside cable ( in the shadow ) to measure outdoor temperatures . the body weight of each student in light clothing and no shoes was measured to the nearest 0.1 kg using an electronic scale ( seca model 826 , bradford , uk ) in the morning in yaound . an 8 hour ( from 7:30 am to 15:30 pm ) symptoms and school performance interview was recorded for monday , tuesday , wednesday , and thursday in yaound ( school 1 and school 2 ) . the interview recall for friday should have been done on saturday but as there is no school on saturday we had to omit these . the students were asked to recall all symptoms that they felt and school performance undertaken during the previous day from 7:30 am to 15:30 pm . this interview recall was about symptoms ( i.e. fatigue , headache ) , hot feeling ( i.e. hot , very hot ) , and school performance ( i.e. writing speed , level of understanding , absentmindedness ) from 7:30 am to 15:30 pm during the previous day . we have previously used the same method for 24-h dietary recall to collect information about food consumption ( 21 ) . descriptive statistics by school and week ( yaound and douala ) were done for fatigue , hot / very hot , fever , vertigo , headache , nausea , thirst , malaria , and fever . scatter plots , linear regression , and correlation ( with pearson correlation coefficient ) were used to investigate the relationship between indoor air temperature and the percentage of schoolchildren who felt very hot , had fatigue , and had headaches in yaound . daily ( monday to friday ) and hourly ( 7:30 am to 15:30 pm ) indoor air temperature , humidity , and dew point in yaound ( week 1 and week 2 ) were computed . the percentage of schoolchildren performance in yaound was computed and included writing speed , lack of understanding , and absentmindedness . outdoor air temperature in yaound and douala as well as indoor air temperature and humidity in douala was described . the mean ages were 12.71.4 years in school 1 , 13.31.2 years in school 2 , and 12.91.3 years in school 3 . girls were heavier than boys , with mean weights ( kg ) of 49.99.2 vs 45.18.3 in school 1 , and 46.89.3 vs 44.77.6 in school 2 . overall , the mean body temperature was 37.6c ; 30 children had a body temperature of at least 38c in yaound . table 1 describes the characteristics of the schoolchildren during the 2-week study period in yaound and douala . school 1 , school 2 , and school 3 are three different groups of schoolchildren . up to 75% of the children reported fatigue , more than 60% were thirsty , and 49% had a headache . more than a third slept in class , 31% felt very hot overall , and 20% felt very hot in the head . prevalences ( % ) were higher among girls vs boys for sickness ( 17 vs 12 ) , thirst ( 63 vs 60 ) , fatigue ( 77 vs 71 ) , vertigo ( 20 vs 15 ) , malaria ( 9 vs 6 ) , headache ( 58 vs 39 ) , very hot overall ( 37 vs 21 ) , very hot in head ( 21 vs 18 ) , and sleeping in class ( 40 vs 32 ) although significant only for headache ( p = 0.010 ) , very hot overall ( p = 0.014 ) , and very hot in head ( p = 0.027 ) . prevalences of all symptoms except for very hot overall and very hot in head were higher during week 2 , school 2 ( march 2225 ) than week 1 , school 1 ( march 1518 ) in yaound ( table 1 ) . characteristics ( percentage in% , with number in brackets ) of schoolchildren percentage ( % ) of girls vs boys . significant differences between boys and girls ( p = 0.010 ; 0.014 ; 0.027 , for headache , very hot overall , and very hot in the head , respectively ) . note . school 1 , school 2 , and school 3 are three different groups of schoolchildren . 24 show that there was a significant and positive correlation between indoor air temperature and the percentages of schoolchildren , who felt very hot , had fatigue , and had headaches in schools 1 and 2 in yaound . the correlation coefficient was strong for very hot ( p<0.001 ) , fatigue ( p = 0.002 ) , and headache ( p = 0.001 ) in school 1 , week 1 and strong for very hot ( p<0.001 ) , weak for fatigue ( p = 0.037 ) , and headache ( p = 0.267 ) in school 2 , week 2 . during the day , percentages of very hot , fatigue , and headaches increase linearly with indoor air temperature in yaound ( figs . scatter plot showing the relationships between the temperature and the percentage of schoolchildren who felt very hot in yaound . the lines are fitted linear regressions with x = temperature in degrees celsius and y = percentage ( % ) of schoolchildren who felt hot from monday to thursday between 7:30 and 15:30 , r is the pearson correlation coefficient between temperature and very hot ( p<0.001 , school 1 and school 2 ) . scatter plot showing the relationships between the temperature and the percentage of schoolchildren who had fatigue in yaound . the lines are fitted linear regressions with x = temperature in degrees celsius and y = percentage ( % ) of schoolchildren who had fatigue from monday to thursday between 7:30 and 15:30 , r is the pearson correlation coefficient between temperature and fatigue ( p = 0.002 , school 1 and p = 0.037 , school 2 ) . scatter plot showing the relationships between the temperature and the percentage of schoolchildren who had headaches in yaound . the lines are fitted linear regressions with x = temperature in degrees celsius and y = percentage ( % ) of schoolchildren who had headaches from monday to thursday between 7:30 and 15:30 and r is the pearson correlation coefficient between temperature and headache ( p = 0.001 , school 1 and p = 0.267 , school 2 ) . . the within day comparisons ( between 12:30 am and 15:30 pm ) indicated that feeling very hot and a headache was associated with indoor temperature and more prevalent on the hottest days especially during week 1 , school 1 ( r = 0.72 and 0.53 , very hot and a headache , respectively , school 1 ) . the highest proportion of students who felt very hot ( 48% ) was observed on march 24 in yaound . the highest proportions of fatigue ( 76% ) and headache ( 38% ) were observed on march 18 and 23 , respectively , in yaound . the proportion of schoolchildren with poor performance increases during the day . up to 62% were absentminded , 45% had slow writing speed , and 25% did not understand the lessons in yaound . the indoor air temperatures , humidity , and dew points in yaound are presented in fig . the indoor air temperatures , humidity , and dew points are presented from monday to friday for week 1 , school 1 ( from 7:38 am to 15:38 pm ) and week 2 , school 2 ( from 7:34 am to 15:34 pm ) in yaound . daily indoor air temperature increases from morning to afternoon . the highest indoor temperature during school hours was 32.5c in yaound and 36.6c in douala . the highest relative humidity in yaound was 87% during week 1 and the lowest was 55.3% and occurred during week 2 . in douala , the highest relative humidity was 85% and the lowest 53% . in yaound , the highest dew point was at 25.3c during week 1 and the lowest was at 17.8c during week 2 . daily outdoor air temperature increases from morning to afternoon , with the highest temperatures of 33.6c in yaound and 35.6c in douala . datalogger of indoor air temperature , humidity , and dew point by hours from monday to friday from 7:30 to 15:30 in classrooms in yaound . temperature , dew point ( an index of absolute humidity ) , and humidity ( percentage relative humidity ) are given by hours from monday to friday . the lines are fitted linear regressions with x = hours : 7:30 to 15:30 and y = the level of each variable in degrees celsius and in percentage of relative humidity . school 1 and school 2 are two different groups of schoolchildren . the present study is both important and needed since data on the impact of heat on self - reported and measured health in cameroon is largely missing , especially among schoolchildren . our study is one of the first to provide data on indoor air temperature in schools and on the health impact of heat on schoolchildren in cameroon . temperature inside the classroom was very high during the present study and was found associated with health symptoms such as fatigue , headaches , and feeling very hot . symptoms of heat exhaustion , including headache , vertigo , fatigue , very hot , slept in class , and thirst were observed among our schoolchildren . these were particularly present during the afternoon and around noon when indoor air temperature and dew point were high . in fact , most people feel comfortable when the indoor air temperature is between 2027c with relative humidity ranges from 3560% ( 22 ) . in this study , most of the time , the temperature in yaound was close to 30c with an increasing dew point . in douala , temperatures were greater than 32c with a high relative humidity close to 70% . this explains why the children felt hot / very hot and reported headaches and fatigue as well as other symptoms . exposure to high temperature has been associated to illnesses and high morbidity in many studies ( 1 , 8 , 10 , 23 ) this is probably because there was no ventilation , no adequate roof , windows , and wall in the classrooms . moreover , there were more than 80 children in a classroom and their uniforms and shoes were not appropriate for the heat . in addition , schoolchildren do not always drink fluids when they are thirsty because there is no water supply inside the classroom . the health impact of heat , as described in the present study , has been reported in many other studies ( 1 , 2 , 4 , 8 , 24 ) . warm environments were related to headaches , feeling bad , and lethargy in a review by wyon ( 14 ) . recently , parsons described how fatigue , heat stroke , and death were related to extreme heat ( 25 ) . girls reported a higher prevalence of headaches and feeling very hot overall and in the head than boys did . girls are probably more vulnerable to high indoor air temperature because of menstruation and body composition . boys sometimes removed their shirts and rinsed their heads when it was hot ( although only during breaks ) . the boys ' short haircuts might be protective , while girls have plaited and tight hairstyles . also , girls had tight uniforms whereas boys had roomy uniforms . sometimes girls have extra clothes / tee - shirt under their uniform . girls were heavier than boys , and this might partially explain why they felt very hot more often than the boys did . gender differences due to heat have been related to adverse health problems in other studies ( 23 ) . the higher prevalence of very hot overall and in the head among schoolchildren during week 1 compared to week 2 ( table 1 ) , as well as the difference between week 1 and week 2 in yaound regarding the relationship between indoor temperature and very hot , fatigue , and headaches among schoolchildren , could be due to the fact that school 2 experienced almost no changes in indoor temperature between days in contrast to school 1 . this difference could also be due to the higher number of girls in school 1 ( 56 girls vs 33 boys ) compared to school 2 ( 47 girls vs 38 boys ) , since more girls felt very hot than did boys . this was probably due to extremely high indoor air temperatures , especially during week 1 . higher temperatures can cause malfunction of body systems ( 2 , 5 , 8) , and vulnerability to heat is linked to intrinsic changes in the regulatory system ( 1 , 4 , 8) . several studies have reported excess mortality due to heat waves . in 1995 , a 13.5% excess in deaths due to heat was reported among children aged 0 to 15 years in london ( 2 , 26 , 27 ) . in the present study , many schoolchildren were absentminded , had low writing speed , and did not understand the lessons especially in the afternoon and around noon probably because of indoor air temperatures of more than 32c in classrooms in yaound . high indoor air temperatures and dew points were related to headache , fatigue , and feeling hot and this might have had negative effects on school performance . in fact , the speed of school work decreases for each 1c increase in indoor air temperature ( 28 ) . some classroom studies in the 1960s found that reading speed , reading comprehension , and multiplication performance of schoolchildren were poorer at temperatures of 2729c ( 29 ) . also , more recent studies found that warm environments affect work performance by increasing spelling , reading , typing , grammatical errors , and a difficulty in thinking clearly and concentrating ( 11 , 14 ) . creative thinking was worse at 30c than at lower temperatures in a study of office work ( 3 ) . longitudinal data analysis studying hourly and daily symptoms , body measurements , and indoor air temperature measures between days in schools are needed to confirm these results and to disentangle trends within and between days from the association between indoor temperature and health status as well as to properly adjust for confounding factors . adverse symptoms such as headache , fatigue , and feeling very hot was found associated with high indoor air temperatures among schoolchildren in cameroon . poor school performance was also observed among our schoolchildren during the warmest period of the day . adequate environmental actions including good ventilation , open doors and windows , and water supplies are needed in schools that experience high heat . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

backgroundhealth impacts related to climate change are potentially an increasing problem in cameroon , especially during hot seasons when there are no means for protective and adaptive actions.objectiveto describe environmental conditions in schools and to evaluate the impact of heat on schoolchildren 's health during school days in the cameroon cities of yaound and douala.methodsschoolchildren ( n = 285 ) aged 1216 years from public secondary schools completed a questionnaire about their background , general symptoms , and hot feelings in a cross - sectional study . in yaound , 50 schoolchildren were individually interviewed during school days about hourly symptoms ( fatigue , headache , and feeling very hot ) and performance . lascar dataloggers were used to measure indoor classroom temperatures and humidity.resultsthere was a significant correlation between daily indoor temperature and the percentages of schoolchildren who felt very hot , had fatigue , and headaches in yaound . a high proportion of schoolchildren felt very hot ( 48% ) , had fatigue ( 76% ) , and headaches ( 38% ) in yaound . prevalences ( % ) were higher among girls than boys for headaches ( 58 vs 39 ) , feeling very hot overall ( 37 vs 21 ) , and very hot in head ( 21 vs 18 ) . up to 62% were absentminded and 45% had slow writing speed . high indoor temperatures of 32.5c in yaound and 36.6c in douala were observed in school.conclusionsheadache , fatigue , and feeling very hot associated with high indoor air temperature were observed among schoolchildren in the present study . longitudinal data in schools are needed to confirm these results . school environmental conditions should be improved in order to enhance learning .

Methodology Settings Weather and climate in the study regions School and classroom description Study population Data collection Data analysis Results Discussion Conclusions Conflict of interest and funding

douala is located in the coastal zone of the central province , the economic center of cameroon , and is one of the hottest cities . douala has more than three million inhabitants ( and is the most densely populated area in cameroon ) , and contains most of the urbanized and industrial areas of the country . the main diseases in douala and yaound are malaria , respiratory diseases , diarrheal diseases , hiv / aids , tuberculosis , hypertension , obesity , and cardiovascular diseases . household furnishings and belongings are characterized by cooking with gas ( 45% ) or an open fire using wood , ownership of televisions , radios ( 81% ) , mobile phones ( 10% ) , and cars ( 19 ) . this problem is exacerbated in the north and southwest provinces and cities of yaound and douala as compared to other provinces and cities . 1 , assembled by kjellstrom and lemke for this project from the us noaa global climate website ( http://www.noaa.gov/ ) , show that both average temperature and average dew point ( absolute humidity ) increased consistently during the last 30 years at the two main cameroon city airports . these data show annual trends and to get a better understanding of the actual heat stress during the hottest periods , monthly and daily data would be required . in douala , the increasing dew point combined with the increasing temperature means a significant increase in heat stress . boys and girls , aged 1216 years , were recruited from three public secondary schools randomly selected in yaound and douala . two schools were randomly selected from yaound ( school 1 , school 2 ) and one was randomly selected from douala ( school 3 ) . ethical permission was obtained from the medical faculty of the university of yaound , cameroon . most of the study personnel were trained during a previous study about schoolchildren 's nutrition . all schoolchildren ( n = 285 ) in yaound ( n = 174 ) and douala ( n = 111 ) completed the questionnaire about their background ( age , sex ) , general symptoms , and hot feeling . the background questionnaire was done at the beginning of the week and the general questionnaire / hot feeling was done at the end of the week . in yaound ( school 1 , march 1519 , 2010 ; school 2 , march 2226 , 2010 ) , 50 of 174 students were individually interviewed about hourly symptoms and performance . a lascar datalogger was used to measure indoor temperature , dew point , and humidity in the school 1 classroom ( from 7:38 am to 15:38 pm ) march 1519 , 2010 and from march 2226 , 2010 in the classroom for school 2 ( from 7:34 am to 14:34 pm ) in yaound . in school 3 , a celsius thermometer was used to measure the daily indoor temperature and relative humidity in classrooms from march 2226 , 2010 in douala ( school 3 ) . the thermometer was placed across the shadow of a window with an outside cable ( in the shadow ) to measure outdoor temperatures . the body weight of each student in light clothing and no shoes was measured to the nearest 0.1 kg using an electronic scale ( seca model 826 , bradford , uk ) in the morning in yaound . an 8 hour ( from 7:30 am to 15:30 pm ) symptoms and school performance interview was recorded for monday , tuesday , wednesday , and thursday in yaound ( school 1 and school 2 ) . this interview recall was about symptoms ( i.e. fatigue , headache ) , hot feeling ( i.e. hot , very hot ) , and school performance ( i.e. we have previously used the same method for 24-h dietary recall to collect information about food consumption ( 21 ) . descriptive statistics by school and week ( yaound and douala ) were done for fatigue , hot / very hot , fever , vertigo , headache , nausea , thirst , malaria , and fever . scatter plots , linear regression , and correlation ( with pearson correlation coefficient ) were used to investigate the relationship between indoor air temperature and the percentage of schoolchildren who felt very hot , had fatigue , and had headaches in yaound . daily ( monday to friday ) and hourly ( 7:30 am to 15:30 pm ) indoor air temperature , humidity , and dew point in yaound ( week 1 and week 2 ) were computed . the percentage of schoolchildren performance in yaound was computed and included writing speed , lack of understanding , and absentmindedness . outdoor air temperature in yaound and douala as well as indoor air temperature and humidity in douala was described . douala is located in the coastal zone of the central province , the economic center of cameroon , and is one of the hottest cities . douala has more than three million inhabitants ( and is the most densely populated area in cameroon ) , and contains most of the urbanized and industrial areas of the country . the main diseases in douala and yaound are malaria , respiratory diseases , diarrheal diseases , hiv / aids , tuberculosis , hypertension , obesity , and cardiovascular diseases . household furnishings and belongings are characterized by cooking with gas ( 45% ) or an open fire using wood , ownership of televisions , radios ( 81% ) , mobile phones ( 10% ) , and cars ( 19 ) . this problem is exacerbated in the north and southwest provinces and cities of yaound and douala as compared to other provinces and cities . 1 , assembled by kjellstrom and lemke for this project from the us noaa global climate website ( http://www.noaa.gov/ ) , show that both average temperature and average dew point ( absolute humidity ) increased consistently during the last 30 years at the two main cameroon city airports . in douala , the increasing dew point combined with the increasing temperature means a significant increase in heat stress . t(ave ) is mean annual temperature , dewpt is mean annual dew point ( an index of absolute humidity ) , tmax and tmin are annual averages of maximum and minimum temperature . the floors and walls are made of cement blocks and the roof is made of cement or tile . boys and girls , aged 1216 years , were recruited from three public secondary schools randomly selected in yaound and douala . ethical permission was obtained from the medical faculty of the university of yaound , cameroon . study personnel consisted of one public health expert , two nurses , and four university students . all schoolchildren ( n = 285 ) in yaound ( n = 174 ) and douala ( n = 111 ) completed the questionnaire about their background ( age , sex ) , general symptoms , and hot feeling . the background questionnaire was done at the beginning of the week and the general questionnaire / hot feeling was done at the end of the week . in yaound ( school 1 , march 1519 , 2010 ; school 2 , march 2226 , 2010 ) , 50 of 174 students were individually interviewed about hourly symptoms and performance . a lascar datalogger was used to measure indoor temperature , dew point , and humidity in the school 1 classroom ( from 7:38 am to 15:38 pm ) march 1519 , 2010 and from march 2226 , 2010 in the classroom for school 2 ( from 7:34 am to 14:34 pm ) in yaound . in school 3 , a celsius thermometer was used to measure the daily indoor temperature and relative humidity in classrooms from march 2226 , 2010 in douala ( school 3 ) . the thermometer was placed across the shadow of a window with an outside cable ( in the shadow ) to measure outdoor temperatures . the body weight of each student in light clothing and no shoes was measured to the nearest 0.1 kg using an electronic scale ( seca model 826 , bradford , uk ) in the morning in yaound . an 8 hour ( from 7:30 am to 15:30 pm ) symptoms and school performance interview was recorded for monday , tuesday , wednesday , and thursday in yaound ( school 1 and school 2 ) . fatigue , headache ) , hot feeling ( i.e. hot , very hot ) , and school performance ( i.e. writing speed , level of understanding , absentmindedness ) from 7:30 am to 15:30 pm during the previous day . we have previously used the same method for 24-h dietary recall to collect information about food consumption ( 21 ) . descriptive statistics by school and week ( yaound and douala ) were done for fatigue , hot / very hot , fever , vertigo , headache , nausea , thirst , malaria , and fever . scatter plots , linear regression , and correlation ( with pearson correlation coefficient ) were used to investigate the relationship between indoor air temperature and the percentage of schoolchildren who felt very hot , had fatigue , and had headaches in yaound . daily ( monday to friday ) and hourly ( 7:30 am to 15:30 pm ) indoor air temperature , humidity , and dew point in yaound ( week 1 and week 2 ) were computed . the percentage of schoolchildren performance in yaound was computed and included writing speed , lack of understanding , and absentmindedness . outdoor air temperature in yaound and douala as well as indoor air temperature and humidity in douala was described . the mean ages were 12.71.4 years in school 1 , 13.31.2 years in school 2 , and 12.91.3 years in school 3 . girls were heavier than boys , with mean weights ( kg ) of 49.99.2 vs 45.18.3 in school 1 , and 46.89.3 vs 44.77.6 in school 2 . table 1 describes the characteristics of the schoolchildren during the 2-week study period in yaound and douala . school 1 , school 2 , and school 3 are three different groups of schoolchildren . up to 75% of the children reported fatigue , more than 60% were thirsty , and 49% had a headache . more than a third slept in class , 31% felt very hot overall , and 20% felt very hot in the head . prevalences ( % ) were higher among girls vs boys for sickness ( 17 vs 12 ) , thirst ( 63 vs 60 ) , fatigue ( 77 vs 71 ) , vertigo ( 20 vs 15 ) , malaria ( 9 vs 6 ) , headache ( 58 vs 39 ) , very hot overall ( 37 vs 21 ) , very hot in head ( 21 vs 18 ) , and sleeping in class ( 40 vs 32 ) although significant only for headache ( p = 0.010 ) , very hot overall ( p = 0.014 ) , and very hot in head ( p = 0.027 ) . prevalences of all symptoms except for very hot overall and very hot in head were higher during week 2 , school 2 ( march 2225 ) than week 1 , school 1 ( march 1518 ) in yaound ( table 1 ) . characteristics ( percentage in% , with number in brackets ) of schoolchildren percentage ( % ) of girls vs boys . significant differences between boys and girls ( p = 0.010 ; 0.014 ; 0.027 , for headache , very hot overall , and very hot in the head , respectively ) . school 1 , school 2 , and school 3 are three different groups of schoolchildren . 24 show that there was a significant and positive correlation between indoor air temperature and the percentages of schoolchildren , who felt very hot , had fatigue , and had headaches in schools 1 and 2 in yaound . the correlation coefficient was strong for very hot ( p<0.001 ) , fatigue ( p = 0.002 ) , and headache ( p = 0.001 ) in school 1 , week 1 and strong for very hot ( p<0.001 ) , weak for fatigue ( p = 0.037 ) , and headache ( p = 0.267 ) in school 2 , week 2 . during the day , percentages of very hot , fatigue , and headaches increase linearly with indoor air temperature in yaound ( figs . scatter plot showing the relationships between the temperature and the percentage of schoolchildren who felt very hot in yaound . the lines are fitted linear regressions with x = temperature in degrees celsius and y = percentage ( % ) of schoolchildren who felt hot from monday to thursday between 7:30 and 15:30 , r is the pearson correlation coefficient between temperature and very hot ( p<0.001 , school 1 and school 2 ) . scatter plot showing the relationships between the temperature and the percentage of schoolchildren who had fatigue in yaound . the lines are fitted linear regressions with x = temperature in degrees celsius and y = percentage ( % ) of schoolchildren who had fatigue from monday to thursday between 7:30 and 15:30 , r is the pearson correlation coefficient between temperature and fatigue ( p = 0.002 , school 1 and p = 0.037 , school 2 ) . scatter plot showing the relationships between the temperature and the percentage of schoolchildren who had headaches in yaound . the lines are fitted linear regressions with x = temperature in degrees celsius and y = percentage ( % ) of schoolchildren who had headaches from monday to thursday between 7:30 and 15:30 and r is the pearson correlation coefficient between temperature and headache ( p = 0.001 , school 1 and p = 0.267 , school 2 ) . the within day comparisons ( between 12:30 am and 15:30 pm ) indicated that feeling very hot and a headache was associated with indoor temperature and more prevalent on the hottest days especially during week 1 , school 1 ( r = 0.72 and 0.53 , very hot and a headache , respectively , school 1 ) . the highest proportion of students who felt very hot ( 48% ) was observed on march 24 in yaound . the highest proportions of fatigue ( 76% ) and headache ( 38% ) were observed on march 18 and 23 , respectively , in yaound . the proportion of schoolchildren with poor performance increases during the day . up to 62% were absentminded , 45% had slow writing speed , and 25% did not understand the lessons in yaound . the indoor air temperatures , humidity , and dew points in yaound are presented in fig . the indoor air temperatures , humidity , and dew points are presented from monday to friday for week 1 , school 1 ( from 7:38 am to 15:38 pm ) and week 2 , school 2 ( from 7:34 am to 15:34 pm ) in yaound . daily indoor air temperature increases from morning to afternoon . the highest indoor temperature during school hours was 32.5c in yaound and 36.6c in douala . the highest relative humidity in yaound was 87% during week 1 and the lowest was 55.3% and occurred during week 2 . in douala , the highest relative humidity was 85% and the lowest 53% . in yaound , the highest dew point was at 25.3c during week 1 and the lowest was at 17.8c during week 2 . daily outdoor air temperature increases from morning to afternoon , with the highest temperatures of 33.6c in yaound and 35.6c in douala . datalogger of indoor air temperature , humidity , and dew point by hours from monday to friday from 7:30 to 15:30 in classrooms in yaound . temperature , dew point ( an index of absolute humidity ) , and humidity ( percentage relative humidity ) are given by hours from monday to friday . school 1 and school 2 are two different groups of schoolchildren . the present study is both important and needed since data on the impact of heat on self - reported and measured health in cameroon is largely missing , especially among schoolchildren . our study is one of the first to provide data on indoor air temperature in schools and on the health impact of heat on schoolchildren in cameroon . temperature inside the classroom was very high during the present study and was found associated with health symptoms such as fatigue , headaches , and feeling very hot . symptoms of heat exhaustion , including headache , vertigo , fatigue , very hot , slept in class , and thirst were observed among our schoolchildren . these were particularly present during the afternoon and around noon when indoor air temperature and dew point were high . in fact , most people feel comfortable when the indoor air temperature is between 2027c with relative humidity ranges from 3560% ( 22 ) . in this study , most of the time , the temperature in yaound was close to 30c with an increasing dew point . in douala , temperatures were greater than 32c with a high relative humidity close to 70% . exposure to high temperature has been associated to illnesses and high morbidity in many studies ( 1 , 8 , 10 , 23 ) this is probably because there was no ventilation , no adequate roof , windows , and wall in the classrooms . the health impact of heat , as described in the present study , has been reported in many other studies ( 1 , 2 , 4 , 8 , 24 ) . warm environments were related to headaches , feeling bad , and lethargy in a review by wyon ( 14 ) . recently , parsons described how fatigue , heat stroke , and death were related to extreme heat ( 25 ) . girls reported a higher prevalence of headaches and feeling very hot overall and in the head than boys did . girls are probably more vulnerable to high indoor air temperature because of menstruation and body composition . girls were heavier than boys , and this might partially explain why they felt very hot more often than the boys did . gender differences due to heat have been related to adverse health problems in other studies ( 23 ) . the higher prevalence of very hot overall and in the head among schoolchildren during week 1 compared to week 2 ( table 1 ) , as well as the difference between week 1 and week 2 in yaound regarding the relationship between indoor temperature and very hot , fatigue , and headaches among schoolchildren , could be due to the fact that school 2 experienced almost no changes in indoor temperature between days in contrast to school 1 . this difference could also be due to the higher number of girls in school 1 ( 56 girls vs 33 boys ) compared to school 2 ( 47 girls vs 38 boys ) , since more girls felt very hot than did boys . this was probably due to extremely high indoor air temperatures , especially during week 1 . higher temperatures can cause malfunction of body systems ( 2 , 5 , 8) , and vulnerability to heat is linked to intrinsic changes in the regulatory system ( 1 , 4 , 8) . in the present study , many schoolchildren were absentminded , had low writing speed , and did not understand the lessons especially in the afternoon and around noon probably because of indoor air temperatures of more than 32c in classrooms in yaound . high indoor air temperatures and dew points were related to headache , fatigue , and feeling hot and this might have had negative effects on school performance . in fact , the speed of school work decreases for each 1c increase in indoor air temperature ( 28 ) . some classroom studies in the 1960s found that reading speed , reading comprehension , and multiplication performance of schoolchildren were poorer at temperatures of 2729c ( 29 ) . creative thinking was worse at 30c than at lower temperatures in a study of office work ( 3 ) . longitudinal data analysis studying hourly and daily symptoms , body measurements , and indoor air temperature measures between days in schools are needed to confirm these results and to disentangle trends within and between days from the association between indoor temperature and health status as well as to properly adjust for confounding factors . adverse symptoms such as headache , fatigue , and feeling very hot was found associated with high indoor air temperatures among schoolchildren in cameroon . adequate environmental actions including good ventilation , open doors and windows , and water supplies are needed in schools that experience high heat .

data from randomized clinical trials have demonstrated that implantable cardioverter defibrillators ( icds ) provide a significant reduction in arrhythmic mortality in patients with ischaemic and non - ischaemic cardiomyopathy , both in primary and secondary prevention . in addition , cardiac resynchronization therapy ( crt ) has been shown to improve symptoms , reduce hospitalizations , and to reduce mortality in patients with refractory heart failure ( hf ) . despite the advances in medical therapy , commonly used risk factors that predict prognosis in icd recipients with hf include nyha functional class , qrs width , and left ventricular ejection fraction ( lvef ) . however , the majority of patients has more than one condition affecting their health state . recent studies suggest that comorbidity as renal failure and diabetes further influence the prognosis of icd recipients . the role of comorbidity in the short- and long - term prognosis of hf patients implanted with an icd capable of cardiac resynchronization therapy ( crt - d ) has not been adequately studied . the charlson comorbidity index ( cci ) the index is based on comorbid conditions and cardiovascular risk factors of known prognostic value with varying assigned weights . the aim of the present study was to determine whether the pre - implant cci predicts long - term survival and whether the cci has the potential to identify those patients who would benefit less from crt - d implantation . two prospective icd registries of the cardiology departments of the erasmus mc and the university hospital of basel were the basis for this study . out of these registries , we identified all patients who received a crt - d . the following selection criteria for crt were applied : symptomatic hf despite optimal drug therapy , impaired lvef ( lvef 35% ) , inter- or intra - ventricular conduction delay ( qrs duration 120 ms ) , and lv end - diastolic diameter > 55 mm . secondary prevention was defined as cardiac arrest without transient or reversible cause or spontaneous symptomatic sustained ventricular arrhythmias . primary prevention as indication for icd therapy was defined as the presence of lvef 35% with ischaemic or non - ischaemic cardiomyopathy in the absence of a history of cardiac arrest or sustained ventricular arrhythmia . as a certain period of follow - up was warranted , only patients who were followed for at least 12 months were eligible for the analysis . the mean ventricular tachycardia detection rate was 353 24 ms , the mean ventricular fibrillation detection rate was 284 15 ms . for all devices , antitachycardia pacing in combination with cardioversion / defibrillation therapy features was activated . for resynchronization therapy , the atrio - ventricular delay was optimized by 2d echocardiography to provide the longest diastolic filling time and the highest left ventricular outflow tract velocity timed integral . comorbidity as present before icd implantation was identified via patient history , laboratory values , and review of patients ' clinical data . for all patients , the renal function was assessed by estimating the baseline glomerular filtration rate ( egfr ) using the abbreviated modification of diet in renal disease ( mdrd ) study equation : egfr ( ml / min/1.73 m of body surface area ) = 186 ( serum creatinine in mg / dl ) ( age ) 0.742 in female subjects . impaired renal function was defined as an egfr < 60 ml / min/1.73 m according to practice guidelines . comorbidity was quantified using the cci , which is based on 17 different categories of comorbidity with varying assigned weights . the comorbid conditions included in our abbreviated cci model were myocardial infarction , cerebrovascular disease , chronic obstructive pulmonary disease , diabetes , peripheral vascular disease , renal failure , and any malignancy excluding metastatic tumours . the comorbidity index was calculated by assigning a weight of 2 to renal failure and any malignancy , and a weight of 1 to the other comorbid conditions . the comorbidity score for each patient is the arithmetic sum of the value assigned to each identified comorbid condition . age is also a risk factor for mortality independent from each comorbid condition . to account for the effects of increasing age , the comorbidity score was adjusted by adding one point to the score for each decade of life over the age of 50 at time of implantation . follow - up started at the time of icd implantation . in basel , patients were followed at 1 , 3 , and 6 months after implantation , and then at 6-month intervals . in rotterdam , patients were seen at 10 days , 3 , 6 , 9 , and 12 months after implantation , and then also at 6-month intervals . all patients were advised to contact the outpatient clinic as soon as possible after symptomatic events . at each visit , appropriate icd therapy was defined as an antitachycardia pacing therapy or shock for an arrhythmia determined to be either ventricular tachycardia or ventricular fibrillation . the secondary endpoint was time from icd implantation until the first appropriate icd therapy or death ( e.g. death without prior appropriate icd therapy ) . all statistics were performed using spss ( version 16.0 ) for windows ( spss inc . , continuous variables were expressed as mean sd , if normally distributed , or otherwise by median and interquartile range ( iqr ) . categorical data were expressed as percentages and compared with fisher 's exact test . simultaneous comparison of > 2 mean values was performed by one - way analysis of variance . baseline clinical variables and previously identified variables associated with mortality ( p < 0.10 ) were entered in the multivariate cox proportional hazards analysis . the proportional hazards assumption was checked graphically by log survival vs. log ( log survival distribution function ) . in addition , the proportional hazards assumption for all variables was tested using schoenfeld residuals . hazard ratios ( hrs ) with corresponding 95% confidence intervals ( cis ) are reported . two prospective icd registries of the cardiology departments of the erasmus mc and the university hospital of basel were the basis for this study . out of these registries , we identified all patients who received a crt - d . the following selection criteria for crt were applied : symptomatic hf despite optimal drug therapy , impaired lvef ( lvef 35% ) , inter- or intra - ventricular conduction delay ( qrs duration 120 ms ) , and lv end - diastolic diameter > 55 mm . secondary prevention was defined as cardiac arrest without transient or reversible cause or spontaneous symptomatic sustained ventricular arrhythmias . primary prevention as indication for icd therapy was defined as the presence of lvef 35% with ischaemic or non - ischaemic cardiomyopathy in the absence of a history of cardiac arrest or sustained ventricular arrhythmia . as a certain period of follow - up was warranted , only patients who were followed for at least 12 months were eligible for the analysis . the mean ventricular tachycardia detection rate was 353 24 ms , the mean ventricular fibrillation detection rate was 284 15 ms . for all devices , antitachycardia pacing in combination with cardioversion / defibrillation therapy features was activated . for resynchronization therapy , the atrio - ventricular delay was optimized by 2d echocardiography to provide the longest diastolic filling time and the highest left ventricular outflow tract velocity timed integral . comorbidity as present before icd implantation was identified via patient history , laboratory values , and review of patients ' clinical data . for all patients , the renal function was assessed by estimating the baseline glomerular filtration rate ( egfr ) using the abbreviated modification of diet in renal disease ( mdrd ) study equation : egfr ( ml / min/1.73 m of body surface area ) = 186 ( serum creatinine in mg / dl ) ( age ) 0.742 in female subjects . impaired renal function was defined as an egfr < 60 ml / min/1.73 m according to practice guidelines . comorbidity was quantified using the cci , which is based on 17 different categories of comorbidity with varying assigned weights . the comorbid conditions included in our abbreviated cci model were myocardial infarction , cerebrovascular disease , chronic obstructive pulmonary disease , diabetes , peripheral vascular disease , renal failure , and any malignancy excluding metastatic tumours . the comorbidity index was calculated by assigning a weight of 2 to renal failure and any malignancy , and a weight of 1 to the other comorbid conditions . the comorbidity score for each patient is the arithmetic sum of the value assigned to each identified comorbid condition . age is also a risk factor for mortality independent from each comorbid condition . to account for the effects of increasing age , the comorbidity score was adjusted by adding one point to the score for each decade of life over the age of 50 at time of implantation . follow - up started at the time of icd implantation . in basel , patients were followed at 1 , 3 , and 6 months after implantation , and then at 6-month intervals . in rotterdam , patients were seen at 10 days , 3 , 6 , 9 , and 12 months after implantation , and then also at 6-month intervals . all patients were advised to contact the outpatient clinic as soon as possible after symptomatic events . at each visit , appropriate icd therapy was defined as an antitachycardia pacing therapy or shock for an arrhythmia determined to be either ventricular tachycardia or ventricular fibrillation . the secondary endpoint was time from icd implantation until the first appropriate icd therapy or death ( e.g. death without prior appropriate icd therapy ) . all statistics were performed using spss ( version 16.0 ) for windows ( spss inc . , chicago , il , usa ) . continuous variables were expressed as mean sd , if normally distributed , or otherwise by median and interquartile range ( iqr ) . categorical data were expressed as percentages and compared with fisher 's exact test . simultaneous comparison of > 2 mean values was performed by one - way analysis of variance . baseline clinical variables and previously identified variables associated with mortality ( p < 0.10 ) were entered in the multivariate cox proportional hazards analysis . the proportional hazards assumption was checked graphically by log survival vs. log ( log survival distribution function ) . in addition , the proportional hazards assumption for all variables was tested using schoenfeld residuals . hazard ratios ( hrs ) with corresponding 95% confidence intervals ( cis ) are reported . the majority of patients was aged < 65 years ( 55% ) with a mean age of 62 11 years . the mean lvef was 24 7% , and the mean qrs duration was 165 30 ms . atrial fibrillation was present in 18% of patients . in the entire study population , the number of comorbid conditions ranged from one to seven per patient . comorbidity was common among hf patients , with 81% of the patients having at least three comorbid conditions . the most common non - cardiac comorbid conditions were renal disease , diabetes mellitus , cerebrovascular disease , and chronic obstructive pulmonary disease ( table 2 ) . the prevalence of comorbidity increased with older age ( all p - value trends < 0.001 ) . the comorbidity index scores ranged from 1 to 9 , with a mean of 3.1 1.5 . table 1baseline clinical characteristics of the study populationtotal population ( n = 463)demographic data , n ( % ) male gender349 ( 75 ) age ( years)62 11clinical data , n ( % ) indication icd therapy primary prevention346 ( 75 ) secondary prevention117 ( 25 ) nyha class ii129 ( 28 ) iii334 ( 72 ) left ventricular ejection fraction ( % ) 24 7 qrs duration ( ms)165 30 creatinine ( mg / dl)1.35 0.70 ischaemic aetiology233 ( 50 ) history of myocardial infarction200 ( 43 ) history of bypass surgery95 ( 21 ) pharmacological treatment amiodarone135 ( 29 ) beta - blockers350 ( 76 ) ace - inhibitors / arbs423 ( 91 ) diuretics388 ( 84 ) digoxin117 ( 25 ) statin245 ( 53 ) table 2prevalence of comorbid conditionscomorbid conditionstotal population ( n = 463)cardiac comorbidities , n ( % ) myocardial infarction200 ( 43)non - cardiac comorbidities , n ( % ) cerebrovascular disease59 ( 13 ) chronic obstructive pulmonary disease47 ( 10 ) diabetes118 ( 26 ) peripheral vascular disease17 ( 4 ) renal disease236 ( 51 ) any malignancy ( excluding metastatic tumours)36 ( 8) baseline clinical characteristics of the study population prevalence of comorbid conditions during a median follow - up of 30.5 months ( iqr : 18.250.7 months ) , a total of 85 patients ( 18% ) reached the primary endpoint . of the 85 deaths , 66 ( 78% ) were classified as cardiac and 7 ( 8% ) as non - cardiac . the overall mortality rates were 6.3 , 12.9 , and 32.3% , at 1 , 2 , and 7 years , respectively . at 7 years , mortality rates were not different between primary and secondary prevention patients ( 31 vs. 36% ) . figure 1cumulative mortality for heart failure patients treated with cardiac resynchronization therapy and defibrillation . survivors and non - survivors did not differ significantly with respect to gender , lvef , qrs duration , and pharmacological treatment ( ace - i , diuretics , beta - blocker , and statin ) . there was a trend towards a higher prevalence of atrial fibrillation , and use of amiodarone and digoxin was higher in patients who died , but the difference was not statistically significant ( p < 0.10 ) . regarding the comorbidity index score , patients who died had a significantly higher cci score compared with those who survived ( 3.9 1.5 vs. 2.9 1.5 ; p < 0.001 ) . patients who died during long - term follow - up were significantly older ( median 67 vs. 62 years , p < 0.05 ) . older age at implant is associated with increased mortality risk . in univariate analysis , the hr for all - cause mortality was 1.92 ( 95% ci 1.252.96 ; p = 0.003 ) in those aged 65 years . accordingly , the cci scores were adjusted for age to account for the effects of increasing age by adding one point to the score for each decade of life over the age of 50 . the mean age - adjusted cci score for patients who died was significantly higher compared with those who survived ( 5.9 1.9 vs. 4.7 2.1 ; p < 0.001 ) . figure 2a presents the difference in 2-year mortality rates among patients according to the cut - off value of age - adjusted cci score . notably , the difference in mortality rate was most prominent among patients with an age - adjusted cci 5 compared with those with age - adjusted cci < 5 , whereas among patients with an age - adjusted cci 7 , mortality was slightly higher compared with those with age - adjusted cci < 7 . the effect of increasing age - adjusted cci score on 2-year mortality rates showed an inverted u - shaped curve ( figure 2b ) . the 7-year mortality rates were significantly higher for patients with age - adjusted cci score 5 compared with those with age - adjusted cci score < 5 ( 42.3 vs. 17.1% ; p < 0.001 ; figure 3 ) . ( a ) two - year kaplan meier mortality rates for the groups dichotomized according to the cut - off value of age - adjusted charlson comorbidity index score . ( b ) the corresponding 2-year mortality increase for patients with age - adjusted charlson comorbidity index score cut - off value . figure 3cumulative mortality rates for the two groups dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 . meier mortality rates for the groups dichotomized according to the cut - off value of age - adjusted charlson comorbidity index score . ( b ) the corresponding 2-year mortality increase for patients with age - adjusted charlson comorbidity index score cut - off value . cumulative mortality rates for the two groups dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 . during follow - up , 149 patients ( 32% ) experienced icd therapy for ventricular tachyarrhythmias . the first appropriate device therapy occurred at median interval of 6.9 months ( iqr : 1.715.0 months ) after implantation . cumulative event rates for appropriate icd therapy were 21.7 , 28.5 , and 50.4% , at 1 , 2 , and 7 years , respectively . patients with a secondary prevention indication were more likely to receive appropriate icd therapy compared with primary prevention ( 56 vs. 24% , p < 0.001 ) . the 7-year event rate of appropriate icd therapy was 66.8% for secondary prevention patients , compared with 39.1% for primary prevention patients ( figure 4 ) . the event rates of appropriate icd therapy were not significantly different in both primary and secondary patients with an age - adjusted cci 5 compared with those with age - adjusted cci < 5 ( figure 5 ) . figure 4cumulative rates of appropriate device therapy for patients with a primary or secondary prevention indication . figure 5cumulative rates of appropriate device therapy for patients dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 and a primary prevention indication ( a ) or secondary prevention indication ( b ) . cumulative rates of appropriate device therapy for patients with a primary or secondary prevention indication . cumulative rates of appropriate device therapy for patients dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 and a primary prevention indication ( a ) or secondary prevention indication ( b ) . a total of 189 patients ( 41% ) reached the secondary endpoint of time to first appropriate icd therapy or death . death without prior appropriate icd therapy was observed in 40 patients , whereas 45 patients received appropriate icd therapy prior to death , and 104 patients received appropriate icd therapy . the estimated proportion of patients free of appropriate icd therapy or death was 40.9% at 7 years . the majority of patients ( 85% ) who died without prior appropriate icd therapy had a primary prevention indication . the 7-year mortality rates between patients with and without prior appropriate icd therapy were significantly different ( 44.9 vs. 21.9% , p < 0.001 ) . in univariate analysis , appropriate icd therapy was associated with an increased risk for all - cause mortality ( hr 2.06 , 95% ci 1.343.17 ; p < 0.001 ) . the multivariate cox proportional hazard regression analysis identified an age - adjusted cci score 5 ( hr 3.69 , 95% ci 2.066.60 ; p < 0.001 ) and appropriate device therapy ( hr 1.80 , 95% ci 1.172.77 ; p = 0.008 ) as predictors of all - cause mortality independent from the prevention indication for icd therapy . atrial fibrillation , use of amiodarone and digoxin was not significantly associated with mortality in the multivariate analysis . the majority of patients was aged < 65 years ( 55% ) with a mean age of 62 11 years . the mean lvef was 24 7% , and the mean qrs duration was 165 30 ms . atrial fibrillation was present in 18% of patients . in the entire study population , the number of comorbid conditions ranged from one to seven per patient . comorbidity was common among hf patients , with 81% of the patients having at least three comorbid conditions . the most common non - cardiac comorbid conditions were renal disease , diabetes mellitus , cerebrovascular disease , and chronic obstructive pulmonary disease ( table 2 ) . the prevalence of comorbidity increased with older age ( all p - value trends < 0.001 ) . the comorbidity index scores ranged from 1 to 9 , with a mean of 3.1 1.5 . table 1baseline clinical characteristics of the study populationtotal population ( n = 463)demographic data , n ( % ) male gender349 ( 75 ) age ( years)62 11clinical data , n ( % ) indication icd therapy primary prevention346 ( 75 ) secondary prevention117 ( 25 ) nyha class ii129 ( 28 ) iii334 ( 72 ) left ventricular ejection fraction ( % ) 24 7 qrs duration ( ms)165 30 creatinine ( mg / dl)1.35 0.70 ischaemic aetiology233 ( 50 ) history of myocardial infarction200 ( 43 ) history of bypass surgery95 ( 21 ) pharmacological treatment amiodarone135 ( 29 ) beta - blockers350 ( 76 ) ace - inhibitors / arbs423 ( 91 ) diuretics388 ( 84 ) digoxin117 ( 25 ) statin245 ( 53 ) table 2prevalence of comorbid conditionscomorbid conditionstotal population ( n = 463)cardiac comorbidities , n ( % ) myocardial infarction200 ( 43)non - cardiac comorbidities , n ( % ) cerebrovascular disease59 ( 13 ) chronic obstructive pulmonary disease47 ( 10 ) diabetes118 ( 26 ) peripheral vascular disease17 ( 4 ) renal disease236 ( 51 ) any malignancy ( excluding metastatic tumours)36 ( 8) baseline clinical characteristics of the study population prevalence of comorbid conditions during a median follow - up of 30.5 months ( iqr : 18.250.7 months ) , a total of 85 patients ( 18% ) reached the primary endpoint . of the 85 deaths , 66 ( 78% ) were classified as cardiac and 7 ( 8% ) as non - cardiac . the overall mortality rates were 6.3 , 12.9 , and 32.3% , at 1 , 2 , and 7 years , respectively . at 7 years , mortality rates were not different between primary and secondary prevention patients ( 31 vs. 36% ) . figure 1cumulative mortality for heart failure patients treated with cardiac resynchronization therapy and defibrillation . cumulative mortality for heart failure patients treated with cardiac resynchronization therapy and defibrillation . survivors and non - survivors did not differ significantly with respect to gender , lvef , qrs duration , and pharmacological treatment ( ace - i , diuretics , beta - blocker , and statin ) . there was a trend towards a higher prevalence of atrial fibrillation , and use of amiodarone and digoxin was higher in patients who died , but the difference was not statistically significant ( p < 0.10 ) . regarding the comorbidity index score , patients who died had a significantly higher cci score compared with those who survived ( 3.9 1.5 vs. 2.9 1.5 ; p < 0.001 ) . patients who died during long - term follow - up were significantly older ( median 67 vs. 62 years , p < 0.05 ) . older age at implant is associated with increased mortality risk . in univariate analysis , the hr for all - cause mortality was 1.92 ( 95% ci 1.252.96 ; p = 0.003 ) in those aged 65 years . accordingly , the cci scores were adjusted for age to account for the effects of increasing age by adding one point to the score for each decade of life over the age of 50 . the mean age - adjusted cci score for patients who died was significantly higher compared with those who survived ( 5.9 1.9 vs. 4.7 2.1 ; p < 0.001 ) . figure 2a presents the difference in 2-year mortality rates among patients according to the cut - off value of age - adjusted cci score . notably , the difference in mortality rate was most prominent among patients with an age - adjusted cci 5 compared with those with age - adjusted cci < 5 , whereas among patients with an age - adjusted cci 7 , mortality was slightly higher compared with those with age - adjusted cci < 7 . the effect of increasing age - adjusted cci score on 2-year mortality rates showed an inverted u - shaped curve ( figure 2b ) . the 7-year mortality rates were significantly higher for patients with age - adjusted cci score 5 compared with those with age - adjusted cci score < 5 ( 42.3 vs. 17.1% ; p < 0.001 ; figure 3 ) . meier mortality rates for the groups dichotomized according to the cut - off value of age - adjusted charlson comorbidity index score . ( b ) the corresponding 2-year mortality increase for patients with age - adjusted charlson comorbidity index score cut - off value . figure 3cumulative mortality rates for the two groups dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 . inverted u - shaped curve for mortality . ( a ) two - year kaplan meier mortality rates for the groups dichotomized according to the cut - off value of age - adjusted charlson comorbidity index score . ( b ) the corresponding 2-year mortality increase for patients with age - adjusted charlson comorbidity index score cut - off value . cumulative mortality rates for the two groups dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 . during follow - up , 149 patients ( 32% ) experienced icd therapy for ventricular tachyarrhythmias . the first appropriate device therapy occurred at median interval of 6.9 months ( iqr : 1.715.0 months ) after implantation . cumulative event rates for appropriate icd therapy were 21.7 , 28.5 , and 50.4% , at 1 , 2 , and 7 years , respectively . patients with a secondary prevention indication were more likely to receive appropriate icd therapy compared with primary prevention ( 56 vs. 24% , p < 0.001 ) . the 7-year event rate of appropriate icd therapy was 66.8% for secondary prevention patients , compared with 39.1% for primary prevention patients ( figure 4 ) . the event rates of appropriate icd therapy were not significantly different in both primary and secondary patients with an age - adjusted cci 5 compared with those with age - adjusted cci < 5 ( figure 5 ) . figure 4cumulative rates of appropriate device therapy for patients with a primary or secondary prevention indication . figure 5cumulative rates of appropriate device therapy for patients dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 and a primary prevention indication ( a ) or secondary prevention indication ( b ) . cumulative rates of appropriate device therapy for patients with a primary or secondary prevention indication . cumulative rates of appropriate device therapy for patients dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 and a primary prevention indication ( a ) or secondary prevention indication ( b ) . a total of 189 patients ( 41% ) reached the secondary endpoint of time to first appropriate icd therapy or death . death without prior appropriate icd therapy was observed in 40 patients , whereas 45 patients received appropriate icd therapy prior to death , and 104 patients received appropriate icd therapy . the estimated proportion of patients free of appropriate icd therapy or death was 40.9% at 7 years . the majority of patients ( 85% ) who died without prior appropriate icd therapy had a primary prevention indication . the 7-year mortality rates between patients with and without prior appropriate icd therapy were significantly different ( 44.9 vs. 21.9% , p < 0.001 ) . in univariate analysis , appropriate icd therapy was associated with an increased risk for all - cause mortality ( hr 2.06 , 95% ci 1.343.17 ; p < 0.001 ) . the multivariate cox proportional hazard regression analysis identified an age - adjusted cci score 5 ( hr 3.69 , 95% ci 2.066.60 ; p < 0.001 ) and appropriate device therapy ( hr 1.80 , 95% ci 1.172.77 ; p = 0.008 ) as predictors of all - cause mortality independent from the prevention indication for icd therapy . atrial fibrillation , use of amiodarone and digoxin was not significantly associated with mortality in the multivariate analysis . the present study evaluated the prognostic impact of comorbidity using an abbreviated modified cci in hf patients who received a crt - d for primary or secondary prevention of sudden cardiac death . we found that comorbidity was common in our study population of hf patients , with 81% of the patients having at least three comorbid conditions . patients with a high comorbidity burden , defined as an age - adjusted cci 5 had an increased risk for mortality , independent from the prevention indication . independent factors were age > 80 years , history of atrial fibrillation , renal dysfunction ( creatinine 1.8 mg / dl ) , and nyha class iii or iv . patients with two or more of these factors had an increased risk for early mortality after device implantation , with renal dysfunction as comorbidity . a recent analysis of the second multicenter automatic defibrillator implantation trial ( madit ii ) demonstrated that a point score based on clinical variables ( age > 65 years , diabetes , nyha class > ii , blood urea nitrogen > 28 mg / dl , and non - sinus rhythm ) can identify patients at high risk for death during long - term follow - up . at 6-years follow - up , patients with high risk ( at least three risk factors ) , medium risk ( one to two risk factors ) , and low risk ( no risk factors ) had a cumulative mortality of 68 , 43 , and 19% , respectively . in a study by bruch et al . the prognostic impact of comorbidities in hf patients treated with an icd was examined . in this study , patients with all of these variables had an event - free survival of 15% at 1400 days follow - up . the previous studies identified renal failure as a predictor of mortality , next to traditional markers as nyha functional class and advanced age . the effect of cardiac and non - cardiac conditions on survival has been evaluated in a large community - based examination of icd recipients . this study revealed that the presence of non - cardiac comorbidity and prior hf was a significant predictor of death . the adjusted hr for death was 2.98 for patients with at least three comorbid conditions . in our study all patients had hf , but consistent results were observed with increased mortality for those with a high comorbidity burden . the results of the present study may have implications for the translation of the expanded indications for icd therapy from randomized trials into clinical practice . evidence from randomized clinical trials indicates that the icd reduces arrhythmic mortality in hf patients with ischaemic and non - ischaemic cardiomyopathy , both in primary and secondary prevention . in addition , the efficacy and survival benefits of crt have been proved in observational studies as well as in randomized trials . taken this together , the addition of defibrillation to crt is reasonable , as the crt - d targets both hf and arrhythmic mortality . despite the effectiveness of terminating ventricular tachyarrhythmias by the implantable defibrillator , competing non - cardiac comorbidity bai et al . , demonstrated that renal failure and diabetes are strong independent predictors of mortality in patients treated with crt . both comorbid conditions are incorporated in the charlson model as applied in the present study . the rate of appropriate icd therapy was twice as likely in secondary prevention compared with primary prevention . , it is of particular interest to identify patients who might not benefit from icd implantation . in our study , the majority of patients who died prior to appropriate icd therapy had a primary prevention indication . taken all together , the findings of this study may assist the clinician contemplating icd insertion as primary prevention . however , it is still a personal decision about how high the risk of death from non - cardiac comorbidity should be before icd therapy is no longer justified . unfortunately , appropriate icd interventions can not be predicted by the comorbidity index . another possible limitation is the assessment of comorbidity , which was partly based on administrative data sources . another possible limitation is the assessment of comorbidity , which was partly based on administrative data sources . these sources have a likelihood of underreporting . however , an analysis of administrative data suggested a high degree of specificity . comorbidity is common in hf patients with a crt - d , which has a major impact on survival . a high comorbidity burden was a significant predictor of death in crt - d recipients . the abbreviated cci is a useful and easy tool to assess the burden of comorbidity at baseline . our findings may assist clinicians contemplating icd insertion as primary prevention in patients with hf in the presence of non - cardiac comorbidity . however , further research is still needed to characterize other specific comorbid conditions as predictors of survival , and to predict who will receive appropriate icd therapy . b.a.s . was supported by a grant from the swiss national fund ( izk0z3 - 12146/1 ) and from st jude medical . funding to pay the open access publication charges was provided by the department of cardiology , thoraxcenter , erasmus mc , rotterdam , the netherlands .

aimscomorbidity , such as myocardial infarction , diabetes , and renal failure , plays a pivotal role in the prognosis of a patient with arrhythmias . however , data on the prognostic impact of comorbiditiy in heart failure patients with cardiac resynchronization therapy and defibrillation ( crt - d ) are scarce . the purpose of this study was to determine the impact of comorbidity on survival in crt - d patients.methods and resultsthe study population consisted of 463 heart failure patients who received a crt - d between 1999 and 2008 in rotterdam and basel . the charlson comorbidity index ( cci ) is often used as an adjusting variable in prognostic models . the cox proportional hazards analysis was performed to determine the independent effect of comorbidity on survival . during a median follow - up of 30.5 months , 85 patients died . mortality rates at 1 and 7 years were 6.3 and 32.3% . cumulative incidence of implantable cardioverter defibrillator ( icd ) therapy at 7 years was 50% , and death without icd therapy was observed in 9% of patients . at least three comorbid conditions were observed in 81% of patients . patients who died had a higher cci score compared with those who survived ( 3.9 1.5 vs. 2.9 1.5 ; p < 0.001 ) . an age - adjusted cci score 5 was a predictor of mortality ( hazard ratio 3.69 , 95% ci 2.066.60 ; p < 0.001 ) independent from indication for icd therapy , and from icd interventions during the clinical course.conclusioncomorbidity is often present in heart failure patients , and a high comorbidity burden was a significant predictor of mortality in crt - d recipients . comorbidity can not predict appropriate icd therapy . death without prior icd therapy occurs in a minor proportion of patients .

Introduction Methods Study population Construction of the comorbidity index Follow-up Statistical analysis Results Patient characteristics Comorbidity and mortality Appropriate implantable cardioverter defibrillator therapy, mortality, and comorbidity Discussion Study limitations Conclusion Funding

data from randomized clinical trials have demonstrated that implantable cardioverter defibrillators ( icds ) provide a significant reduction in arrhythmic mortality in patients with ischaemic and non - ischaemic cardiomyopathy , both in primary and secondary prevention . in addition , cardiac resynchronization therapy ( crt ) has been shown to improve symptoms , reduce hospitalizations , and to reduce mortality in patients with refractory heart failure ( hf ) . the role of comorbidity in the short- and long - term prognosis of hf patients implanted with an icd capable of cardiac resynchronization therapy ( crt - d ) has not been adequately studied . the charlson comorbidity index ( cci ) the index is based on comorbid conditions and cardiovascular risk factors of known prognostic value with varying assigned weights . the aim of the present study was to determine whether the pre - implant cci predicts long - term survival and whether the cci has the potential to identify those patients who would benefit less from crt - d implantation . out of these registries , we identified all patients who received a crt - d . primary prevention as indication for icd therapy was defined as the presence of lvef 35% with ischaemic or non - ischaemic cardiomyopathy in the absence of a history of cardiac arrest or sustained ventricular arrhythmia . as a certain period of follow - up was warranted , only patients who were followed for at least 12 months were eligible for the analysis . the comorbid conditions included in our abbreviated cci model were myocardial infarction , cerebrovascular disease , chronic obstructive pulmonary disease , diabetes , peripheral vascular disease , renal failure , and any malignancy excluding metastatic tumours . the comorbidity index was calculated by assigning a weight of 2 to renal failure and any malignancy , and a weight of 1 to the other comorbid conditions . death without prior appropriate icd therapy ) . baseline clinical variables and previously identified variables associated with mortality ( p < 0.10 ) were entered in the multivariate cox proportional hazards analysis . out of these registries , we identified all patients who received a crt - d . primary prevention as indication for icd therapy was defined as the presence of lvef 35% with ischaemic or non - ischaemic cardiomyopathy in the absence of a history of cardiac arrest or sustained ventricular arrhythmia . as a certain period of follow - up was warranted , only patients who were followed for at least 12 months were eligible for the analysis . the comorbid conditions included in our abbreviated cci model were myocardial infarction , cerebrovascular disease , chronic obstructive pulmonary disease , diabetes , peripheral vascular disease , renal failure , and any malignancy excluding metastatic tumours . the comorbidity index was calculated by assigning a weight of 2 to renal failure and any malignancy , and a weight of 1 to the other comorbid conditions . baseline clinical variables and previously identified variables associated with mortality ( p < 0.10 ) were entered in the multivariate cox proportional hazards analysis . comorbidity was common among hf patients , with 81% of the patients having at least three comorbid conditions . the prevalence of comorbidity increased with older age ( all p - value trends < 0.001 ) . table 1baseline clinical characteristics of the study populationtotal population ( n = 463)demographic data , n ( % ) male gender349 ( 75 ) age ( years)62 11clinical data , n ( % ) indication icd therapy primary prevention346 ( 75 ) secondary prevention117 ( 25 ) nyha class ii129 ( 28 ) iii334 ( 72 ) left ventricular ejection fraction ( % ) 24 7 qrs duration ( ms)165 30 creatinine ( mg / dl)1.35 0.70 ischaemic aetiology233 ( 50 ) history of myocardial infarction200 ( 43 ) history of bypass surgery95 ( 21 ) pharmacological treatment amiodarone135 ( 29 ) beta - blockers350 ( 76 ) ace - inhibitors / arbs423 ( 91 ) diuretics388 ( 84 ) digoxin117 ( 25 ) statin245 ( 53 ) table 2prevalence of comorbid conditionscomorbid conditionstotal population ( n = 463)cardiac comorbidities , n ( % ) myocardial infarction200 ( 43)non - cardiac comorbidities , n ( % ) cerebrovascular disease59 ( 13 ) chronic obstructive pulmonary disease47 ( 10 ) diabetes118 ( 26 ) peripheral vascular disease17 ( 4 ) renal disease236 ( 51 ) any malignancy ( excluding metastatic tumours)36 ( 8) baseline clinical characteristics of the study population prevalence of comorbid conditions during a median follow - up of 30.5 months ( iqr : 18.250.7 months ) , a total of 85 patients ( 18% ) reached the primary endpoint . the overall mortality rates were 6.3 , 12.9 , and 32.3% , at 1 , 2 , and 7 years , respectively . figure 1cumulative mortality for heart failure patients treated with cardiac resynchronization therapy and defibrillation . there was a trend towards a higher prevalence of atrial fibrillation , and use of amiodarone and digoxin was higher in patients who died , but the difference was not statistically significant ( p < 0.10 ) . regarding the comorbidity index score , patients who died had a significantly higher cci score compared with those who survived ( 3.9 1.5 vs. 2.9 1.5 ; p < 0.001 ) . patients who died during long - term follow - up were significantly older ( median 67 vs. 62 years , p < 0.05 ) . the mean age - adjusted cci score for patients who died was significantly higher compared with those who survived ( 5.9 1.9 vs. 4.7 2.1 ; p < 0.001 ) . figure 2a presents the difference in 2-year mortality rates among patients according to the cut - off value of age - adjusted cci score . notably , the difference in mortality rate was most prominent among patients with an age - adjusted cci 5 compared with those with age - adjusted cci < 5 , whereas among patients with an age - adjusted cci 7 , mortality was slightly higher compared with those with age - adjusted cci < 7 . the effect of increasing age - adjusted cci score on 2-year mortality rates showed an inverted u - shaped curve ( figure 2b ) . the 7-year mortality rates were significantly higher for patients with age - adjusted cci score 5 compared with those with age - adjusted cci score < 5 ( 42.3 vs. 17.1% ; p < 0.001 ; figure 3 ) . ( a ) two - year kaplan meier mortality rates for the groups dichotomized according to the cut - off value of age - adjusted charlson comorbidity index score . ( b ) the corresponding 2-year mortality increase for patients with age - adjusted charlson comorbidity index score cut - off value . figure 3cumulative mortality rates for the two groups dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 . meier mortality rates for the groups dichotomized according to the cut - off value of age - adjusted charlson comorbidity index score . ( b ) the corresponding 2-year mortality increase for patients with age - adjusted charlson comorbidity index score cut - off value . cumulative mortality rates for the two groups dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 . cumulative event rates for appropriate icd therapy were 21.7 , 28.5 , and 50.4% , at 1 , 2 , and 7 years , respectively . patients with a secondary prevention indication were more likely to receive appropriate icd therapy compared with primary prevention ( 56 vs. 24% , p < 0.001 ) . the 7-year event rate of appropriate icd therapy was 66.8% for secondary prevention patients , compared with 39.1% for primary prevention patients ( figure 4 ) . the event rates of appropriate icd therapy were not significantly different in both primary and secondary patients with an age - adjusted cci 5 compared with those with age - adjusted cci < 5 ( figure 5 ) . figure 5cumulative rates of appropriate device therapy for patients dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 and a primary prevention indication ( a ) or secondary prevention indication ( b ) . cumulative rates of appropriate device therapy for patients dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 and a primary prevention indication ( a ) or secondary prevention indication ( b ) . death without prior appropriate icd therapy was observed in 40 patients , whereas 45 patients received appropriate icd therapy prior to death , and 104 patients received appropriate icd therapy . the estimated proportion of patients free of appropriate icd therapy or death was 40.9% at 7 years . the majority of patients ( 85% ) who died without prior appropriate icd therapy had a primary prevention indication . the 7-year mortality rates between patients with and without prior appropriate icd therapy were significantly different ( 44.9 vs. 21.9% , p < 0.001 ) . in univariate analysis , appropriate icd therapy was associated with an increased risk for all - cause mortality ( hr 2.06 , 95% ci 1.343.17 ; p < 0.001 ) . the multivariate cox proportional hazard regression analysis identified an age - adjusted cci score 5 ( hr 3.69 , 95% ci 2.066.60 ; p < 0.001 ) and appropriate device therapy ( hr 1.80 , 95% ci 1.172.77 ; p = 0.008 ) as predictors of all - cause mortality independent from the prevention indication for icd therapy . comorbidity was common among hf patients , with 81% of the patients having at least three comorbid conditions . table 1baseline clinical characteristics of the study populationtotal population ( n = 463)demographic data , n ( % ) male gender349 ( 75 ) age ( years)62 11clinical data , n ( % ) indication icd therapy primary prevention346 ( 75 ) secondary prevention117 ( 25 ) nyha class ii129 ( 28 ) iii334 ( 72 ) left ventricular ejection fraction ( % ) 24 7 qrs duration ( ms)165 30 creatinine ( mg / dl)1.35 0.70 ischaemic aetiology233 ( 50 ) history of myocardial infarction200 ( 43 ) history of bypass surgery95 ( 21 ) pharmacological treatment amiodarone135 ( 29 ) beta - blockers350 ( 76 ) ace - inhibitors / arbs423 ( 91 ) diuretics388 ( 84 ) digoxin117 ( 25 ) statin245 ( 53 ) table 2prevalence of comorbid conditionscomorbid conditionstotal population ( n = 463)cardiac comorbidities , n ( % ) myocardial infarction200 ( 43)non - cardiac comorbidities , n ( % ) cerebrovascular disease59 ( 13 ) chronic obstructive pulmonary disease47 ( 10 ) diabetes118 ( 26 ) peripheral vascular disease17 ( 4 ) renal disease236 ( 51 ) any malignancy ( excluding metastatic tumours)36 ( 8) baseline clinical characteristics of the study population prevalence of comorbid conditions during a median follow - up of 30.5 months ( iqr : 18.250.7 months ) , a total of 85 patients ( 18% ) reached the primary endpoint . the overall mortality rates were 6.3 , 12.9 , and 32.3% , at 1 , 2 , and 7 years , respectively . figure 1cumulative mortality for heart failure patients treated with cardiac resynchronization therapy and defibrillation . cumulative mortality for heart failure patients treated with cardiac resynchronization therapy and defibrillation . there was a trend towards a higher prevalence of atrial fibrillation , and use of amiodarone and digoxin was higher in patients who died , but the difference was not statistically significant ( p < 0.10 ) . regarding the comorbidity index score , patients who died had a significantly higher cci score compared with those who survived ( 3.9 1.5 vs. 2.9 1.5 ; p < 0.001 ) . patients who died during long - term follow - up were significantly older ( median 67 vs. 62 years , p < 0.05 ) . the mean age - adjusted cci score for patients who died was significantly higher compared with those who survived ( 5.9 1.9 vs. 4.7 2.1 ; p < 0.001 ) . figure 2a presents the difference in 2-year mortality rates among patients according to the cut - off value of age - adjusted cci score . notably , the difference in mortality rate was most prominent among patients with an age - adjusted cci 5 compared with those with age - adjusted cci < 5 , whereas among patients with an age - adjusted cci 7 , mortality was slightly higher compared with those with age - adjusted cci < 7 . the effect of increasing age - adjusted cci score on 2-year mortality rates showed an inverted u - shaped curve ( figure 2b ) . the 7-year mortality rates were significantly higher for patients with age - adjusted cci score 5 compared with those with age - adjusted cci score < 5 ( 42.3 vs. 17.1% ; p < 0.001 ; figure 3 ) . meier mortality rates for the groups dichotomized according to the cut - off value of age - adjusted charlson comorbidity index score . ( b ) the corresponding 2-year mortality increase for patients with age - adjusted charlson comorbidity index score cut - off value . figure 3cumulative mortality rates for the two groups dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 . ( a ) two - year kaplan meier mortality rates for the groups dichotomized according to the cut - off value of age - adjusted charlson comorbidity index score . ( b ) the corresponding 2-year mortality increase for patients with age - adjusted charlson comorbidity index score cut - off value . cumulative mortality rates for the two groups dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 . cumulative event rates for appropriate icd therapy were 21.7 , 28.5 , and 50.4% , at 1 , 2 , and 7 years , respectively . patients with a secondary prevention indication were more likely to receive appropriate icd therapy compared with primary prevention ( 56 vs. 24% , p < 0.001 ) . the 7-year event rate of appropriate icd therapy was 66.8% for secondary prevention patients , compared with 39.1% for primary prevention patients ( figure 4 ) . the event rates of appropriate icd therapy were not significantly different in both primary and secondary patients with an age - adjusted cci 5 compared with those with age - adjusted cci < 5 ( figure 5 ) . figure 5cumulative rates of appropriate device therapy for patients dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 and a primary prevention indication ( a ) or secondary prevention indication ( b ) . cumulative rates of appropriate device therapy for patients dichotomized according to an age - adjusted charlson comorbidity index with cut - off value 5 and a primary prevention indication ( a ) or secondary prevention indication ( b ) . death without prior appropriate icd therapy was observed in 40 patients , whereas 45 patients received appropriate icd therapy prior to death , and 104 patients received appropriate icd therapy . the estimated proportion of patients free of appropriate icd therapy or death was 40.9% at 7 years . the majority of patients ( 85% ) who died without prior appropriate icd therapy had a primary prevention indication . the 7-year mortality rates between patients with and without prior appropriate icd therapy were significantly different ( 44.9 vs. 21.9% , p < 0.001 ) . in univariate analysis , appropriate icd therapy was associated with an increased risk for all - cause mortality ( hr 2.06 , 95% ci 1.343.17 ; p < 0.001 ) . the multivariate cox proportional hazard regression analysis identified an age - adjusted cci score 5 ( hr 3.69 , 95% ci 2.066.60 ; p < 0.001 ) and appropriate device therapy ( hr 1.80 , 95% ci 1.172.77 ; p = 0.008 ) as predictors of all - cause mortality independent from the prevention indication for icd therapy . the present study evaluated the prognostic impact of comorbidity using an abbreviated modified cci in hf patients who received a crt - d for primary or secondary prevention of sudden cardiac death . we found that comorbidity was common in our study population of hf patients , with 81% of the patients having at least three comorbid conditions . patients with a high comorbidity burden , defined as an age - adjusted cci 5 had an increased risk for mortality , independent from the prevention indication . a recent analysis of the second multicenter automatic defibrillator implantation trial ( madit ii ) demonstrated that a point score based on clinical variables ( age > 65 years , diabetes , nyha class > ii , blood urea nitrogen > 28 mg / dl , and non - sinus rhythm ) can identify patients at high risk for death during long - term follow - up . at 6-years follow - up , patients with high risk ( at least three risk factors ) , medium risk ( one to two risk factors ) , and low risk ( no risk factors ) had a cumulative mortality of 68 , 43 , and 19% , respectively . this study revealed that the presence of non - cardiac comorbidity and prior hf was a significant predictor of death . the adjusted hr for death was 2.98 for patients with at least three comorbid conditions . the rate of appropriate icd therapy was twice as likely in secondary prevention compared with primary prevention . in our study , the majority of patients who died prior to appropriate icd therapy had a primary prevention indication . comorbidity is common in hf patients with a crt - d , which has a major impact on survival . a high comorbidity burden was a significant predictor of death in crt - d recipients . however , further research is still needed to characterize other specific comorbid conditions as predictors of survival , and to predict who will receive appropriate icd therapy .

the online version of this article ( doi:10.1007/s12014 - 010 - 9050 - 3 ) contains supplementary material , which is available to authorized users . squamous cell carcinoma ( scc ) is a common type of cancer originating in epithelial cells . survival rates of sccs of the upper respiratory tract 5 years post - diagnosis are about 50% . however , early detection and treatment results in substantially better prognosis . currently , the most accurate diagnostic test for all sccs is a histological examination of a tissue biopsy . a less invasive test that still maintains sensitivity and selectivity , from a readily available bodily fluid like plasma or saliva , would be greatly beneficial . one potential test would be based on specific differential protein expression , or protein biomarkers of scc . a promising source of possible biomarkers of scc is tumor interstitial fluid ( tif ) . the production of tif results from buildup of fluid derived from cell secretions due to a lack of a fully formed vascular and lymphatic system within the tumor . because this fluid is directly associated with the tumor , it may be a rich source of tumor - specific biomarkers . in the case of scc of the head and neck ( and potentially scc at other anatomical locations ) , tif may infiltrate other easily sampled bio - fluids such as plasma or saliva . thus , knowledge of tif proteomes may help to prioritize putative protein biomarkers found in plasma or saliva to guide validation studies . despite its potential value as a source of tumor biomarkers , representative catalogs of tif proteomes currently , a couple of different analysis strategies for studying interstitial fluid have been employed . first , some studies have used indirect extraction of tif from biopsies where resected tissue is soaked in buffer and proteins diffused into the buffer over time are analyzed [ 3 , 4 ] . other studies have used cell culture models to study tif - like proteomes where conditioned media are collected after a period of incubation and shed / secreted proteins are analyzed [ 5 , 6 ] . these collections may not be true representations of tif due to the ex vivo and in vitro sampling techniques employed . in this study , tif from a head and neck scc tumor was obtained directly in situ without biopsy using a novel ultrafiltration catheter , and its proteome was characterized via immunodepletion , trypsin digestion , three - dimensional peptide fractionation , and mass spectrometry . this represents the first study of tif collected in situ and the first study of tif obtained from head and neck squamous cell carcinoma ( hnscc ) . our collection technique , sample preparation , and resulting proteomic catalog should be a valuable resource to others investigating tumor progression mechanisms as well as those seeking to identify tumor - specific biomarkers of scc . in situ scc tif collection tif was collected from the tumor of a patient with stage iv head and neck cancer with informed consent using an ultrafiltration catheter previously described . briefly , the catheter is constructed with hollow fibers composed of polysulfone with 0.45-m pores throughout the walls of the fiber . it is slightly smaller than a 27-gauge needle , and the proximal shaft of the catheter is the size of a 24-gauge needle to limit disturbances from seeding . tif was collected from the right and left side of the tumor , was mixed with phosphate buffered saline , and was immediately stored at 80c . depletion of high abundance proteins from tif to remove high abundance proteins from tif for enhancement of proteomic coverage , a multiple affinity removal system ( mars , agilent technologies , inc . the mars column consists of immobilized antibodies directed against albumin , igg , iga , transferrin , haptoglobin , and antitrypsin . protein from tif ( 0.4 mg ) was applied to the mars column , and the column was used according to manufacturer s instructions using manufacturer - supplied buffers . fractions consisting of flow - through of unbound and eluted bound proteins were collected separately . trypsin digestion and fractionation of tif peptides both undepleted ( 100 g ) and mars - depleted ( 150 g ) tif protein samples were independently digested and used for proteomics analysis . as a denaturant , sds was added to tif samples to a final concentration of 0.5% w / v . dithiothreitol ( sigma ) was added at 10 mm and incubated for 60 min at 56c . resulting free sulfhydryl groups were alkylated with addition of 50 mm iodoacetamide ( sigma ) for 45 min at room temperature in the dark . samples were diluted tenfold with 10 mm ammonium bicarbonate buffer ( ph 8.0 ) to reduce the sds concentration to 0.05% w / v . sequencing grade trypsin ( promega , madison , wi ) was added to a final enzyme / substrate ratio of 1:50 , and the reaction was incubated at 37c overnight . the next day , samples were cleaned with an oasis mcx solid phase extraction cartridge ( waters , milford , ma ) according to manufacturer s protocol , and the eluted peptides were dried by vacuum centrifugation.peptide isoelectric focusing ( ief ) was performed using an agilent 3100 offgel fractionator ( agilent ) using the 24-well setup with ph 310 ipg strips according to manufacturer s instructions . briefly , peptides resulting from trypsin digestion of tif proteins were dissolved in 3.6 ml of offgel peptide sample solution consisting of appropriate ampholytes . the ipg strip was rehydrated with the peptide ipg strip rehydration solution for 15 min . the peptide solutions ( either from the non - depleted or depleted sample ) were separately loaded in equal volumes ( 150 l ) into each of the wells . the peptides were then electrofocused at a maximum limit of 50 a until 50 kvh was reached . after completion , peptide ief fractions from the non - depleted samples were acidified to ph ~2 with tfa , purified using stage tips composed of 2 22-gauge needle punches of empore extraction disks ( 3 m , st . paul , mn ) , dried by vacuum centrifugation , and stored pending mass spectrometric analysis . the mars depleted samples were further fractionated with offline strong cation exchange ( scx ) hplc as described previously . capillary lc - ms / ms dried peptides were resuspended in 5 l of load buffer ( 98:2:0.1 water / acetonitrile / formic acid ) . theses samples were loaded onto an in - house poured reversed phase capillary column at 1,000 nl / min using an eksigent microas autosampler and 1dlc nano - hplc . the column consisted of fused silica tubing packed with magic c18aq5-m , 200- pore size resin ( michrom bioresources ) to dimensions of 13 cm 100 m , and the outlet of the column was a pulled tip . reversed phase chromatography followed a gradient of 240% acetonitrile in 0.1% formic acid over 90 min at 250 nl / min . the tip of the column was in line with an ltq - orbitrap xl mass spectrometer ( thermofisher scientific ) . spray voltage was set to 1.75 kv , and the capillary ion transfer tube was set to 160c . a series of four separate full scans were acquired from the orbital trap ( m / z 380520 ; 515690 ; 685975 ; 9702,000 ) at a resolution of 60,000 at m / z 400 with agc settings of 1e6 ions or 500 ms . the top three intense ions from each individual full scan fraction were subjected to fragmentation by cid at a normalized collision energy of 35% and scanned out using the ltq ion trap . charge state screening was enabled for full scans so that unassigned charge states and singly charged ions were rejected for cid . exclusion time was set to 60 s with a mass tolerance window of 0.6 to 1.2 amu . spectral data were acquired and saved using xcalibur software . cheek cell brush biopsy collection and analysis the inside of the cheek of a healthy volunteer was dried with a cotton swab to minimize salivary contamination , and buccal epithelial cells were obtained with brushing using an oralcdx brush test kit ( oralcdx laboratories , inc . , after that , the oral brush was rotated 360 for three times on the tissue surface . the brush head was placed in 0.25 ml of lysis buffer ( 4% sds , 10% 2-mercaptoethanol , 100 mm tris hcl , ph 6.8 , and 1 protease inhibitors ( complete mini , roche applied science , indianapolis , in , usa ) ) , and the exfoliated epithelial cells were lysed with continuous vortexing for 15 min at room temperature . the protein lysate ( 0.25 ml ) was chilled on ice prior to addition of 1 ml of ice - cold acetone to precipitate proteins overnight at 20c . precipitated protein was centrifuged at 6,000 rpm for 10 min at 4c , and the protein pellet was dissolved in 50 mm tris an aliquot containing 0.1 mg of protein was digested with sequencing grade trypsin ( promega , madison , wi ) at a 1:100 enzyme / substrate ratio . digestion occurred overnight at 37c , and resulting peptides were subjected to scx as described above . a total of nine fractions were analyzed by reversed phase nlc ms / ms essentially as described database searching and data processing mass spectral data were searched using maxquant software . parameters included full trypsin specificity with up to two missed cleavages and oxidized methionine as a variable modification . the msm files were searched against a composite of the ipi human database v3.52 and its reversed compliment including common contaminant proteins using mascot with 7 ppm precursor and 0.5 amu fragment ion mass tolerances , respectively . identification parameters included minimum pep score of 1 , at least two unique peptides for identification , and protein false discovery rate ( fdr ) < 1% . comparison of tif to other proteomes the salivary protein dataset was generated from .raw files obtained from and was searched using maxquant and parameters as above . protein datasets from conditioned media of the ovarian cancer cell lines : htb-75 , rmug - s , tov-112d , and tov-21 g were obtained from . identifications were filtered based on 95% peptide probability and 80% protein probability for peptideprophet and proteinprophet , respectively . the liver interstitial fluid ( if ) proteomes were from , and each identification had at least two unique peptide matches . comparison using gene ontology terms proteins identified from tif and epithelial brush biopsy cell lysate were compared by the cellular components gene ontology ( go ) term . cytosolic , cytoplasmic , or cytoskeletol were counted and grouped as names that referred to organelles such as the endoplasmic reticulum , the nucleus , mitochondria , the golgi complex , peroxisomes , or lysosomes were counted and grouped as in situ collection of tif for our study , a unique ultrafiltration catheter [ 7 , 16 ] was used for in situ tif collection from a patient with a stage iv neck scc . the catheter , originally designed to deliver therapeutics to localized sites , was composed of a fibrous wall 3-mm long with an outer diameter of 380 m similar to that of a 28-gauge needle . the walls were made of semi - permeable hydrophilic polysulfone fiber with 0.45-m pores to prevent solid material from passing through . the porous fiber also had a relatively large surface area to dissipate the force generated from suction , thereby providing gentle extraction . in all , the miniature design with large surface area was intended to limit tissue damage . the distal end was enclosed , and the proximal end was connected to a vacutainer , providing suction for removal and storage of the tif . sample preparation and proteomic analysis of tif tif was collected from two bilateral regional metastases of a stage iv hnscc in a single patient with informed consent . the extracted fluids were mixed with phosphate - buffered saline and immediately stored at 80c until further use . the coomassie staining pattern after sds - page showed that the two tif collections were very similar ( fig . 1 , lanes 1 and 2 ) , suggesting that the tif was homogenous throughout the tumor and that extraction was consistent and repeatable . the detection of only a few intense bands by coomassie staining indicated the possible presence of several proteins at much higher abundance compared to other proteins in the sample , similar to the large dynamic range of protein abundances observed in other bio - fluids like plasma . tif ( 10 g ) was separated by sds - page and stained with coomassie brilliant blue . the two lanes labeled right and left represent tif collected from two separate extractions using the ultrafiltration catheter device of a head and neck tumor in situto get an initial assessment of the protein composition of tif , a small portion was digested with trypsin , and the peptides were analyzed via tandem mass spectrometry ( ms / ms ) . the seven identified proteins that had the highest number of assigned spectra ( starting with the highest in descending order ) were albumin , hemoglobin ( beta and alpha subunits ) , transferrin , complement component 3 precursor , haptoglobin isoform 2 preprotein , alpha-2-macroglobulin , and alpha-1-antitrypsin . 1 at ~70 and ~15 kda match the expected molecular weight of albumin and alpha / beta globin , respectively . except for the two hemoglobin subunits , the other six proteins are among the most abundant proteins found in plasma [ 17 , 18].seeking to expand our identification of lower abundance proteins , we chose to remove the high abundance proteins through immunodepletion using a mars column designed to remove the abundant plasma proteins we found in tif . after starting with ~0.4 mg , the depleted flow - through from the mars column contained 0.15 mg of total protein . these proteins were digested with trypsin , and the peptides were separated based on isoelectric point . we then incorporated scx chromatography as depicted in fig . 2 , which outlines the sample preparation and analysis scheme . scx had previously been shown to be an effective second fractionation step after isoelectric focusing for complex samples [ 10 , 19 ] . specifically , the isoelectric point peptide fractions were combined in groups of six to eight fractions creating three separate pools , each of which was subjected to scx hplc . a total of 1215 fractions from each pooled sample were selected based on signal from uv chromatograms of the associated scx fractionation . each of these fractions was individually subjected to lc - ms / ms analysis , which resulted in identification of 525 proteins with at least two unique peptide sequences . all of the identified peptides are listed in supplementary table 1 , and the corresponding proteins are listed in supplementary table 2 . the peptides were subjected to three dimensions of fractionation using isoelectric focusing , strong cation exchange chromatography , and reversed - phase chromatography prior to tandem mass spectrometry analysis . ms / ms data was searched against a human database using maxquant sds - page analysis of tif . tif ( 10 g ) was separated by sds - page and stained with coomassie brilliant blue . the two lanes labeled right and left represent tif collected from two separate extractions using the ultrafiltration catheter device of a head and neck tumor in situ proteomic analysis strategy for identifying proteins from tif . the peptides were subjected to three dimensions of fractionation using isoelectric focusing , strong cation exchange chromatography , and reversed - phase chromatography prior to tandem mass spectrometry analysis . ms / ms data was searched against a human database using maxquant comparison of hnscc tif to other proteomes with a novel , high confidence protein catalog in hand , we next sought to compare it with other proteomes to further characterize the hnscc tif proteome . since hnscc tif contained several high abundance plasma proteins , we initially compared it against the plasma proteome to determine the degree of similarity between these two proteomes . the human plasma proteome was obtained from the hupo plasma proteome project ( http://www.bioinformatics.med.umich.edu/hupo/ppp ) and contained 3,020 proteins with at least two unique peptide identifications . unexpectedly , hnscc tif had a relatively small overlap of common proteins with plasma ( 40% overlap , fig . in fact , hnscc tif shared more in common with the proteome of whole saliva ( 76% overlap , fig . this finding was even more striking considering that the plasma proteome had about 1,000 more proteins than saliva . the relatively low amount of overlap with plasma supported that the hnscc tif was collected with little or no accidental contamination from blood . venn diagrams showing common and unique proteins from hnscc tif to a plasma , 40% in common ; b saliva , 76% in common ; c hepatocyte cell carcinoma tif , 41% in common ; and d conditioned media from tov-112d ovarian carcinoma cell line , 34% in commonwe next sought a more direct comparison of the hnscc tif proteome to other if or if - like proteomes . recently , the ifs were derived from tissue biopsies and obtained ex vivo from proteins released into buffer . these samples included tif from a hepatocellular carcinoma ( hcc ) , if from a non - tumor part of the same liver , and if from a separate healthy liver . not surprisingly , the hcc tif proteome had the most in common with the hnscc tif proteome with 41% overlap ( fig . 3c ) , whereas the if from a non - tumor part of the same liver and the if from a separate healthy liver had 38% and 35% of proteins in common . even though there was 41% overlap , the hcc and hnscc tif proteomes had large numbers of non - shared proteins suggesting that they were quite distinct.finally , the hnscc tif was compared to secreted proteins from four different ovarian cancer cell lines . the proteomic datasets were obtained in a previous study where an extensive analysis was performed using conditioned media after incubation with cells in order to obtain shed or secreted proteins , similar in principle to those found in tif . this type of rigorous proteomics analysis has not been performed for hnscc - based cell models , and despite differences , an ovarian cancer model seemed appropriate since both cancer types are epithelial in nature . hnscc tif had 34% proteins in common with media from tov-112d cells ( fig . there was somewhat less overlap with media from htb-75 cells ( 28% ) , tov-21 g cells ( 29% ) , and rmug - s ( 18% ; data not shown ) . once again , the hnscc tif proteome had a high amount of non - overlapping proteins further demonstrating that our in situ collected hnscc tif proteome was distinct . venn diagrams showing common and unique proteins from hnscc tif to a plasma , 40% in common ; b saliva , 76% in common ; c hepatocyte cell carcinoma tif , 41% in common ; and d conditioned media from tov-112d ovarian carcinoma cell line , 34% in common the identified hnscc tif proteins were classified according to the gene ontology term cellular components to determine cellular localizations . cytoplasmic , and proteins associated with the endoplasmic reticulum , golgi complex , mitochondrion , lysosome , peroxisome , or nucleus were grouped as organellar . in cases of ambiguity , proteins were grouped into more than one category . in order to assess potential cell lysis caused by our in situ collection technique , the cellular localization of the hnscc tif proteome ( 525 proteins ) was compared to a similar sized proteome ( 524 proteins ) identified from lysed cells gathered from brushing the cheek buccal epithelium of a healthy volunteer ( fig . if our collection technique caused a significant amount of cell lysis , we expected that the hnscc tif proteome would show a similar proportion of cytoplasmic and organellar proteins as compared to the epithelial cell proteome . as expected , the cellular lysate contained a large amount of cytoplasmic ( 421 ) and organellar ( 374 ) proteins , whereas only 61 proteins were classified as extracellular . on the other hand , hnscc tif proteome had lower amounts of proteins classified as cytoplasmic and organellar , 339 and 269 , respectively , but a comparatively higher proportion of proteins grouped as extracellular ( 203 total ) . the overall percentage of proteins grouped as extracellular for hnscc tif was high even compared to known extracellular fluids like saliva ( data not shown ) . in all , this data was in agreement with a low level cell lysis associated with the hnscc tif sample . 4comparison of hnscc tif and healthy buccal epithelial cell lysate from brush biopsy using the cellular components gene ontology term . identified proteins of hnscc tif ( solid bars ) and epithelial cell lysate ( open bars ) were categorized into different cellular localizations . proteins with localizations for the nucleus , mitochondria , the endoplasmic reticulum , the golgi complex , peroxisomes , or lysosomes were grouped as organellar . proteins with no gene ontology information or no clear localization were grouped as unclassified comparison of hnscc tif and healthy buccal epithelial cell lysate from brush biopsy using the cellular components gene ontology term . identified proteins of hnscc tif ( solid bars ) and epithelial cell lysate ( open bars ) were categorized into different cellular localizations . proteins with localizations for the nucleus , mitochondria , the endoplasmic reticulum , the golgi complex , peroxisomes , or lysosomes were grouped as organellar . proteins with no gene ontology information or no clear localization were grouped as unclassified potential protein biomarkers found in hnscc tif the proteins of hnscc tif were examined to see if any had been previously reported as a potential biomarker for hnscc . in a recent review , schaaij - visser et al . compiled a list of putative biomarkers for scc from several different proteomics - based studies . the list , shown in table 1 , was modified to contain biomarkers specific for cancer or cancer progression , which came from in vivo sources of bio - fluid and were up - regulated compared to healthy states . we identified several putative up - regulated protein biomarkers that were identified in hnscc tif . some were identified with several unique peptides and high sequence coverage such as catalase ( 60% coverage ) , s100a9 ( 56.1% coverage ) , and profilin ( 59.3% coverage ) . this suggests that tif may be a bountiful source of tumor - related biomarkers . table 1overlay of hnscc tif identified proteins with previously determined protein biomarkers for hnscc assayed from bio - fluidssource of protein biomarker discoveryproteomics approach ( separation and quantification)verified biomarkers ( accession no.)biomarker verification methods / statistical analysesbiomarker potential characteristicsref.no . of unique peptides identified in hnscc tifmolecular weight ( kda)sequence coverage ( % ) orthotopic xenograft mouse model oscc / serum analysisdige and itraq/2dlcegfr ( a8k2t7)elisaserum biomarkers for oscc tissue and tumor invasion01340western blotihcoscc salivascx / rp hplc and 2d gemac-2 binding protein ( q08380)saliva biomarkers for oscc665.310.4s100-a9 ( p06702)elisa513.256.1cd59 ( p13987)western blot114.29.4profilin ( p07737)receiver operator characteristics analysis715.159.3catalase ( p04040)3159.860hnscc saliva2d diges100-a9 ( p06702)western blotearly detection of hnscc513.256.1western blotihcoscc cell line secretome1d sds - pagemac-2 binding protein ( q08380)elisa on serummac-2 bp as serum biomarker665.310.4receiver operator characteristics analysismac-2 bp sirna and assay cell proliferation , invasion , and migration overlay of hnscc tif identified proteins with previously determined protein biomarkers for hnscc assayed from bio - fluids this study describes the first report to our knowledge of the proteomic characterization of hnscc tif . initial characterization showed that hnscc tif had a large dynamic range of protein abundance ( see fig . 1 ) . many of the high abundance proteins were the same high abundance proteins found in plasma . as a result , we chose to use mars depletion coupled with three - dimensional peptide fractionation in our sample preparation , which enabled deeper proteomic coverage . the scheme employed here , as depicted in fig . 2 , may serve as a template for future tif proteomic studies . in all , 525 proteins were identified with high confidence . as a whole , our results indicated that the ultrafiltration catheter effectively collected tif in situ and preserved its integrity by limiting contamination from other sources . first , sds - page analysis of two separate collections of tif from the same tumor showed that each collection was consistent . if the samples were contaminated with proteins due to cell lysis or blood vessel damage , we would have expected more random protein patterns detected between the samples . second , hnscc tif identified proteins had a relatively small overlap to proteins identified from plasma ( fig . this demonstrated that the catheter did not accidentally damage blood vessels leading to introduction of blood proteins into our sample . gene ontology term revealed that the tif proteome had a high proportion of proteins grouped as extracellular and a low proportion of proteins classified as cytoplasmic and organellar compared to a similar sized dataset of an epithelial cell lysate ( fig . the localization of proteins identified from tif was consistent with that of an extracellular fluid and further suggested that the insertion of the catheter caused minimal cell damage . the tif proteome characterized here was quite distinct when compared to other relevant proteomes as shown in fig . there was a relatively low overlap of identified proteins when compared to interstitial fluids derived from liver and when compared to proteins secreted into conditioned media from various ovarian cancer cell lines . part of the low overlap is likely due to the different biological sources of the fluids . however , the distinctiveness of the hnscc tif is also probably a result of the unique sample collection technique . previous studies of tif have relied on excision of tissue biopsies followed by soaking of the extracted tissue over time in buffer . this method is comparably quite destructive and has invariably led to identification of contaminating proteins , which may negatively affect biomarker studies . the unique technique used in this study allowed for gentle collection of tif in situ , without a biopsy or time delay . overall , our in situ technique reliably collects high fidelity tif samples , and it should prove beneficial for further studies characterizing fluid from other types of tumors . tif protein catalogs obtained from high fidelity samples , such as the hnscc catalog presented here , have potential value in prioritization of potential biomarkers found in other studies . there has been a large push in the field of proteomics to find protein biomarkers from easily accessible fluids such as plasma or saliva . it is difficult to validate all of these potential biomarkers because each validation requires it own specific and sensitive test , which together is rate - limiting . consequently , prioritization is usually necessary , enabling validation of the most promising potential biomarkers . given the rich source of potential biomarkers provided by the catalog of tif proteins , it should serve as a valuable guide to help prioritize potential biomarkers discovered in other bodily fluids for validation . indeed , a number of proteins identified in our study were previously shown to be up - regulated in scc progression ( table 1 ) .

introductiontumors lack normal drainage of secreted fluids and consequently build up tumor interstitial fluid ( tif ) . unlike other bodily fluids , tif likely contains a high proportion of tumor - specific proteins with potential as biomarkers.methodshere , we evaluated a novel technique using a unique ultrafiltration catheter for in situ collection of tif and used it to generate the first catalog of tif proteins from a head and neck squamous cell carcinoma ( hnscc ) . to maximize proteomic coverage , tif was immunodepleted for high abundance proteins and digested with trypsin , and peptides were fractionated in three dimensions prior to mass spectrometry.resultswe identified 525 proteins with high confidence . the hnscc tif proteome was distinct compared to proteomes of other bodily fluids . it contained a relatively high proportion of proteins annotated by gene ontology as extracellular compared to other secreted fluid and cellular proteomes , indicating minimal cell lysis from our in situ collection technique . several proteins identified are putative biomarkers of hnscc , supporting our catalog s value as a source of potential biomarkers.conclusionsin all , we demonstrate a reliable new technique for in situ tif collection and provide the first hnscc tif protein catalog with value as a guide for others seeking to develop tumor biomarkers.electronic supplementary materialthe online version of this article ( doi:10.1007/s12014 - 010 - 9050 - 3 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Experimental Methods Results Cellular Localization of HNSCC TIF Proteins Conclusions Electronic Supplementary Material

the online version of this article ( doi:10.1007/s12014 - 010 - 9050 - 3 ) contains supplementary material , which is available to authorized users . squamous cell carcinoma ( scc ) is a common type of cancer originating in epithelial cells . one potential test would be based on specific differential protein expression , or protein biomarkers of scc . a promising source of possible biomarkers of scc is tumor interstitial fluid ( tif ) . because this fluid is directly associated with the tumor , it may be a rich source of tumor - specific biomarkers . in the case of scc of the head and neck ( and potentially scc at other anatomical locations ) , tif may infiltrate other easily sampled bio - fluids such as plasma or saliva . thus , knowledge of tif proteomes may help to prioritize putative protein biomarkers found in plasma or saliva to guide validation studies . despite its potential value as a source of tumor biomarkers , representative catalogs of tif proteomes currently , a couple of different analysis strategies for studying interstitial fluid have been employed . first , some studies have used indirect extraction of tif from biopsies where resected tissue is soaked in buffer and proteins diffused into the buffer over time are analyzed [ 3 , 4 ] . these collections may not be true representations of tif due to the ex vivo and in vitro sampling techniques employed . in this study , tif from a head and neck scc tumor was obtained directly in situ without biopsy using a novel ultrafiltration catheter , and its proteome was characterized via immunodepletion , trypsin digestion , three - dimensional peptide fractionation , and mass spectrometry . this represents the first study of tif collected in situ and the first study of tif obtained from head and neck squamous cell carcinoma ( hnscc ) . our collection technique , sample preparation , and resulting proteomic catalog should be a valuable resource to others investigating tumor progression mechanisms as well as those seeking to identify tumor - specific biomarkers of scc . in situ scc tif collection tif was collected from the tumor of a patient with stage iv head and neck cancer with informed consent using an ultrafiltration catheter previously described . it is slightly smaller than a 27-gauge needle , and the proximal shaft of the catheter is the size of a 24-gauge needle to limit disturbances from seeding . tif was collected from the right and left side of the tumor , was mixed with phosphate buffered saline , and was immediately stored at 80c . depletion of high abundance proteins from tif to remove high abundance proteins from tif for enhancement of proteomic coverage , a multiple affinity removal system ( mars , agilent technologies , inc . protein from tif ( 0.4 mg ) was applied to the mars column , and the column was used according to manufacturer s instructions using manufacturer - supplied buffers . trypsin digestion and fractionation of tif peptides both undepleted ( 100 g ) and mars - depleted ( 150 g ) tif protein samples were independently digested and used for proteomics analysis . samples were diluted tenfold with 10 mm ammonium bicarbonate buffer ( ph 8.0 ) to reduce the sds concentration to 0.05% w / v . the next day , samples were cleaned with an oasis mcx solid phase extraction cartridge ( waters , milford , ma ) according to manufacturer s protocol , and the eluted peptides were dried by vacuum centrifugation.peptide isoelectric focusing ( ief ) was performed using an agilent 3100 offgel fractionator ( agilent ) using the 24-well setup with ph 310 ipg strips according to manufacturer s instructions . briefly , peptides resulting from trypsin digestion of tif proteins were dissolved in 3.6 ml of offgel peptide sample solution consisting of appropriate ampholytes . the peptides were then electrofocused at a maximum limit of 50 a until 50 kvh was reached . the column consisted of fused silica tubing packed with magic c18aq5-m , 200- pore size resin ( michrom bioresources ) to dimensions of 13 cm 100 m , and the outlet of the column was a pulled tip . cheek cell brush biopsy collection and analysis the inside of the cheek of a healthy volunteer was dried with a cotton swab to minimize salivary contamination , and buccal epithelial cells were obtained with brushing using an oralcdx brush test kit ( oralcdx laboratories , inc . the protein lysate ( 0.25 ml ) was chilled on ice prior to addition of 1 ml of ice - cold acetone to precipitate proteins overnight at 20c . precipitated protein was centrifuged at 6,000 rpm for 10 min at 4c , and the protein pellet was dissolved in 50 mm tris an aliquot containing 0.1 mg of protein was digested with sequencing grade trypsin ( promega , madison , wi ) at a 1:100 enzyme / substrate ratio . digestion occurred overnight at 37c , and resulting peptides were subjected to scx as described above . comparison of tif to other proteomes the salivary protein dataset was generated from .raw files obtained from and was searched using maxquant and parameters as above . protein datasets from conditioned media of the ovarian cancer cell lines : htb-75 , rmug - s , tov-112d , and tov-21 g were obtained from . the liver interstitial fluid ( if ) proteomes were from , and each identification had at least two unique peptide matches . comparison using gene ontology terms proteins identified from tif and epithelial brush biopsy cell lysate were compared by the cellular components gene ontology ( go ) term . cytosolic , cytoplasmic , or cytoskeletol were counted and grouped as names that referred to organelles such as the endoplasmic reticulum , the nucleus , mitochondria , the golgi complex , peroxisomes , or lysosomes were counted and grouped as in situ collection of tif for our study , a unique ultrafiltration catheter [ 7 , 16 ] was used for in situ tif collection from a patient with a stage iv neck scc . the porous fiber also had a relatively large surface area to dissipate the force generated from suction , thereby providing gentle extraction . sample preparation and proteomic analysis of tif tif was collected from two bilateral regional metastases of a stage iv hnscc in a single patient with informed consent . 1 , lanes 1 and 2 ) , suggesting that the tif was homogenous throughout the tumor and that extraction was consistent and repeatable . the detection of only a few intense bands by coomassie staining indicated the possible presence of several proteins at much higher abundance compared to other proteins in the sample , similar to the large dynamic range of protein abundances observed in other bio - fluids like plasma . the two lanes labeled right and left represent tif collected from two separate extractions using the ultrafiltration catheter device of a head and neck tumor in situto get an initial assessment of the protein composition of tif , a small portion was digested with trypsin , and the peptides were analyzed via tandem mass spectrometry ( ms / ms ) . except for the two hemoglobin subunits , the other six proteins are among the most abundant proteins found in plasma [ 17 , 18].seeking to expand our identification of lower abundance proteins , we chose to remove the high abundance proteins through immunodepletion using a mars column designed to remove the abundant plasma proteins we found in tif . these proteins were digested with trypsin , and the peptides were separated based on isoelectric point . 2 , which outlines the sample preparation and analysis scheme . each of these fractions was individually subjected to lc - ms / ms analysis , which resulted in identification of 525 proteins with at least two unique peptide sequences . all of the identified peptides are listed in supplementary table 1 , and the corresponding proteins are listed in supplementary table 2 . the peptides were subjected to three dimensions of fractionation using isoelectric focusing , strong cation exchange chromatography , and reversed - phase chromatography prior to tandem mass spectrometry analysis . the two lanes labeled right and left represent tif collected from two separate extractions using the ultrafiltration catheter device of a head and neck tumor in situ proteomic analysis strategy for identifying proteins from tif . the peptides were subjected to three dimensions of fractionation using isoelectric focusing , strong cation exchange chromatography , and reversed - phase chromatography prior to tandem mass spectrometry analysis . ms / ms data was searched against a human database using maxquant comparison of hnscc tif to other proteomes with a novel , high confidence protein catalog in hand , we next sought to compare it with other proteomes to further characterize the hnscc tif proteome . since hnscc tif contained several high abundance plasma proteins , we initially compared it against the plasma proteome to determine the degree of similarity between these two proteomes . the human plasma proteome was obtained from the hupo plasma proteome project ( http://www.bioinformatics.med.umich.edu/hupo/ppp ) and contained 3,020 proteins with at least two unique peptide identifications . unexpectedly , hnscc tif had a relatively small overlap of common proteins with plasma ( 40% overlap , fig . the relatively low amount of overlap with plasma supported that the hnscc tif was collected with little or no accidental contamination from blood . venn diagrams showing common and unique proteins from hnscc tif to a plasma , 40% in common ; b saliva , 76% in common ; c hepatocyte cell carcinoma tif , 41% in common ; and d conditioned media from tov-112d ovarian carcinoma cell line , 34% in commonwe next sought a more direct comparison of the hnscc tif proteome to other if or if - like proteomes . these samples included tif from a hepatocellular carcinoma ( hcc ) , if from a non - tumor part of the same liver , and if from a separate healthy liver . not surprisingly , the hcc tif proteome had the most in common with the hnscc tif proteome with 41% overlap ( fig . 3c ) , whereas the if from a non - tumor part of the same liver and the if from a separate healthy liver had 38% and 35% of proteins in common . even though there was 41% overlap , the hcc and hnscc tif proteomes had large numbers of non - shared proteins suggesting that they were quite distinct.finally , the hnscc tif was compared to secreted proteins from four different ovarian cancer cell lines . this type of rigorous proteomics analysis has not been performed for hnscc - based cell models , and despite differences , an ovarian cancer model seemed appropriate since both cancer types are epithelial in nature . there was somewhat less overlap with media from htb-75 cells ( 28% ) , tov-21 g cells ( 29% ) , and rmug - s ( 18% ; data not shown ) . once again , the hnscc tif proteome had a high amount of non - overlapping proteins further demonstrating that our in situ collected hnscc tif proteome was distinct . venn diagrams showing common and unique proteins from hnscc tif to a plasma , 40% in common ; b saliva , 76% in common ; c hepatocyte cell carcinoma tif , 41% in common ; and d conditioned media from tov-112d ovarian carcinoma cell line , 34% in common the identified hnscc tif proteins were classified according to the gene ontology term cellular components to determine cellular localizations . in order to assess potential cell lysis caused by our in situ collection technique , the cellular localization of the hnscc tif proteome ( 525 proteins ) was compared to a similar sized proteome ( 524 proteins ) identified from lysed cells gathered from brushing the cheek buccal epithelium of a healthy volunteer ( fig . if our collection technique caused a significant amount of cell lysis , we expected that the hnscc tif proteome would show a similar proportion of cytoplasmic and organellar proteins as compared to the epithelial cell proteome . as expected , the cellular lysate contained a large amount of cytoplasmic ( 421 ) and organellar ( 374 ) proteins , whereas only 61 proteins were classified as extracellular . on the other hand , hnscc tif proteome had lower amounts of proteins classified as cytoplasmic and organellar , 339 and 269 , respectively , but a comparatively higher proportion of proteins grouped as extracellular ( 203 total ) . the overall percentage of proteins grouped as extracellular for hnscc tif was high even compared to known extracellular fluids like saliva ( data not shown ) . in all , this data was in agreement with a low level cell lysis associated with the hnscc tif sample . 4comparison of hnscc tif and healthy buccal epithelial cell lysate from brush biopsy using the cellular components gene ontology term . identified proteins of hnscc tif ( solid bars ) and epithelial cell lysate ( open bars ) were categorized into different cellular localizations . proteins with localizations for the nucleus , mitochondria , the endoplasmic reticulum , the golgi complex , peroxisomes , or lysosomes were grouped as organellar . proteins with no gene ontology information or no clear localization were grouped as unclassified comparison of hnscc tif and healthy buccal epithelial cell lysate from brush biopsy using the cellular components gene ontology term . identified proteins of hnscc tif ( solid bars ) and epithelial cell lysate ( open bars ) were categorized into different cellular localizations . proteins with localizations for the nucleus , mitochondria , the endoplasmic reticulum , the golgi complex , peroxisomes , or lysosomes were grouped as organellar . proteins with no gene ontology information or no clear localization were grouped as unclassified potential protein biomarkers found in hnscc tif the proteins of hnscc tif were examined to see if any had been previously reported as a potential biomarker for hnscc . the list , shown in table 1 , was modified to contain biomarkers specific for cancer or cancer progression , which came from in vivo sources of bio - fluid and were up - regulated compared to healthy states . some were identified with several unique peptides and high sequence coverage such as catalase ( 60% coverage ) , s100a9 ( 56.1% coverage ) , and profilin ( 59.3% coverage ) . this suggests that tif may be a bountiful source of tumor - related biomarkers . table 1overlay of hnscc tif identified proteins with previously determined protein biomarkers for hnscc assayed from bio - fluidssource of protein biomarker discoveryproteomics approach ( separation and quantification)verified biomarkers ( accession no. of unique peptides identified in hnscc tifmolecular weight ( kda)sequence coverage ( % ) orthotopic xenograft mouse model oscc / serum analysisdige and itraq/2dlcegfr ( a8k2t7)elisaserum biomarkers for oscc tissue and tumor invasion01340western blotihcoscc salivascx / rp hplc and 2d gemac-2 binding protein ( q08380)saliva biomarkers for oscc665.310.4s100-a9 ( p06702)elisa513.256.1cd59 ( p13987)western blot114.29.4profilin ( p07737)receiver operator characteristics analysis715.159.3catalase ( p04040)3159.860hnscc saliva2d diges100-a9 ( p06702)western blotearly detection of hnscc513.256.1western blotihcoscc cell line secretome1d sds - pagemac-2 binding protein ( q08380)elisa on serummac-2 bp as serum biomarker665.310.4receiver operator characteristics analysismac-2 bp sirna and assay cell proliferation , invasion , and migration overlay of hnscc tif identified proteins with previously determined protein biomarkers for hnscc assayed from bio - fluids this study describes the first report to our knowledge of the proteomic characterization of hnscc tif . initial characterization showed that hnscc tif had a large dynamic range of protein abundance ( see fig . many of the high abundance proteins were the same high abundance proteins found in plasma . as a result , we chose to use mars depletion coupled with three - dimensional peptide fractionation in our sample preparation , which enabled deeper proteomic coverage . in all , 525 proteins were identified with high confidence . as a whole , our results indicated that the ultrafiltration catheter effectively collected tif in situ and preserved its integrity by limiting contamination from other sources . if the samples were contaminated with proteins due to cell lysis or blood vessel damage , we would have expected more random protein patterns detected between the samples . second , hnscc tif identified proteins had a relatively small overlap to proteins identified from plasma ( fig . gene ontology term revealed that the tif proteome had a high proportion of proteins grouped as extracellular and a low proportion of proteins classified as cytoplasmic and organellar compared to a similar sized dataset of an epithelial cell lysate ( fig . the localization of proteins identified from tif was consistent with that of an extracellular fluid and further suggested that the insertion of the catheter caused minimal cell damage . the tif proteome characterized here was quite distinct when compared to other relevant proteomes as shown in fig . there was a relatively low overlap of identified proteins when compared to interstitial fluids derived from liver and when compared to proteins secreted into conditioned media from various ovarian cancer cell lines . however , the distinctiveness of the hnscc tif is also probably a result of the unique sample collection technique . this method is comparably quite destructive and has invariably led to identification of contaminating proteins , which may negatively affect biomarker studies . the unique technique used in this study allowed for gentle collection of tif in situ , without a biopsy or time delay . overall , our in situ technique reliably collects high fidelity tif samples , and it should prove beneficial for further studies characterizing fluid from other types of tumors . tif protein catalogs obtained from high fidelity samples , such as the hnscc catalog presented here , have potential value in prioritization of potential biomarkers found in other studies . it is difficult to validate all of these potential biomarkers because each validation requires it own specific and sensitive test , which together is rate - limiting . given the rich source of potential biomarkers provided by the catalog of tif proteins , it should serve as a valuable guide to help prioritize potential biomarkers discovered in other bodily fluids for validation . indeed , a number of proteins identified in our study were previously shown to be up - regulated in scc progression ( table 1 ) .

in summary , we linked t1d and drp data from the swedish national patient register ( npr ) ( 12 ) with nationwide cd data from small intestinal biopsy reports . this register contains data on inpatient health care since 1964 ( nationwide coverage since 1987 ) and hospital - based outpatient care since 2001 ( 12 ) . t1d was defined as having a relevant icd code : icd-7 , 260 ; icd-8 , 250 ; icd-9 , 250 ; and icd-10 : e10 ) . in earlier icd versions ( icd-7 , -8 , and -9 ) therefore , in this study , we restricted our study sample to individuals diagnosed with diabetes who were 30 years of age . this tid definition has been used before and has a high positive predictive value ( 13 ) . initially , the swedish national board of health and welfare identified 42,806 individuals with a diagnosis of t1d . we then excluded 31 individuals because of data irregularities . finally , we excluded 981 individuals ( 2.3% ) who had a diagnosis of drp before the first recorded diagnosis of t1d . 947 ( 2.3% ) had a diagnosis of cd before 31 december 2009 ( hence , 40,619 were not diagnosed with cd ) . cd was defined as duodenal / jejunal villous atrophy ( marsh stage 3 ) ( 14 ) according to biopsy reports from all 28 pathology departments in sweden . the biopsies had been performed from 1969 to 2008 ( 15 ) , but our data collection took place 20062008 . we originally had data on 29,096 individuals with biopsy - verified cd ( 15 ) . some 95% of individuals with villous atrophy have cd ( 16 ) , and non - cd diagnoses seldom explain villous atrophy ( 0.3% of individuals with villous atrophy had indications of concomitant inflammatory bowel disease ) ( 16 ) . drp was defined based on relevant icd codes in the swedish npr ( inpatient and hospital - based outpatient data ) ( icd-7 , 388.22 ; icd-8 , 250.02 ; icd-9 , 250e and 362a ; and icd-10 , h36 ) . severe drp was defined as having a drp code and requiring retinal laser therapy ( surgical codes 1600 , 1637 , ckc10 , and ckc15 ) . we used cox regression analysis with cd modeled as a time - dependent covariate to examine the risk of drp in patients with t1d and cd versus those with only t1d . follow - up began on the date of first t1d diagnosis and ended with the diagnosis of drp , emigration , death , or end of the study period ( 31 december 2009)whichever occurred first . the risk of drp was evaluated by years since cd diagnosis ( < 5 years , 5 to < 10 years , 10 to < 15 years , and 15 years ) . other predefined subgroup analyses included stratification by sex , calendar year at t1d diagnosis ( 19641975 , 19761987 , 19881999 , and 20002009 ) , and age at t1d diagnosis ( 09 , 1019 , and 2030 years ) . we chose this age categorization ( i.e. , 09 , 1019 , and 2030 years ) because puberty in swedish children seldom starts before age 10 years . the risk of drp in the above subgroups was analyzed in two time strata : < 10 years since cd diagnosis and 10 years since cd diagnosis ( table 2 ) . stratification for time since cd diagnosis was deemed necessary because the proportional hazards assumption was not fulfilled ( hence , no overall hr for drp in individuals with t1d and cd was calculated in this study ) . the incidence rates ( absolute risk ) of drp in t1d and cd were estimated by dividing the number of first drp events with the number of person - years at risk in the cohort . the number of expected events was calculated as the number of observed events divided by the hr . in all adjusted analyses , the following covariates were considered : age at t1d onset ( three categories ) , calendar period ( four categories ) , and sex . in a separate analysis , we adjusted for country of birth ( nordic versus non - nordic country ) because cd ( 17 ) and t1d ( 18 ) may vary by geographic origin . in a first sensitivity analysis , we excluded individuals with a record of oral antidiabetic medication ( atc codes a10b+a10 ) in the prescribed drug register ( 19 ) ( such patients may have type 2 diabetes even though they have an icd-10 code consistent with insulin - dependent diabetes [ e10 ] ) . in a second analysis , we excluded women who received their first diagnosis of t1d 09 months before giving birth because these women may have suffered from gestational diabetes mellitus rather than t1d ( data on pregnancy duration were obtained from the medical birth register ) . in a third sensitivity analysis , we restricted study participants to those with an inpatient diagnosis of t1d ( n = 39,612 ; 95.3% ) . we also performed a subanalysis in which we restricted the outcome to severe drp requiring retinal laser therapy . this project ( 2011/841 - 31/3 ) was approved on 15 june 2011 by the ethics review board , stockholm , sweden . this register contains data on inpatient health care since 1964 ( nationwide coverage since 1987 ) and hospital - based outpatient care since 2001 ( 12 ) . t1d was defined as having a relevant icd code : icd-7 , 260 ; icd-8 , 250 ; icd-9 , 250 ; and icd-10 : e10 ) . in earlier icd versions ( icd-7 , -8 , and -9 ) therefore , in this study , we restricted our study sample to individuals diagnosed with diabetes who were 30 years of age . this tid definition has been used before and has a high positive predictive value ( 13 ) . initially , the swedish national board of health and welfare identified 42,806 individuals with a diagnosis of t1d . finally , we excluded 981 individuals ( 2.3% ) who had a diagnosis of drp before the first recorded diagnosis of t1d . 947 ( 2.3% ) had a diagnosis of cd before 31 december 2009 ( hence , 40,619 were not diagnosed with cd ) . cd was defined as duodenal / jejunal villous atrophy ( marsh stage 3 ) ( 14 ) according to biopsy reports from all 28 pathology departments in sweden . the biopsies had been performed from 1969 to 2008 ( 15 ) , but our data collection took place 20062008 . we originally had data on 29,096 individuals with biopsy - verified cd ( 15 ) . some 95% of individuals with villous atrophy have cd ( 16 ) , and non - cd diagnoses seldom explain villous atrophy ( 0.3% of individuals with villous atrophy had indications of concomitant inflammatory bowel disease ) ( 16 ) . drp was defined based on relevant icd codes in the swedish npr ( inpatient and hospital - based outpatient data ) ( icd-7 , 388.22 ; icd-8 , 250.02 ; icd-9 , 250e and 362a ; and icd-10 , h36 ) . severe drp was defined as having a drp code and requiring retinal laser therapy ( surgical codes 1600 , 1637 , ckc10 , and ckc15 ) . we used cox regression analysis with cd modeled as a time - dependent covariate to examine the risk of drp in patients with t1d and cd versus those with only t1d . follow - up began on the date of first t1d diagnosis and ended with the diagnosis of drp , emigration , death , or end of the study period ( 31 december 2009)whichever occurred first . the risk of drp was evaluated by years since cd diagnosis ( < 5 years , 5 to < 10 years , 10 to < 15 years , and 15 years ) . other predefined subgroup analyses included stratification by sex , calendar year at t1d diagnosis ( 19641975 , 19761987 , 19881999 , and 20002009 ) , and age at t1d diagnosis ( 09 , 1019 , and 2030 years ) . we chose this age categorization ( i.e. , 09 , 1019 , and 2030 years ) because puberty in swedish children seldom starts before age 10 years . the risk of drp in the above subgroups was analyzed in two time strata : < 10 years since cd diagnosis and 10 years since cd diagnosis ( table 2 ) . stratification for time since cd diagnosis was deemed necessary because the proportional hazards assumption was not fulfilled ( hence , no overall hr for drp in individuals with t1d and cd was calculated in this study ) . the incidence rates ( absolute risk ) of drp in t1d and cd were estimated by dividing the number of first drp events with the number of person - years at risk in the cohort . the number of expected events was calculated as the number of observed events divided by the hr . in all adjusted analyses , the following covariates were considered : age at t1d onset ( three categories ) , calendar period ( four categories ) , and sex . in a separate analysis , we adjusted for country of birth ( nordic versus non - nordic country ) because cd ( 17 ) and t1d ( 18 ) may vary by geographic origin . in a first sensitivity analysis , we excluded individuals with a record of oral antidiabetic medication ( atc codes a10b+a10 ) in the prescribed drug register ( 19 ) ( such patients may have type 2 diabetes even though they have an icd-10 code consistent with insulin - dependent diabetes [ e10 ] ) . in a second analysis , we excluded women who received their first diagnosis of t1d 09 months before giving birth because these women may have suffered from gestational diabetes mellitus rather than t1d ( data on pregnancy duration were obtained from the medical birth register ) . in a third sensitivity analysis , we restricted study participants to those with an inpatient diagnosis of t1d ( n = 39,612 ; 95.3% ) . we also performed a subanalysis in which we restricted the outcome to severe drp requiring retinal laser therapy . this project ( 2011/841 - 31/3 ) was approved on 15 june 2011 by the ethics review board , stockholm , sweden . . characteristics of the study participants the risk of drp correlated with duration of cd . with adjustment for age , sex , and calendar period , individuals with t1d and cd were initially at a lower risk of drp during the first 5 years after cd diagnosis ( adjusted hazard ratio [ ahr ] 0.57 ) , followed by a neutral risk during years 5 to < 10 ( 1.03 ) ( supplementary table 1 ) . the ahr then increased substantially 10 to < 15 years after cd diagnosis ( 2.83 ) , followed by a threefold increased risk of drp > 15 years after cd diagnosis ( 3.01 ) ( supplementary table 1 ) . the absolute risk of drp during the first 5 years of follow - up in patients with t1d and cd was 289/100,000 person - years ( compared with 507/100,000 person - years in the t1d cohort ) ( excess risk : 218/100,000 person - years ) . the absolute risk increased over time , and after > 15 years of follow - up the absolute risk of drp was 2,769/100,000 person - years in patients with t1d and cd versus 920/100,000 person - years in the t1d cohort ( excess risk of 1,849/100,000 person - years ) ( supplementary table 1 ) . adjusting for country of birth did not change our risk estimates ( data not shown ) . the overall ahr for drp during the first 10 years after cd diagnosis was low ( 0.75 ) ( table 2 ) , and in this time stratum there were no interactions between cd and sex , age , or calendar period at t1d diagnosis ( data not shown ) . because of a lack of drp events in patients with t1d and cd aged 2030 years at t1d diagnosis , we were unable to calculate a hazard ratio ( hr ) in this group ( only 120 patients with t1d and cd had t1d onset between 20 and 30 years of age ) ( tables 1 and 2 ) . subgroup analyses in relation to risk of drp according to duration of cd the overall ahr for drp beyond 10 years of cd diagnosis was increased ( 2.87 ) ( table 2 ) , and drp risks did not differ according to sex , age , or calendar period at t1d diagnosis ( data not shown ) . we were not able to estimate hr for the last calendar period ( 20002009 ) because no study participant had 10 years of follow - up before the end of the study ( 31 december 2009 ) . excluding women who had their first t1d diagnosis during pregnancy ( these women could potentially have gestational diabetes mellitus ) or those with a record of oral antidiabetes medication did not influence the hrs ( supplementary table 2 ) . the risk estimate also did not change when we restricted our dataset to inpatients with t1d ( supplementary table 2 ) . by restricting the outcome to severe drp ( drp requiring retinal laser therapy ) , we found the same pattern of low initial risk followed by an increased hr after 10 years with cd ( < 5 years with cd , hr 0.56 [ 95% ci 0.181.74 ] ; 59.99 years , 0.43 [ 0.111.73 ] ; 1014.99 years , 2.49 [ 1.185.25 ] ; and 15 years , 2.01 [ 0.508.06 ] ) . the risk of drp correlated with duration of cd . with adjustment for age , sex , and calendar period , individuals with t1d and cd were initially at a lower risk of drp during the first 5 years after cd diagnosis ( adjusted hazard ratio [ ahr ] 0.57 ) , followed by a neutral risk during years 5 to < 10 ( 1.03 ) ( supplementary table 1 ) . the ahr then increased substantially 10 to < 15 years after cd diagnosis ( 2.83 ) , followed by a threefold increased risk of drp > 15 years after cd diagnosis ( 3.01 ) ( supplementary table 1 ) . the absolute risk of drp during the first 5 years of follow - up in patients with t1d and cd was 289/100,000 person - years ( compared with 507/100,000 person - years in the t1d cohort ) ( excess risk : 218/100,000 person - years ) . the absolute risk increased over time , and after > 15 years of follow - up the absolute risk of drp was 2,769/100,000 person - years in patients with t1d and cd versus 920/100,000 person - years in the t1d cohort ( excess risk of 1,849/100,000 person - years ) ( supplementary table 1 ) . adjusting for country of birth did not change our risk estimates ( data not shown ) . the overall ahr for drp during the first 10 years after cd diagnosis was low ( 0.75 ) ( table 2 ) , and in this time stratum there were no interactions between cd and sex , age , or calendar period at t1d diagnosis ( data not shown ) . because of a lack of drp events in patients with t1d and cd aged 2030 years at t1d diagnosis , we were unable to calculate a hazard ratio ( hr ) in this group ( only 120 patients with t1d and cd had t1d onset between 20 and 30 years of age ) ( tables 1 and 2 ) . subgroup analyses in relation to risk of drp according to duration of cd the overall ahr for drp beyond 10 years of cd diagnosis was increased ( 2.87 ) ( table 2 ) , and drp risks did not differ according to sex , age , or calendar period at t1d diagnosis ( data not shown ) . we were not able to estimate hr for the last calendar period ( 20002009 ) because no study participant had 10 years of follow - up before the end of the study ( 31 december 2009 ) . excluding women who had their first t1d diagnosis during pregnancy ( these women could potentially have gestational diabetes mellitus ) or those with a record of oral antidiabetes medication did not influence the hrs ( supplementary table 2 ) . the risk estimate also did not change when we restricted our dataset to inpatients with t1d ( supplementary table 2 ) . by restricting the outcome to severe drp ( drp requiring retinal laser therapy ) , we found the same pattern of low initial risk followed by an increased hr after 10 years with cd ( < 5 years with cd , hr 0.56 [ 95% ci 0.181.74 ] ; 59.99 years , 0.43 [ 0.111.73 ] ; 1014.99 years , 2.49 [ 1.185.25 ] ; and 15 years , 2.01 [ 0.508.06 ] ) . in this large population - based cohort study , duration of cd proved to be a strong predictor of future drp development . the association between t1d and cd is well recognized and may be due to shared risk factors ( 21 ) . research has largely focused on studying the prevalence of cd in t1d ( 8,9 ) , as well as the benefits of starting a gluten - free diet in asymptomatic cd within the t1d population ( 9,22 ) . few studies have examined the risk of complications in patients with both conditions ( 10,11,23 ) , and none have thus far been able to determine time - specific risks for t1d complications . study ) ( 11 ) in which advanced retinopathy was seen in 58.3% of patients with t1d and cd versus in 25% of patients with t1d without cd ( 11 ) . study could mirror different patient characteristics and t1d - management traditions in sweden and the u.k . study were selected from a tertiary diabetes center ( possibly with higher rates of complications because of selecting patients with severe t1d ) , whereas the current ( swedish ) study was based on all patients with a recorded diagnosis of t1d . study could also be due to malnutrition in the cd plus t1d group , since they were thinner than the t1d - only group . research evidence suggests that patients with t1d screened for cd and subsequently prescribed a gluten - free diet improve in their clinical parameters , including growth and metabolic control , compared with t1d patients untreated for cd ( 9,22 ) . study on retinopathy a 1-year gluten - free diet did not influence the prevalence of retinopathy ( 11 ) . although the majority of young patients with cd seem to adhere well to a gluten - free diet ( 24 ) , we can not rule out that the addition of yet another condition ( i.e. , t1d ) affected dietary adherence negatively . in a random subset of patients with cd in our dataset , 83% adhered to a gluten - free diet ( 16 ) . in the current study , we lack individual - based information on gluten - free diet , but one can speculate that the highest degree of dietary adherence was noted just after diagnosis , when the risk of drp is lower . in a recent multicenter study ( 25 ) , the effect of biopsy - proven cd on metabolic control in patients with t1d was examined over time . after 5 years of follow - up , patients with t1d and cd had lower weight and height than patients with only t1d ( 25 ) . however , no differences in bmi and hba1c levels were observed between the groups after the 5-year follow - up . if patients with t1d and cd have worse nutritional status than t1d patients without cd , the former 's risk of drp development could be increased ( 26 ) . one explanation for the lower risk of drp at baseline in patients with t1d and cd is the lower levels of cholesterol and blood pressure found in cd patients ( 27 ) . 10 ) demonstrated that patients with t1d and cd had lower levels of hba1c , triglycerides , and cholesterol than patients with only t1d . these researchers ( 10 ) found no signs of retinal or renal abnormalities in patients with t1d and cd ( 10 ) , but the study was cross - sectional without follow - up . in fact , anti - inflammatory drugs have been suggested as potential new therapies against drp ( 7 ) . when the carotid intima - media thickness was examined in italian patients with t1d and cd ( 23 ) ( as a measure of subclinical atherosclerosis ) , these patients had greater carotid intima - media thickness than patients with only t1d ( 23 ) . the positive association between cd and subclinical atherosclerosis could signal microvascular damage ( drp ) . patients with cd are at increased risk of cardiovascular death ( 15 ) and incident ischemic heart disease ( 28 ) . another possible mechanism for the increased risk of drp seen over time is that of persistent low - grade inflammation . the intestinal mucosa in patients with cd can take a long time to fully recover , even after initiation of a gluten - free diet . studies show that chronic , low - grade inflammation plays an important role in the pathogenesis of drp ( 29 ) . having low - grade intestinal inflammation or cd with little symptoms might also affect the patient s adherence to a strict gluten - free diet , which in turn could potentially affect the risk of future drp . the pattern of increasing risk of drp seen over time was also present in our subgroup analyses in which we found lower risk estimates for drp during < 10 years duration of cd diagnosis and higher risk during 10 years cd duration . the nonsignificant differences in drp risk across calendar periods may be due to longer t1d duration before the end of follow - up in patients diagnosed in earlier calendar periods ( many years at risk for drp in each patient ) . in contrast , patients diagnosed in the latest calendar period were ( for study design reasons ) only at risk just after t1d diagnosis , and because the follow - up time was short , most patients did not develop drp . the number of drp events after 2000 was low , with a wide 95% ci ( 0.111.80 ) . the differences in calendar period specific risk estimates may also reflect the changes made in t1d care and management in sweden over time as well as the diagnostic methods used for identification of cd . the major strengths of this study are the population - based design , the definition of cd ( all cases were biopsy verified ) , and that our study included all patients with t1d in sweden . the nationwide identification of cd from all pathology departments in sweden ( 16 ) minimized the risk of selection bias . although we did not use positive cd serology for the diagnosis of cd , 88% of those with available data on cd serology had positive antibodies before biopsy ( 16 ) . another strength is the large number of participants and statistical power : because > 900 patients had t1d and cd , we could perform stratified analyses . additional data on pregnancy and medication allowed us to conduct sensitivity analyses and minimize potential misclassification . even when we restricted our outcome to drp requiring retinal laser therapy , we found the same pattern of low hr in early cd followed by an increased hr over time ( longer duration of cd ) . because of fewer positive events in this subanalysis , only the hr in patients with cd for 1014.99 years was statistically significant . this study is limited by the absence of information on metabolic control ( hba1c , insulin dosage , and bmi ) in patients with t1d . in addition , the 41,566 patients with t1d were not screened for cd specifically for this study ; therefore , the clinical presentation may vary among our cd patients . today , all swedish children and adolescents with t1d are screened for cd ( routine care ) , but that may not have been the case in the beginning of the study period . in the 1990s , two - thirds ( 29 of 44 ) of all pediatric departments regularly screened all t1d patients for cd , with the remaining departments opting for cd testing on clinical suspicion ( 30 ) . hence , we can not dismiss the possibility that there are individuals with undiagnosed cd in our t1d - only cohort . still , their presence will not affect our risk estimate more than marginally because patients with t1d and undiagnosed cd are unlikely to make up more than a small percentage of our reference category ( t1d only ) . furthermore , if undiagnosed cd would have any effect , it would probably dilute existing associations . our results indicate that cd is a strong predictor for simplex and severe laser - treated drp in patients with t1d . we suggest that the lower effect of drp in early cd is due to drp - protective characteristics of patients with cd ( lower cholesterol and bmi ) . long - standing cd , however , increased the risk of drp by > 200% ( ahr 3.01 ) and thus merits closer monitoring of drp in patients with t1d .

objectiveceliac disease ( cd ) is associated with type 1 diabetes ( t1d ) . in the current study , we examined whether cd affects the risk of diabetic retinopathy ( drp ) in patients with t1d.research design and methodsthis was a population - based cohort study . through the swedish national patient register , we identified 41,566 patients diagnosed with diabetes in 19642009 and who were 30 years of age at diagnosis . cd was defined as having villous atrophy ( marsh stage 3 ) according to small intestinal biopsies performed between 1969 and 2008 , with biopsy reports obtained from sweden s 28 pathology departments . during follow - up , 947 t1d patients had a diagnosis of cd . we used cox regression analysis with cd as a time - dependent covariate to estimate adjusted hazard ratios ( ahrs ) for drp in patients with t1d and cd and compared them with patients with t1d but no cd.resultsduration of cd correlated with the risk of drp . when results were stratified by time since cd diagnosis , individuals with t1d and cd were at a lower risk of drp in the first 5 years after cd diagnosis ( ahr 0.57 [ 95% ci 0.360.91 ] ) , followed by a neutral risk in years 5 to < 10 ( 1.03 [ 0.681.57 ] ) . with longer follow - up , coexisting cd was a risk factor for drp ( 10 to < 15 years of follow - up , ahr 2.83 [ 95% ci 1.954.11 ] ; 15 years of follow - up , 3.01 [ 1.436.32]).conclusionshaving a diagnosis of cd for > 10 years is a risk factor for the development of drp in t1d . long - standing cd in patients with t1d merits intense monitoring of drp .

RESEARCH DESIGN AND METHODS T1D CD Outcome measure Statistical analyses and covariates Ethics RESULTS DRP risk relative to duration of CD Stratified analyses Sensitivity analyses CONCLUSIONS Supplementary Material

in summary , we linked t1d and drp data from the swedish national patient register ( npr ) ( 12 ) with nationwide cd data from small intestinal biopsy reports . t1d was defined as having a relevant icd code : icd-7 , 260 ; icd-8 , 250 ; icd-9 , 250 ; and icd-10 : e10 ) . in earlier icd versions ( icd-7 , -8 , and -9 ) therefore , in this study , we restricted our study sample to individuals diagnosed with diabetes who were 30 years of age . initially , the swedish national board of health and welfare identified 42,806 individuals with a diagnosis of t1d . finally , we excluded 981 individuals ( 2.3% ) who had a diagnosis of drp before the first recorded diagnosis of t1d . 947 ( 2.3% ) had a diagnosis of cd before 31 december 2009 ( hence , 40,619 were not diagnosed with cd ) . cd was defined as duodenal / jejunal villous atrophy ( marsh stage 3 ) ( 14 ) according to biopsy reports from all 28 pathology departments in sweden . some 95% of individuals with villous atrophy have cd ( 16 ) , and non - cd diagnoses seldom explain villous atrophy ( 0.3% of individuals with villous atrophy had indications of concomitant inflammatory bowel disease ) ( 16 ) . drp was defined based on relevant icd codes in the swedish npr ( inpatient and hospital - based outpatient data ) ( icd-7 , 388.22 ; icd-8 , 250.02 ; icd-9 , 250e and 362a ; and icd-10 , h36 ) . severe drp was defined as having a drp code and requiring retinal laser therapy ( surgical codes 1600 , 1637 , ckc10 , and ckc15 ) . we used cox regression analysis with cd modeled as a time - dependent covariate to examine the risk of drp in patients with t1d and cd versus those with only t1d . follow - up began on the date of first t1d diagnosis and ended with the diagnosis of drp , emigration , death , or end of the study period ( 31 december 2009)whichever occurred first . the risk of drp was evaluated by years since cd diagnosis ( < 5 years , 5 to < 10 years , 10 to < 15 years , and 15 years ) . the risk of drp in the above subgroups was analyzed in two time strata : < 10 years since cd diagnosis and 10 years since cd diagnosis ( table 2 ) . stratification for time since cd diagnosis was deemed necessary because the proportional hazards assumption was not fulfilled ( hence , no overall hr for drp in individuals with t1d and cd was calculated in this study ) . the incidence rates ( absolute risk ) of drp in t1d and cd were estimated by dividing the number of first drp events with the number of person - years at risk in the cohort . in a first sensitivity analysis , we excluded individuals with a record of oral antidiabetic medication ( atc codes a10b+a10 ) in the prescribed drug register ( 19 ) ( such patients may have type 2 diabetes even though they have an icd-10 code consistent with insulin - dependent diabetes [ e10 ] ) . t1d was defined as having a relevant icd code : icd-7 , 260 ; icd-8 , 250 ; icd-9 , 250 ; and icd-10 : e10 ) . in earlier icd versions ( icd-7 , -8 , and -9 ) therefore , in this study , we restricted our study sample to individuals diagnosed with diabetes who were 30 years of age . initially , the swedish national board of health and welfare identified 42,806 individuals with a diagnosis of t1d . finally , we excluded 981 individuals ( 2.3% ) who had a diagnosis of drp before the first recorded diagnosis of t1d . 947 ( 2.3% ) had a diagnosis of cd before 31 december 2009 ( hence , 40,619 were not diagnosed with cd ) . cd was defined as duodenal / jejunal villous atrophy ( marsh stage 3 ) ( 14 ) according to biopsy reports from all 28 pathology departments in sweden . some 95% of individuals with villous atrophy have cd ( 16 ) , and non - cd diagnoses seldom explain villous atrophy ( 0.3% of individuals with villous atrophy had indications of concomitant inflammatory bowel disease ) ( 16 ) . drp was defined based on relevant icd codes in the swedish npr ( inpatient and hospital - based outpatient data ) ( icd-7 , 388.22 ; icd-8 , 250.02 ; icd-9 , 250e and 362a ; and icd-10 , h36 ) . severe drp was defined as having a drp code and requiring retinal laser therapy ( surgical codes 1600 , 1637 , ckc10 , and ckc15 ) . we used cox regression analysis with cd modeled as a time - dependent covariate to examine the risk of drp in patients with t1d and cd versus those with only t1d . follow - up began on the date of first t1d diagnosis and ended with the diagnosis of drp , emigration , death , or end of the study period ( 31 december 2009)whichever occurred first . the risk of drp was evaluated by years since cd diagnosis ( < 5 years , 5 to < 10 years , 10 to < 15 years , and 15 years ) . other predefined subgroup analyses included stratification by sex , calendar year at t1d diagnosis ( 19641975 , 19761987 , 19881999 , and 20002009 ) , and age at t1d diagnosis ( 09 , 1019 , and 2030 years ) . the risk of drp in the above subgroups was analyzed in two time strata : < 10 years since cd diagnosis and 10 years since cd diagnosis ( table 2 ) . stratification for time since cd diagnosis was deemed necessary because the proportional hazards assumption was not fulfilled ( hence , no overall hr for drp in individuals with t1d and cd was calculated in this study ) . the incidence rates ( absolute risk ) of drp in t1d and cd were estimated by dividing the number of first drp events with the number of person - years at risk in the cohort . in a first sensitivity analysis , we excluded individuals with a record of oral antidiabetic medication ( atc codes a10b+a10 ) in the prescribed drug register ( 19 ) ( such patients may have type 2 diabetes even though they have an icd-10 code consistent with insulin - dependent diabetes [ e10 ] ) . in a second analysis , we excluded women who received their first diagnosis of t1d 09 months before giving birth because these women may have suffered from gestational diabetes mellitus rather than t1d ( data on pregnancy duration were obtained from the medical birth register ) . characteristics of the study participants the risk of drp correlated with duration of cd . with adjustment for age , sex , and calendar period , individuals with t1d and cd were initially at a lower risk of drp during the first 5 years after cd diagnosis ( adjusted hazard ratio [ ahr ] 0.57 ) , followed by a neutral risk during years 5 to < 10 ( 1.03 ) ( supplementary table 1 ) . the ahr then increased substantially 10 to < 15 years after cd diagnosis ( 2.83 ) , followed by a threefold increased risk of drp > 15 years after cd diagnosis ( 3.01 ) ( supplementary table 1 ) . the absolute risk of drp during the first 5 years of follow - up in patients with t1d and cd was 289/100,000 person - years ( compared with 507/100,000 person - years in the t1d cohort ) ( excess risk : 218/100,000 person - years ) . the absolute risk increased over time , and after > 15 years of follow - up the absolute risk of drp was 2,769/100,000 person - years in patients with t1d and cd versus 920/100,000 person - years in the t1d cohort ( excess risk of 1,849/100,000 person - years ) ( supplementary table 1 ) . the overall ahr for drp during the first 10 years after cd diagnosis was low ( 0.75 ) ( table 2 ) , and in this time stratum there were no interactions between cd and sex , age , or calendar period at t1d diagnosis ( data not shown ) . because of a lack of drp events in patients with t1d and cd aged 2030 years at t1d diagnosis , we were unable to calculate a hazard ratio ( hr ) in this group ( only 120 patients with t1d and cd had t1d onset between 20 and 30 years of age ) ( tables 1 and 2 ) . subgroup analyses in relation to risk of drp according to duration of cd the overall ahr for drp beyond 10 years of cd diagnosis was increased ( 2.87 ) ( table 2 ) , and drp risks did not differ according to sex , age , or calendar period at t1d diagnosis ( data not shown ) . we were not able to estimate hr for the last calendar period ( 20002009 ) because no study participant had 10 years of follow - up before the end of the study ( 31 december 2009 ) . by restricting the outcome to severe drp ( drp requiring retinal laser therapy ) , we found the same pattern of low initial risk followed by an increased hr after 10 years with cd ( < 5 years with cd , hr 0.56 [ 95% ci 0.181.74 ] ; 59.99 years , 0.43 [ 0.111.73 ] ; 1014.99 years , 2.49 [ 1.185.25 ] ; and 15 years , 2.01 [ 0.508.06 ] ) . the risk of drp correlated with duration of cd . with adjustment for age , sex , and calendar period , individuals with t1d and cd were initially at a lower risk of drp during the first 5 years after cd diagnosis ( adjusted hazard ratio [ ahr ] 0.57 ) , followed by a neutral risk during years 5 to < 10 ( 1.03 ) ( supplementary table 1 ) . the ahr then increased substantially 10 to < 15 years after cd diagnosis ( 2.83 ) , followed by a threefold increased risk of drp > 15 years after cd diagnosis ( 3.01 ) ( supplementary table 1 ) . the absolute risk of drp during the first 5 years of follow - up in patients with t1d and cd was 289/100,000 person - years ( compared with 507/100,000 person - years in the t1d cohort ) ( excess risk : 218/100,000 person - years ) . the absolute risk increased over time , and after > 15 years of follow - up the absolute risk of drp was 2,769/100,000 person - years in patients with t1d and cd versus 920/100,000 person - years in the t1d cohort ( excess risk of 1,849/100,000 person - years ) ( supplementary table 1 ) . the overall ahr for drp during the first 10 years after cd diagnosis was low ( 0.75 ) ( table 2 ) , and in this time stratum there were no interactions between cd and sex , age , or calendar period at t1d diagnosis ( data not shown ) . because of a lack of drp events in patients with t1d and cd aged 2030 years at t1d diagnosis , we were unable to calculate a hazard ratio ( hr ) in this group ( only 120 patients with t1d and cd had t1d onset between 20 and 30 years of age ) ( tables 1 and 2 ) . subgroup analyses in relation to risk of drp according to duration of cd the overall ahr for drp beyond 10 years of cd diagnosis was increased ( 2.87 ) ( table 2 ) , and drp risks did not differ according to sex , age , or calendar period at t1d diagnosis ( data not shown ) . we were not able to estimate hr for the last calendar period ( 20002009 ) because no study participant had 10 years of follow - up before the end of the study ( 31 december 2009 ) . by restricting the outcome to severe drp ( drp requiring retinal laser therapy ) , we found the same pattern of low initial risk followed by an increased hr after 10 years with cd ( < 5 years with cd , hr 0.56 [ 95% ci 0.181.74 ] ; 59.99 years , 0.43 [ 0.111.73 ] ; 1014.99 years , 2.49 [ 1.185.25 ] ; and 15 years , 2.01 [ 0.508.06 ] ) . in this large population - based cohort study , duration of cd proved to be a strong predictor of future drp development . research has largely focused on studying the prevalence of cd in t1d ( 8,9 ) , as well as the benefits of starting a gluten - free diet in asymptomatic cd within the t1d population ( 9,22 ) . few studies have examined the risk of complications in patients with both conditions ( 10,11,23 ) , and none have thus far been able to determine time - specific risks for t1d complications . study ) ( 11 ) in which advanced retinopathy was seen in 58.3% of patients with t1d and cd versus in 25% of patients with t1d without cd ( 11 ) . study were selected from a tertiary diabetes center ( possibly with higher rates of complications because of selecting patients with severe t1d ) , whereas the current ( swedish ) study was based on all patients with a recorded diagnosis of t1d . research evidence suggests that patients with t1d screened for cd and subsequently prescribed a gluten - free diet improve in their clinical parameters , including growth and metabolic control , compared with t1d patients untreated for cd ( 9,22 ) . although the majority of young patients with cd seem to adhere well to a gluten - free diet ( 24 ) , we can not rule out that the addition of yet another condition ( i.e. in a random subset of patients with cd in our dataset , 83% adhered to a gluten - free diet ( 16 ) . in the current study , we lack individual - based information on gluten - free diet , but one can speculate that the highest degree of dietary adherence was noted just after diagnosis , when the risk of drp is lower . in a recent multicenter study ( 25 ) , the effect of biopsy - proven cd on metabolic control in patients with t1d was examined over time . after 5 years of follow - up , patients with t1d and cd had lower weight and height than patients with only t1d ( 25 ) . if patients with t1d and cd have worse nutritional status than t1d patients without cd , the former 's risk of drp development could be increased ( 26 ) . one explanation for the lower risk of drp at baseline in patients with t1d and cd is the lower levels of cholesterol and blood pressure found in cd patients ( 27 ) . 10 ) demonstrated that patients with t1d and cd had lower levels of hba1c , triglycerides , and cholesterol than patients with only t1d . these researchers ( 10 ) found no signs of retinal or renal abnormalities in patients with t1d and cd ( 10 ) , but the study was cross - sectional without follow - up . when the carotid intima - media thickness was examined in italian patients with t1d and cd ( 23 ) ( as a measure of subclinical atherosclerosis ) , these patients had greater carotid intima - media thickness than patients with only t1d ( 23 ) . patients with cd are at increased risk of cardiovascular death ( 15 ) and incident ischemic heart disease ( 28 ) . another possible mechanism for the increased risk of drp seen over time is that of persistent low - grade inflammation . studies show that chronic , low - grade inflammation plays an important role in the pathogenesis of drp ( 29 ) . the pattern of increasing risk of drp seen over time was also present in our subgroup analyses in which we found lower risk estimates for drp during < 10 years duration of cd diagnosis and higher risk during 10 years cd duration . the nonsignificant differences in drp risk across calendar periods may be due to longer t1d duration before the end of follow - up in patients diagnosed in earlier calendar periods ( many years at risk for drp in each patient ) . in contrast , patients diagnosed in the latest calendar period were ( for study design reasons ) only at risk just after t1d diagnosis , and because the follow - up time was short , most patients did not develop drp . the number of drp events after 2000 was low , with a wide 95% ci ( 0.111.80 ) . the major strengths of this study are the population - based design , the definition of cd ( all cases were biopsy verified ) , and that our study included all patients with t1d in sweden . the nationwide identification of cd from all pathology departments in sweden ( 16 ) minimized the risk of selection bias . although we did not use positive cd serology for the diagnosis of cd , 88% of those with available data on cd serology had positive antibodies before biopsy ( 16 ) . another strength is the large number of participants and statistical power : because > 900 patients had t1d and cd , we could perform stratified analyses . even when we restricted our outcome to drp requiring retinal laser therapy , we found the same pattern of low hr in early cd followed by an increased hr over time ( longer duration of cd ) . because of fewer positive events in this subanalysis , only the hr in patients with cd for 1014.99 years was statistically significant . this study is limited by the absence of information on metabolic control ( hba1c , insulin dosage , and bmi ) in patients with t1d . in addition , the 41,566 patients with t1d were not screened for cd specifically for this study ; therefore , the clinical presentation may vary among our cd patients . today , all swedish children and adolescents with t1d are screened for cd ( routine care ) , but that may not have been the case in the beginning of the study period . in the 1990s , two - thirds ( 29 of 44 ) of all pediatric departments regularly screened all t1d patients for cd , with the remaining departments opting for cd testing on clinical suspicion ( 30 ) . hence , we can not dismiss the possibility that there are individuals with undiagnosed cd in our t1d - only cohort . still , their presence will not affect our risk estimate more than marginally because patients with t1d and undiagnosed cd are unlikely to make up more than a small percentage of our reference category ( t1d only ) . our results indicate that cd is a strong predictor for simplex and severe laser - treated drp in patients with t1d . we suggest that the lower effect of drp in early cd is due to drp - protective characteristics of patients with cd ( lower cholesterol and bmi ) . long - standing cd , however , increased the risk of drp by > 200% ( ahr 3.01 ) and thus merits closer monitoring of drp in patients with t1d .

glaucoma is group of optic neuropathies associated with characteristic structural damage to the optic nerve as a result of various pathological processes that lead to visual dysfunction . glaucoma is a debilitating disease that causes blindness if left untreated ; thus , early diagnosis is crucial . although early detection and treatment of glaucoma may halt progression of the disease , patients with early glaucoma often do not seek medical help because they do not notice changes in their vision . thus , recent studies have focused on the early detection and treatment of this debilitating disease . in general , diagnostic tests for glaucoma include the measurement of intraocular pressure ( iop ) , the visual field test , and examination of the optic nerve head . although elevated iop is one of the most important risk factors for glaucoma , the damage threshold varies among individuals and diurnal fluctuations can conceal iop spikes . the visual field test is limited as an early diagnostic tool because defects in the visual field occur only following significant ganglion cell loss [ 24 ] . furthermore , examination of the optic nerve head has disadvantages such as high interobserver variability and low reproducibility . several new methods for the evaluation of the optic nerve head have been proposed to overcome these limitations . the disc damage likelihood scale ( ddls ) incorporates the size of the disc and the radial width of the neuroretinal rim into the evaluation of the optic nerve head . the ddls has been reported to provide a more accurate assessment of optic disc damage than the conventional cup / disc ( c / d ) ratio measurement , and a high correlation has been found between the ddls and various indices obtained from heidelberg retina tomography and optical coherence tomography ( oct ) in patients with glaucoma [ 7 , 8 ] . nevertheless , interobserver variability is a potential limitation of ddls and training on the system is essential for low interobserver variability and high reproducibility . recently , a three - dimensional ( 3d ) stereographic camera ( kowa nonmyd wx 3d , kowa , tokyo , japan ) that provides an objective recording of the optic nerve head was developed . this device provides a software algorithm that automatically displays ddls in its final report output . the shape of optic nerve can be visualized on a 3d display , which provides a significant advantage in terms of evaluating the depth distribution of the optic cup and neuroretinal rim . the 3d analysis option is expected to decrease interobserver variability and increase reproducibility compared with standard optic nerve head photographs . we evaluated the validity of the ddls option provided by this 3d stereographic camera and assessed the correlations between various stereometric optic disc parameters obtained by the 3d stereographic camera and optic disc cube parameters measured using oct . moreover , we provide the first report , to our knowledge , of interobserver agreement on the ddls using kappa statistics and evidence of the usefulness of a training module that includes a detailed introduction and clinical skills for measuring ddls . the research was conducted according to the principles of the declaration of helsinki and was approved by the institutional review board of incheon st . all subjects underwent comprehensive ophthalmological examinations including medical , ocular , family history , and visual acuity testing and refraction . standard achromatic perimetry performed with a humphrey field analyzer ( zeiss humphrey systems , dublin , ca , usa ) using the 24 - 2 full - threshold test , iop measurement , dilated slit - lamp biomicroscopy , 3d optic disc photography ( kowa nonmyd wx 3d , kowa , tokyo , japan ) , optic nerve head , and retinal nerve fiber layer ( rnfl ) analysis using cirrus oct ( carl zeiss meditec inc . , dublin , ca , usa ) were performed during the same visit for each patient between march 2011 and may 2013 . following pupil dilation using tropicamide 0.5% and phenylephrine 5% , a glaucoma specialist ( k.d.k ) calculated the vertical and horizontal c / d ratios ( uncorrected for optic disc size ) using direct ophthalmoscope ( welch allyn , skaneateles falls , ny , usa ) . the optic cup was defined on the basis of contour and the course of the small blood vessels on the disc and not on the basis of pallor . the optic disc border was defined as the inner border of the peripapillary scleral ring . the 3d stereoscopic analysis of the optic nerve head was performed according the manufacturer 's instructions . briefly , following pupil dilation , photographic stereo pairs were captured and displayed on a 3d monitor . the optic nerve head was viewed using prism glasses , and the c / d ratio was plotted manually to determine the contour line of each structure . all oct scans were acquired with a cirrus hdoct ( version 3.0.0.64 ) using the optic disc cube 200 200 protocol , which is designed to position the cube scan on the optic nerve head and is primarily used for glaucoma analysis . only scans of good quality ( signal strength better than 6 , without rnfl discontinuity or misalignment , involuntary saccade , or blinking artifacts , and absence of rnfl algorithm segmentation failure without misalignment or movement artefacts ) were included in the analysis . the 190 patients were divided into three diagnostic groups : normal , glaucoma suspect , and glaucoma . the criteria for the normal group were ( 1 ) healthy subjects with no history or presence of glaucoma , no retinal pathological findings , and no intraocular surgery including laser therapy ; ( 2 ) iop 21 mm hg on each visit ; ( 3 ) normal humphrey 24 - 2 visual field test ; ( 4 ) best corrected visual acuity of 20/40 or better with refractive error between + 1.00 and 3.00 diopters ; ( 5 ) open angles by gonioscopy ; and ( 6 ) normal - appearing optic nerve head . the criteria for glaucoma suspect eyes were ( 1 ) no history or presence of retinal pathology and no intraocular surgery including laser therapy ; ( 2 ) iop between 22 and 30 mmhg ; and ( 3 ) normal visual field test . patients who had the following findings were also categorized as glaucoma suspect , ( a ) asymmetric optic nerve head cupping ( difference in vertical c / d ratio 0.2 between the eyes in the presence of a similar optic disc size ) or ( b ) increased cupping ( vertical c / d ratio > 0.6 ) . the diagnostic criteria for glaucoma were ( 1 ) an abnormal visual field defined as the presence of at least two of the following . ( a ) a glaucoma hemifield test outside normal limits , ( b ) p < 5% for corrected pattern standard deviation , or ( c ) a cluster of at least three contiguous points with p < 5% , including at least one of these with p < 1% in the pattern deviation plot . ( 2 ) one or more papillary sign as follows : ( a ) presence of a localized loss or thinning of the neuroretinal rim , ( b ) optic disk excavation , ( c ) vertical or horizontal c / d ratio > 0.6 , and ( d ) c / d asymmetry between the two eyes 0.2 . patients with a previous history of intraocular surgery , including laser therapy or undergoing a systemic therapy that could interfere with ocular hydrodynamics were excluded from the glaucoma subgroup . residents at different levels of training evaluated the eyes of the patients using the ddls . as the ophthalmology residency in korea involves 4 years training in an accredited institution , we recruited three residents from each postgraduate year ( pgy ) for our study ( 12 residents in total ) . the residents were asked to evaluate each patient using the ddls with no prior training and only the ddls index as a reference . six residents evaluated patients on their initial visit to the clinic and the remaining six evaluated patients on their second visit ( usually 3 months after the initial visit ) . a glaucoma specialist ( k.d.k ) used the ddls to evaluate the patients on their initial visit . following the enrollment of all subjects , a 2-h ddls training program designed to provide detailed information about the grading system was conducted for all residents . it included a 1-h objective structured clinical examination ( osce ) followed by a 1-h lecture . the lecture consisted of an introduction to the system , instructions on its use , and a discussion of difficulties involved in scoring ( such as multiplying the size of the disc by the corresponding corrective factors when using 60 or 90 diopter lenses ) . following the training program , the residents were asked to reevaluate the ddls of the participating subjects . at this point , the residents were not aware of their previous ddls scores . additionally , all measurements were made using a superfield nc lens ( volk optical , miami , fl , usa ) to avoid magnification correction errors , and when analyzing the results , we incorporated the 0a and 0b stages initially proposed by spaeth et al . into one stage , 0 the level of interobserver agreement was measured using the weighted kappa statistic because it is an appropriate chance - adjusted measure of agreement between two observers when there are more than two ordered categories of classification . an analysis of variance ( anova ) was used to evaluate the differences among the glaucoma , glaucoma suspect , and normal groups on the various parameters , and the post hoc analysis was conducted using scheffe 's test . the correlations between the ddls value and the cirrus oct and 3d optic disc photography measurements were assessed using pearson 's coefficient of correlation . the statistical tests were conducted using stata / ic ( version 11.2 ; statacorp lp , college station , tx , usa ) , and a p value < 0.05 was deemed to indicate statistical significance . of the 190 eyes under study , 80 were diagnosed with glaucoma , 70 were glaucoma suspect , and 40 were without glaucoma . the average 3d stereographic ddls scores for the normal , glaucoma suspect , and glaucoma groups were 0.58 0.50 , 2.03 0.66 , and 4.23 1.23 , respectively . the average ddls scores obtained by the glaucoma specialist for each group were 1.47 0.56 ( normal ) , 2.39 0.78 ( glaucoma suspect ) , and 4.91 1.42 ( glaucoma ) . refraction error and disc diameter did not differ among the patient groups ( one - way anova ) . the mean deviation of visual field test was 2.35 3.28 for the normal , 2.47 2.52 for the glaucoma suspect , and 8.57 8.78 for the glaucoma groups . the weighted kappa value for interobserver agreement on the ddls between the 3d optic disc photography and glaucoma specialist assessments was 0.59 0.03 . the interobserver agreement between the glaucoma specialist and the residents was analyzed according to each of the 12 possible pairs ( glaucoma specialist ( a ) versus each resident [ b , c , d , e , f , g , h , i , j , k , l , and m , ] ; figure 1(a ) ) before ( white circle ) and after ( black circle ) the ddls training module . the weighted kappa values for interobserver agreement were higher after the training module in all pairs ( figure 1(a ) ) . although the variation in weighted kappa values among the pairs was substantial , the increase in interobserver agreement was higher in the junior than in the senior residents . figure 1(b ) shows the ddls concordance among residents before ( white circles ) and after ( black circles ) the training module . concordance was defined as the percentage of agreement among the 12 residents on the ddls score for each eye compared with the gold standard value ( the glaucoma specialist 's ddls score ) . for example , 50 ( 6/12 ) percent concordance was achieved when 6 of the 12 residents were in complete agreement with the ddls evaluation of the glaucoma specialist . concordance values after the training program showed a right shift toward improved interobserver concordance . the x - axis shows ddls stages ( 07 ) and the y - axis shows the percentage concordance among residents . the degree of concordance was the lowest on stages 5 and 6 , whereas good concordance was observed on stages 04 . the mean values for the ophthalmoscopic examination , 3d optic disc photography , and cirrus oct according to diagnostic group are shown in table 2 . the average vertical c / d ratios of the normal , glaucoma suspect , and glaucoma groups were 0.48 0.11 , 0.57 0.03 , and 0.64 0.11 , respectively . the average c / d area ratios ( obtained by 3d optic disc photography ) of the normal , glaucoma suspect , and glaucoma groups were 0.25 0.10 , 0.33 0.65 , and 0.42 0.12 , respectively . the anova revealed statistically significant differences among all parameters obtained by 3d optic disc photography with the exception of the height variation contour ( p = 0.234 ) . the post hoc analysis of the intervariable mean differences between the normal and glaucoma suspect groups revealed that all variables were significantly different , with the exception of the superior rim width ( p = 0.182 ) , inferior rim width ( p = 0.361 ) , and height variation contour ( p = 0.281 ) . furthermore , the post hoc analysis of the intervariable mean differences between the glaucoma suspect and glaucoma groups revealed significant differences for all variables with the exception of the disc area ( p = 0.657 ) , cup volume ( p = 0.285 ) , disc volume ( p = 0.352 ) , mean cup depth ( p = 0.486 ) , maximum cup depth ( p = 0.326 ) , and height variation contour ( p = 0.994 ) . the vertical c / d ratio , cup area , c / d area ratio , and cup volume obtained by 3d optic disc photography were positively correlated with the ddls score of the glaucoma specialist ( pearson 's correlation , table 3 ) . among the parameters measured by 3d optic disc photography , the vertical c / d ratio was the most highly correlated with the ddls ( r = 0.623 , p < 0.001 ) . the superior rim width , inferior rim width , rim area , rim disc area ratio , and rim volume were negatively correlated with the ddls ( p < 0.001 ) , and disc area , disc volume , mean cup depth , maximum cup depth , and height variation were not correlated with the ddls . all of the parameters acquired by cirrus oct were significantly correlated with the ddls with the exception of disc area . the area under the receiver operating characteristic ( roc ) curve was calculated to assess diagnostic probability ( table 4 , figure 2 ) . when the glaucoma group was compared with the combined glaucoma suspect and normal groups , the ddls of the glaucoma specialist had the best predictive power ( 0.941 ) , followed by the ddls obtained by 3d optic disc photography ( 0.931 ) . the predictive powers of the vertical c / d ratio and c / d area ratio ( obtained by 3d optic disc photography ) were 0.842 and 0.832 , respectively , revealing the overall high predictability of these parameters . the high predictive power of the glaucoma specialist and 3d optic disc photography ddls evaluations remained when the glaucoma suspect group was removed and the glaucoma and the normal groups were compared and when the combined glaucoma suspect and glaucoma groups were compared with the normal group ( table 4 ) . rim area measured by 3d optic disc photography and oct was negatively correlated with ddls ; thus , the area under the curve was calculated from 1 minus the original value , resulting in values of 0.805 and 0.871 , respectively . of the cirrus oct parameters , the rim area had the highest predictability ( 0.871 ) followed by average rnfl thickness ( 0.832 ) . our findings demonstrate that the interobserver agreement on the ddls is acceptable among trainees and specialists alike , suggesting that the grading system is a reliable indicator of the morphologic characteristics of the optic nerve head . furthermore , ddls measured automatically by 3d optic disc photography was reliable and showed moderate agreement ( weighted kappa value , 0.59 ) with that of the glaucoma specialist . the automatic calculation of ddls together with various parameters obtained by 3d optic disc photography showed good predictability in the diagnosis of glaucoma . moreover , we incorporated a supervised teaching module into our training program to increase the interobserver agreement on the ddls among residents . our results showed that the training module increased the interobserver agreement kappa statistic , and the effect was greater in the junior ( pgy 1 or 2 ) than in the senior ( pgy 3 or 4 ) residents . we found that agreement among examiners was the poorest on ddls stages 5 and 6 , whereas the concordance was acceptable on stages 04 suggesting that caution is required when evaluating the thinnest point of the optic disc rim when using this grading system . moreover , as the optic disc size must be measured before using the ddls , care should be taken to measure the optic disc size as accurately as possible . a previous study found that reliable interobserver agreement on the ddls was greater than that for c / d ratio on 3d optic disc photographs . however , the sample size in that study was small , and the grading was performed by a glaucoma specialist whose evaluation was likely to have been highly reproducible . moreover , the previous study used a stand - mounted stereo viewer rather than a 3d display , which is more commonly used at present . our study was conducted in a clinical setting where the procedure was performed under slit - lamp biomicroscopy to provide more detailed information for the ddls . our findings suggest that , unlike c / d ratio , the ddls grading system takes the thickness of the neural rim and optic disc size into consideration , which may enhance the assessment of the glaucomatous change of the optic nerve head . c / d ratio assessment using stereographic tests or ophthalmoscopic examination relies heavily on the subjective judgment of the examiner and may limit sensitivity in the detection of microscopic or local changes in the optic disc . individual variations in the optic disc constitute a further challenge in differentiating between normal and glaucomatous optic discs . moreover , the conventional ophthalmoscopic method of measuring the c / d ratio can not differentiate between optic discs with the same c / d ratio but different neural rim thickness and symmetry . thus , optic disc evaluation using a systematic grading system such as the ddls increases objectivity and improves diagnostic accuracy for glaucoma . the 3d retinal camera is a 12-megapixel single - lens reflex camera with three modes : normal , small pupil ( non - mydriatic ) , and stereographic . in the stereographic mode , the picture is taken in the range of 34 degrees , and a 3d image can be captured in a single shot without changing the position of the camera . the 3d images can be visualized on a 2d monitor and both automatic and manual settings of optic nerve and cup boundaries are possible . to our knowledge , no previous study has evaluated the performance of the automated ddls calculation using the camera itself . our findings indicate that the automated ddls calculation showed good agreement with the glaucoma specialist and has excellent predictability for the diagnosis of glaucoma . furthermore , vertical c / d ratio and rim disc area ratio measured by 3d optic disc photography showed good predictability for the diagnosis of glaucoma . bayer et al . found a strong correlation between the ddls and glaucomatous visual field damage . abdul majid et al . reported that the ddls was a useful index for the diagnosis of glaucoma and was highly correlated with indices measured by the visual field test , c / d ratio , and oct . in a study comparing the optic disc parameters of 3d optic disc photography and heidelberg retina tomography , januschowski et al our study extends previous findings by demonstrating that the indices obtained by 3d optic disc photography were significantly correlated with the ddls evaluation by a glaucoma specialist ; thus , providing further support to the usefulness of stereometric parameters in the diagnosis of glaucoma . choi and lee found that 2d optic disc photography relied on the course of the small vessels and shadowing of adjacent structures to measure cup depth and , as such , is prone to subjective findings . however , 3d optic disc photography is relatively free of subjective error because it enables direct visualization of depth . reported that the diagnostic accuracy of the quantitative optic disc analysis was considerably higher in 3d optic disc photography than in 2d photography . the software package used in our study automatically calculated the cup length , depth , and ratio , the results of which were more accurate than the bare eye alone . we found that the area under the roc curve for vertical c / d ratio ( 0.842 ) , rim disc area ratio ( 0.833 ) , and c / d area ratio ( 0.832 ) obtained by the 3d optic disc photography were high . moreover , the ddls calculated by 3d optic disc photography had excellent diagnostic predictability for glaucoma which was almost equal to that of the glaucoma specialist . a noteworthy finding was that the weighted kappa values of all residents , regardless of pgy , improved after attending a training module . although there were poor performers in both junior and senior groups , the positive effect of the training program was apparent regardless of level of residency training , suggesting that mandatory attendance in the teaching module would likely improve the reliability and interobserver agreement among ophthalmologists . we found that before attending the teaching module , several of the residents did not measure the size of the optic disc before describing the ddls . moreover , when the optic disc size was measured , some residents did not multiply it by the corrective factor , which is necessary when using a 60 or 90 diopter lenses . however , our findings indicate that these issues can be partially overcome by participation in a structured training program . in our study , the ddls was scored by a glaucoma specialist and by residents at different levels of training , which is often the case in busy outpatient clinics faced with limited resources . moreover , the evaluation of time - dependent changes in the optic nerve head requires good interobserver agreement on the ddls . bearing this in mind , our study showed that a well - structured training program can increase the weighted kappa values for agreement on the ddls between residents and a glaucoma specialist . we did not initially plan to include a training module in our retrospective study ; however , while this study was ongoing , we speculated on the value of a structured training program to improve interobserver agreement on the ddls among residents . introduction of such a program was possible because the study was conducted at a tertiary referral center that provides comprehensive training programs for junior doctors . our findings highlight the importance of a supervised training program when assessing glaucoma patients using the ddls and demonstrate the validity of the various parameters measured using 3d optic disc photography for the diagnosis of glaucoma . the various parameters obtained from this camera showed good correlation with those of more commonly used instruments , such as oct . future studies are necessary to investigate intraobserver agreement on the ddls among glaucoma specialists , particularly for cases of stable or progressive glaucoma .

purpose . to compare stereometric parameters obtained by three - dimensional ( 3d ) optic disc photography and optical coherence tomography ( oct ) and assess interobserver agreement on the disc damage likelihood scale ( ddls ) . methods . this retrospective study included 190 eyes from 190 patients classified as normal , glaucoma suspect , or glaucomatous . residents at different levels of training completed the ddls for each patient before and after attending a training module . 3d optic disc photography and oct were performed on each eye , and correlations between the ddls and various parameters obtained by each device were calculated . results . we found moderate agreement ( weighted kappa value , 0.59 0.03 ) between ddls scores obtained by 3d optic disc photography and the glaucoma specialist . the weighted kappa values for agreement and interobserver concordance increased among residents after the training module . interobserver concordance was the poorest at ddls stages 5 and 6 . the ddls scored by the glaucoma specialist had the highest predictability value ( 0.941 ) . conclusions . the ddls obtained by 3d optic disc photography is a useful diagnostic tool for glaucoma . a supervised teaching program increased trainee interobserver agreement on the ddls . ddls stages 5 and 6 showed the poorest interobserver agreement , suggesting that caution is required when recording these stages .

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

glaucoma is a debilitating disease that causes blindness if left untreated ; thus , early diagnosis is crucial . in general , diagnostic tests for glaucoma include the measurement of intraocular pressure ( iop ) , the visual field test , and examination of the optic nerve head . the disc damage likelihood scale ( ddls ) incorporates the size of the disc and the radial width of the neuroretinal rim into the evaluation of the optic nerve head . the ddls has been reported to provide a more accurate assessment of optic disc damage than the conventional cup / disc ( c / d ) ratio measurement , and a high correlation has been found between the ddls and various indices obtained from heidelberg retina tomography and optical coherence tomography ( oct ) in patients with glaucoma [ 7 , 8 ] . nevertheless , interobserver variability is a potential limitation of ddls and training on the system is essential for low interobserver variability and high reproducibility . recently , a three - dimensional ( 3d ) stereographic camera ( kowa nonmyd wx 3d , kowa , tokyo , japan ) that provides an objective recording of the optic nerve head was developed . we evaluated the validity of the ddls option provided by this 3d stereographic camera and assessed the correlations between various stereometric optic disc parameters obtained by the 3d stereographic camera and optic disc cube parameters measured using oct . moreover , we provide the first report , to our knowledge , of interobserver agreement on the ddls using kappa statistics and evidence of the usefulness of a training module that includes a detailed introduction and clinical skills for measuring ddls . standard achromatic perimetry performed with a humphrey field analyzer ( zeiss humphrey systems , dublin , ca , usa ) using the 24 - 2 full - threshold test , iop measurement , dilated slit - lamp biomicroscopy , 3d optic disc photography ( kowa nonmyd wx 3d , kowa , tokyo , japan ) , optic nerve head , and retinal nerve fiber layer ( rnfl ) analysis using cirrus oct ( carl zeiss meditec inc . , dublin , ca , usa ) were performed during the same visit for each patient between march 2011 and may 2013 . following pupil dilation using tropicamide 0.5% and phenylephrine 5% , a glaucoma specialist ( k.d.k ) calculated the vertical and horizontal c / d ratios ( uncorrected for optic disc size ) using direct ophthalmoscope ( welch allyn , skaneateles falls , ny , usa ) . the optic cup was defined on the basis of contour and the course of the small blood vessels on the disc and not on the basis of pallor . the optic disc border was defined as the inner border of the peripapillary scleral ring . the optic nerve head was viewed using prism glasses , and the c / d ratio was plotted manually to determine the contour line of each structure . all oct scans were acquired with a cirrus hdoct ( version 3.0.0.64 ) using the optic disc cube 200 200 protocol , which is designed to position the cube scan on the optic nerve head and is primarily used for glaucoma analysis . only scans of good quality ( signal strength better than 6 , without rnfl discontinuity or misalignment , involuntary saccade , or blinking artifacts , and absence of rnfl algorithm segmentation failure without misalignment or movement artefacts ) were included in the analysis . the 190 patients were divided into three diagnostic groups : normal , glaucoma suspect , and glaucoma . the criteria for the normal group were ( 1 ) healthy subjects with no history or presence of glaucoma , no retinal pathological findings , and no intraocular surgery including laser therapy ; ( 2 ) iop 21 mm hg on each visit ; ( 3 ) normal humphrey 24 - 2 visual field test ; ( 4 ) best corrected visual acuity of 20/40 or better with refractive error between + 1.00 and 3.00 diopters ; ( 5 ) open angles by gonioscopy ; and ( 6 ) normal - appearing optic nerve head . the criteria for glaucoma suspect eyes were ( 1 ) no history or presence of retinal pathology and no intraocular surgery including laser therapy ; ( 2 ) iop between 22 and 30 mmhg ; and ( 3 ) normal visual field test . patients who had the following findings were also categorized as glaucoma suspect , ( a ) asymmetric optic nerve head cupping ( difference in vertical c / d ratio 0.2 between the eyes in the presence of a similar optic disc size ) or ( b ) increased cupping ( vertical c / d ratio > 0.6 ) . the diagnostic criteria for glaucoma were ( 1 ) an abnormal visual field defined as the presence of at least two of the following . ( 2 ) one or more papillary sign as follows : ( a ) presence of a localized loss or thinning of the neuroretinal rim , ( b ) optic disk excavation , ( c ) vertical or horizontal c / d ratio > 0.6 , and ( d ) c / d asymmetry between the two eyes 0.2 . residents at different levels of training evaluated the eyes of the patients using the ddls . the residents were asked to evaluate each patient using the ddls with no prior training and only the ddls index as a reference . six residents evaluated patients on their initial visit to the clinic and the remaining six evaluated patients on their second visit ( usually 3 months after the initial visit ) . a glaucoma specialist ( k.d.k ) used the ddls to evaluate the patients on their initial visit . the lecture consisted of an introduction to the system , instructions on its use , and a discussion of difficulties involved in scoring ( such as multiplying the size of the disc by the corresponding corrective factors when using 60 or 90 diopter lenses ) . following the training program , the residents were asked to reevaluate the ddls of the participating subjects . at this point , the residents were not aware of their previous ddls scores . into one stage , 0 the level of interobserver agreement was measured using the weighted kappa statistic because it is an appropriate chance - adjusted measure of agreement between two observers when there are more than two ordered categories of classification . an analysis of variance ( anova ) was used to evaluate the differences among the glaucoma , glaucoma suspect , and normal groups on the various parameters , and the post hoc analysis was conducted using scheffe 's test . the correlations between the ddls value and the cirrus oct and 3d optic disc photography measurements were assessed using pearson 's coefficient of correlation . of the 190 eyes under study , 80 were diagnosed with glaucoma , 70 were glaucoma suspect , and 40 were without glaucoma . the average 3d stereographic ddls scores for the normal , glaucoma suspect , and glaucoma groups were 0.58 0.50 , 2.03 0.66 , and 4.23 1.23 , respectively . the average ddls scores obtained by the glaucoma specialist for each group were 1.47 0.56 ( normal ) , 2.39 0.78 ( glaucoma suspect ) , and 4.91 1.42 ( glaucoma ) . the mean deviation of visual field test was 2.35 3.28 for the normal , 2.47 2.52 for the glaucoma suspect , and 8.57 8.78 for the glaucoma groups . the weighted kappa value for interobserver agreement on the ddls between the 3d optic disc photography and glaucoma specialist assessments was 0.59 0.03 . the interobserver agreement between the glaucoma specialist and the residents was analyzed according to each of the 12 possible pairs ( glaucoma specialist ( a ) versus each resident [ b , c , d , e , f , g , h , i , j , k , l , and m , ] ; figure 1(a ) ) before ( white circle ) and after ( black circle ) the ddls training module . the weighted kappa values for interobserver agreement were higher after the training module in all pairs ( figure 1(a ) ) . although the variation in weighted kappa values among the pairs was substantial , the increase in interobserver agreement was higher in the junior than in the senior residents . figure 1(b ) shows the ddls concordance among residents before ( white circles ) and after ( black circles ) the training module . concordance was defined as the percentage of agreement among the 12 residents on the ddls score for each eye compared with the gold standard value ( the glaucoma specialist 's ddls score ) . for example , 50 ( 6/12 ) percent concordance was achieved when 6 of the 12 residents were in complete agreement with the ddls evaluation of the glaucoma specialist . concordance values after the training program showed a right shift toward improved interobserver concordance . the x - axis shows ddls stages ( 07 ) and the y - axis shows the percentage concordance among residents . the degree of concordance was the lowest on stages 5 and 6 , whereas good concordance was observed on stages 04 . the mean values for the ophthalmoscopic examination , 3d optic disc photography , and cirrus oct according to diagnostic group are shown in table 2 . the average vertical c / d ratios of the normal , glaucoma suspect , and glaucoma groups were 0.48 0.11 , 0.57 0.03 , and 0.64 0.11 , respectively . the average c / d area ratios ( obtained by 3d optic disc photography ) of the normal , glaucoma suspect , and glaucoma groups were 0.25 0.10 , 0.33 0.65 , and 0.42 0.12 , respectively . the anova revealed statistically significant differences among all parameters obtained by 3d optic disc photography with the exception of the height variation contour ( p = 0.234 ) . the post hoc analysis of the intervariable mean differences between the normal and glaucoma suspect groups revealed that all variables were significantly different , with the exception of the superior rim width ( p = 0.182 ) , inferior rim width ( p = 0.361 ) , and height variation contour ( p = 0.281 ) . furthermore , the post hoc analysis of the intervariable mean differences between the glaucoma suspect and glaucoma groups revealed significant differences for all variables with the exception of the disc area ( p = 0.657 ) , cup volume ( p = 0.285 ) , disc volume ( p = 0.352 ) , mean cup depth ( p = 0.486 ) , maximum cup depth ( p = 0.326 ) , and height variation contour ( p = 0.994 ) . the vertical c / d ratio , cup area , c / d area ratio , and cup volume obtained by 3d optic disc photography were positively correlated with the ddls score of the glaucoma specialist ( pearson 's correlation , table 3 ) . among the parameters measured by 3d optic disc photography , the vertical c / d ratio was the most highly correlated with the ddls ( r = 0.623 , p < 0.001 ) . the superior rim width , inferior rim width , rim area , rim disc area ratio , and rim volume were negatively correlated with the ddls ( p < 0.001 ) , and disc area , disc volume , mean cup depth , maximum cup depth , and height variation were not correlated with the ddls . all of the parameters acquired by cirrus oct were significantly correlated with the ddls with the exception of disc area . when the glaucoma group was compared with the combined glaucoma suspect and normal groups , the ddls of the glaucoma specialist had the best predictive power ( 0.941 ) , followed by the ddls obtained by 3d optic disc photography ( 0.931 ) . the predictive powers of the vertical c / d ratio and c / d area ratio ( obtained by 3d optic disc photography ) were 0.842 and 0.832 , respectively , revealing the overall high predictability of these parameters . the high predictive power of the glaucoma specialist and 3d optic disc photography ddls evaluations remained when the glaucoma suspect group was removed and the glaucoma and the normal groups were compared and when the combined glaucoma suspect and glaucoma groups were compared with the normal group ( table 4 ) . rim area measured by 3d optic disc photography and oct was negatively correlated with ddls ; thus , the area under the curve was calculated from 1 minus the original value , resulting in values of 0.805 and 0.871 , respectively . of the cirrus oct parameters , the rim area had the highest predictability ( 0.871 ) followed by average rnfl thickness ( 0.832 ) . our findings demonstrate that the interobserver agreement on the ddls is acceptable among trainees and specialists alike , suggesting that the grading system is a reliable indicator of the morphologic characteristics of the optic nerve head . furthermore , ddls measured automatically by 3d optic disc photography was reliable and showed moderate agreement ( weighted kappa value , 0.59 ) with that of the glaucoma specialist . the automatic calculation of ddls together with various parameters obtained by 3d optic disc photography showed good predictability in the diagnosis of glaucoma . moreover , we incorporated a supervised teaching module into our training program to increase the interobserver agreement on the ddls among residents . our results showed that the training module increased the interobserver agreement kappa statistic , and the effect was greater in the junior ( pgy 1 or 2 ) than in the senior ( pgy 3 or 4 ) residents . we found that agreement among examiners was the poorest on ddls stages 5 and 6 , whereas the concordance was acceptable on stages 04 suggesting that caution is required when evaluating the thinnest point of the optic disc rim when using this grading system . moreover , as the optic disc size must be measured before using the ddls , care should be taken to measure the optic disc size as accurately as possible . a previous study found that reliable interobserver agreement on the ddls was greater than that for c / d ratio on 3d optic disc photographs . however , the sample size in that study was small , and the grading was performed by a glaucoma specialist whose evaluation was likely to have been highly reproducible . our findings suggest that , unlike c / d ratio , the ddls grading system takes the thickness of the neural rim and optic disc size into consideration , which may enhance the assessment of the glaucomatous change of the optic nerve head . c / d ratio assessment using stereographic tests or ophthalmoscopic examination relies heavily on the subjective judgment of the examiner and may limit sensitivity in the detection of microscopic or local changes in the optic disc . thus , optic disc evaluation using a systematic grading system such as the ddls increases objectivity and improves diagnostic accuracy for glaucoma . the 3d retinal camera is a 12-megapixel single - lens reflex camera with three modes : normal , small pupil ( non - mydriatic ) , and stereographic . in the stereographic mode , the picture is taken in the range of 34 degrees , and a 3d image can be captured in a single shot without changing the position of the camera . our findings indicate that the automated ddls calculation showed good agreement with the glaucoma specialist and has excellent predictability for the diagnosis of glaucoma . furthermore , vertical c / d ratio and rim disc area ratio measured by 3d optic disc photography showed good predictability for the diagnosis of glaucoma . found a strong correlation between the ddls and glaucomatous visual field damage . reported that the ddls was a useful index for the diagnosis of glaucoma and was highly correlated with indices measured by the visual field test , c / d ratio , and oct . in a study comparing the optic disc parameters of 3d optic disc photography and heidelberg retina tomography , januschowski et al our study extends previous findings by demonstrating that the indices obtained by 3d optic disc photography were significantly correlated with the ddls evaluation by a glaucoma specialist ; thus , providing further support to the usefulness of stereometric parameters in the diagnosis of glaucoma . choi and lee found that 2d optic disc photography relied on the course of the small vessels and shadowing of adjacent structures to measure cup depth and , as such , is prone to subjective findings . however , 3d optic disc photography is relatively free of subjective error because it enables direct visualization of depth . reported that the diagnostic accuracy of the quantitative optic disc analysis was considerably higher in 3d optic disc photography than in 2d photography . the software package used in our study automatically calculated the cup length , depth , and ratio , the results of which were more accurate than the bare eye alone . we found that the area under the roc curve for vertical c / d ratio ( 0.842 ) , rim disc area ratio ( 0.833 ) , and c / d area ratio ( 0.832 ) obtained by the 3d optic disc photography were high . moreover , the ddls calculated by 3d optic disc photography had excellent diagnostic predictability for glaucoma which was almost equal to that of the glaucoma specialist . a noteworthy finding was that the weighted kappa values of all residents , regardless of pgy , improved after attending a training module . although there were poor performers in both junior and senior groups , the positive effect of the training program was apparent regardless of level of residency training , suggesting that mandatory attendance in the teaching module would likely improve the reliability and interobserver agreement among ophthalmologists . we found that before attending the teaching module , several of the residents did not measure the size of the optic disc before describing the ddls . moreover , when the optic disc size was measured , some residents did not multiply it by the corrective factor , which is necessary when using a 60 or 90 diopter lenses . in our study , the ddls was scored by a glaucoma specialist and by residents at different levels of training , which is often the case in busy outpatient clinics faced with limited resources . moreover , the evaluation of time - dependent changes in the optic nerve head requires good interobserver agreement on the ddls . bearing this in mind , our study showed that a well - structured training program can increase the weighted kappa values for agreement on the ddls between residents and a glaucoma specialist . we did not initially plan to include a training module in our retrospective study ; however , while this study was ongoing , we speculated on the value of a structured training program to improve interobserver agreement on the ddls among residents . our findings highlight the importance of a supervised training program when assessing glaucoma patients using the ddls and demonstrate the validity of the various parameters measured using 3d optic disc photography for the diagnosis of glaucoma . the various parameters obtained from this camera showed good correlation with those of more commonly used instruments , such as oct . future studies are necessary to investigate intraobserver agreement on the ddls among glaucoma specialists , particularly for cases of stable or progressive glaucoma .

worldwide , there have been significant changes in life history traits of fish stocks that are consistent with a response to fishing pressure ( jrgensen et al . however , in most cases it remains inconclusive whether these changes reflect an evolutionary response to fishing mortality or merely phenotypic plasticity ( marshall and browman 2007 ; kuparinen and meril 2008 ) . evidence in support of contemporary fisheries - induced evolution has accumulated from both experimental ( reviewed by conover and baumann 2009 ) and retrospective studies ( walsh et al . the latter include investigations of long - term trends in empirical field data ( edeline et al . 2007 ; kendall et al . 2009 ) , which have most frequently utilized observations on the age and size at which individuals mature ( jrgensen et al . 2007 ) . the analysis of such data has been facilitated by the development of the probabilistic maturation reaction norm ( pmrn ) approach ( heino et al . 2004 ) , a statistical tool developed to help disentangle genetic variation in maturation from phenotypic plasticity , resulting from variation in growth and survival ( heino and dieckmann 2008 ) . indeed , the major difficulty faced by investigations of fisheries - induced evolution in natural populations is that genetic changes and phenotypic plasticity are confounded in the phenotypic trait upon which most previous studies have had to rely . the need for a direct genetic approach has been noted ( kuparinen and meril 2007 ; allendorf et al . 2008 ) , but unambiguous genetic evidence of contemporary evolution in marine fish populations has proven elusive because of the limited availability of adaptive variation estimates , as well as historical molecular data sets ( conover et al . population genetic analyses of historical samples can provide invaluable insights into human - induced changes in the genetic composition of fish populations ( nielsen and hansen 2008 ; hansen et al . , attention has thus been drawn to archived collections of otoliths and scales as a source of dna for long - term temporal genetic studies of natural fish populations ( nielsen and hansen 2008 ; palstra et al . although genetic studies have commonly focused on neutral variation , recent approaches have shown that non - neutral or functional markers ( loci subject to selection ) are more likely than neutral markers to reveal processes leading to changes in allele frequencies over short temporal scales ( conover et al . 2006 ) . indeed , because of the large effective population sizes of exploited marine fishes , neutral markers are unlikely to exhibit changes in a signal over a relatively short time period , while loci under selection might readily respond to changes in selection regime . like most cod ( gadus morhua ) stocks in the north atlantic , icelandic cod has experienced a drastic reduction in abundance and spawning stock biomass ( ssb ) during the last few decades . ssb has declined from about 1 million tonnes in the early 1950s to < 200 000 tonnes at the beginning of this century ( fig . , estimates of ssb have remained below the long - term average , rarely exceeding 200 000 tonnes . concurrently , there has been a long - term increase in fishing mortality ( f ) , with exploitation rates exceeding 0.8 during three different time periods ( fig . 1 ) . these levels of f are considerably higher than the target of 0.30.4 , which would enable a sustainable fishery that is within biologically safe limits ( marine research institute 2007 ) . estimated total spawning stock biomass ( histogram bars ) and average fishing mortality of age groups 510 ( line ) of cod in icelandic waters during the period 19482002 ( marine research institute 2009 ) . prior to world war ii ( wwii ) , both international trawlers and the icelandic fleet focused their fishing effort on the relatively shallow inshore areas around iceland ( r 2005 ) , with landings of cod reaching a maximum of just short of 52 000 tonnes in 1933 ( marine research institute 2009 ) . during the war , the international fleet was absent from icelandic waters and therefore fishing pressure was considerably lower . since wwii there have been substantial spatial and seasonal changes in the exploitation pattern of icelandic cod . immediately after the war , the inshore cod fishery increased again because of the continued exploitation by the national fleet and return of international vessels , resulting in a peak catch of 548 000 tonnes in 1954 ( marine research institute 2009 ) . in 1952 , icelandic authorities banned the use of bottom trawls and danish seine within four nautical miles of the shoreline . this regulation was followed by a gradual extension of the national fishery jurisdiction , culminating at 200 nautical miles in 1976 ( ministry of fisheries 2004 ; r 2005 ) . this , combined with the stricter inshore fishing regulations , improvements in fishing technology , and the introduction of larger vessels , resulted in the redirection of fishing effort into deeper offshore waters . concurrently , trawlers were able to follow the seasonal migration of cod ( jnsson 1996 ) , rather than being restricted to inshore breeding grounds during the spawning season . alongside the declines in stock abundance , there have been notable alterations in the life history of icelandic cod , whereby far fewer fish now survive beyond 7 years of age , resulting in severely truncated age and size distributions ( schopka 1994 ; marteinsdttir and thorarinsson 1998 ) . furthermore , icelandic cod now reach maturity at younger ages and smaller sizes ( marteinsdttir and begg 2002 ) . a recent study , which estimated pmrns for 36 cohorts of icelandic cod , found evidence that a shift towards maturation at smaller sizes and younger ages has occurred independently of changes in growth , condition and temperature ( pardoe et al . this change in the maturation schedule of icelandic cod , along with the loss of older and larger repeat spawners , raises concerns for the stock 's reproductive potential , and consequently its ability to withstand harvesting and future environmental change ( law 2007 ; rnason et al . an investigation into whether the fishery has also had a long - term effect on genetic variation in the icelandic cod stock is thus imperative . in this study , we used archived otoliths , collected between 1948 and 2002 , to jointly examine phenotypic and genetic data . our goal was to examine historical trends in the genetic composition of the icelandic cod stock using both selected and neutral markers . temporal stability in the allele frequency distribution of neutral markers would reflect stability in population structure and minimal drift , as is expected when effective population sizes are large . when such stability at neutral markers is accompanied by temporal changes in allele frequency distributions at functional loci then one can infer the population has been subjected to changes in selection regimes . we examined polymorphism at the pantophysin ( pan i ) locus ( pogson et al . 1995 ; pogson 2001 ) , a genetic marker known to be under selection or closely linked to a gene under selection , as well as a suite of six microsatellite loci assumed to be neutral . the two main allele classes within the pan i locus , pan i and pan i , are differentiated by a dra i restriction site ( pogson 2001 ) . the distribution of this diallelic system has been the focus of numerous population genetics studies in cod ( pampoulie et al . we found clear evidence that major changes in genetic variation at the pan i locus of icelandic cod have occurred alongside those in fishing patterns and age composition of the stock , whereas no temporal changes were detected with neutral genetic markers . these results strongly demonstrate the importance of long - term genetic data , and more specifically the use of both functional and neutral genes , when studying anthropogenic disturbances in a highly dynamic system such as that characterizing the icelandic cod stock . a total of 16 samples , comprising 1471 spawning or near - spawning individuals , were collected from three relatively shallow locations known to be major spawning grounds for icelandic cod ( south west = sw , faxafli bay = fax and the west coast bay , breidafjrdur = bre ; see table 1 for exact details on location ) . otoliths collected during the period 19481996 were recovered from the archived collections at the icelandic marine research institute ( mri ) . otoliths collected prior to 2000 were stored in dry paper envelopes , while the more recent historical samples ( collected in 2000 ) were stored in specially designed plastic storage wells . a contemporary sample ( 2002 ) the choice of sample years was based on the availability of individuals ( sample size ) stratified by years of known high and low stock size . biological information , i.e. total length , age , sex and maturity status , for all individuals used in the genetic analyses was retrieved directly from the mri database . individuals were pooled into 10-year cohort classes based on their year of birth ( 19311940 through to 19912000 ) . overview of icelandic cod samples used in this study : year , date and subarea of sampling ( see note for corresponding coordinates ) , sampling depth ( meters ; entries with asterisks show depth distributions [ 1090% quantiles ] within the subarea for those samples where actual depth of the catch was not available ) , number of individuals ( n ) , amplification success ( asucc ) , proportion of females ( pfem ) , mean total length ( cm ; tlmean ) , length range ( cm ; tlrange ) , mean age ( years ; amean ) , age range ( years ; arange ) . for all samples , dna was derived from archived otoliths with the exception of 2002 for which dna was obtained from fresh gills note : exact coordinates and mean depth of subareas are as follows : subarea south west ( sw ) : 63.564n , 2021w ; subarea faxafli bay ( fax ) : 6464.5n , 2223w ; subarea west coast bay , breiafjrur ( bre ) : 6565.5n , 2324w . dna was extracted from the otolith surface using a chelex / proteinase k protocol ( estoup et al . the dna was preserved at 4c during this study and subsequently moved to a 80c freezer for long - term storage . primers specially designed to amplify a short fragment ( 142 bp ) of the pan i locus from dna that has experienced considerable degradation ( nielsen et al . 2007 ; forward : 5-ggcaaatgaaacccagaaaa , rev : 5-atgacacttgtggcaagcag ) were used for the polymerase chain reaction ( pcr ) . pcr was performed in a 17 l volume containing 3 l of dna product , 1.7 l 10 buffer , 0.51 l of 1.5 mm mgcl2 , 1.7 l of 2.5 mm dntp , 0.25 l of each 10 m primer solution , 0.25 l of 20 mg / ml bsa ( fermentas ) and 0.5 units of dynazyme polymerase ( finnzymes oy , espoo , finland ) . touchdown procedures as follows : initial denaturation step of 2 min at 95c followed by 10 cycles of 30 s at 94c , 45 s of annealing temperature that decreases in each cycle by 0.5c until 55c was reached and 30 s of 70c . this was followed by 25 cycles of 30 s at 94c , 50 s at 55c and 30 s at 70c . a final elongation step of 5 min at 72c was performed . immediately after the pcr , a restriction analysis was carried out on the pcr product ( 17 l ) using 18 units of the enzyme drai ( fermentas ) and accompanying buffer ( 2 l ) . drai cuts at the diagnostic restriction sites in the pcr product , discriminating between the pan i and the pan i alleles . as expected , the digestion pattern was similar to that described by nielsen et al . ( 2007 ) : a single band at 142 bp for the pan i homozygote , two bands of 40 and 102 for the pan i homozygote and all three bands for the heterozygotes . we also analysed genetic variation at six microsatellite loci assumed to be neutral using a subset of those individuals from the pan analysis : gmo2 ( brooker et al . pcr were performed in 10 l volumes containing 2 l of dna , 1 l of 10 buffer , 1 l of 2.5 mm dntp , 0.20.4 units of dynazyme dna polymerase ( finnzymes ) and 12 m of each primer . pcr were performed on a geneamp2700 thermal block using the touchdown procedure described earlier . pcr products were multiplexed and detected on an abi - automatic sequencer ( abi 377 ; applied biosystem ) using genescan 3.1.2 ( biosystems 2000 ) and scored using genemapper 3.7 ( biosystems 2004 ) . the occurrence of genotypic errors resulting from technical artefacts ( null alleles ) or dna quality ( large allele dropouts ) was assessed using the program micro - checker ( van oosterhout et al . samples were processed in a designated area that was decontaminated between procedures using a chlorine solution and uv light . negative controls were run , and comparisons to microsatellite readings were conducted to test for possible contaminations between neighbouring dna wells . changes in the age distribution between sampling years were tested using chi - square tests . in situations of low expected values ( < 5 ) , samples from cod aged 6 years or younger , and eleven years or older , were pooled together . the changes in pan i genotype frequencies between age groups and cohort classes were also tested using chi - square tests . for these tests , individuals aged 5 years or younger , and aged twelve years or older , were pooled , while all intermediate age classes were kept separate . to investigate the long - term consistency of genotypic change by age , we pooled the samples in three time periods . the earliest period consisted of samples collected in 1948 , 1957 , 1959 and 1966 when average fishing mortality is estimated to have been < 0.3 . the middle period , characterized by an average fishing mortality of 0.6 , contained samples from 1972 , 1973 , 1976 and 1979 . the latest period comprised samples collected in 1985 , 1996 , 2000 and 2002 and featured several peaks of very high fishing mortality ( > 0.8 ) . we used a multinomial log - linear model ( venables and ripley 2002 ) to explore changes in genotype frequencies with time . the purpose of the model was to describe the long - term changes in genotype frequencies of cohorts 19321999 , after taking into account that some cohorts were only sampled at a younger age and other cohorts only at an older age . in a single model , we fitted the frequencies of pan i genotypes pan i , pan i and pan i as a smooth function of both cohort and age . the smooth functions for cohort and age were natural cubic splines , each with 3 degrees of freedom . the multinomial model ensures that the predicted relative frequencies of the three pan i genotypes sum to one within each cohort - age stratum . potential differences in growth between the pan i genotypes were investigated by analysing length - at - age data , combined for all years . the analyses included cod aged 79 years only since those age classes contained sufficient individuals from all three pan i genotypes in all time periods . to test whether differences in growth of icelandic cod were related to their pan i genotype , mean length was modelled using analysis of covariance ( ancova ) with pan i genotype as the categorical independent variable and age as continuous covariate . for the suite of six microsatellite loci , observed ( ho ) and expected ( he ) heterozygosity were calculated in genetix 4.03 ( belkhir et al . tests for deviations from hardy weinberg equilibrium ( hwe ) were conducted for both the pan i locus and the suite of six microsatellite loci using the exact test in genepop 3.1 ( raymond and rousset 1995 ) . genetic differentiation between sampling sites and all pairs of populations was estimated with pairwise fst estimates following weir and cockerham ( 1984 ) , and 95% confidence intervals were determined by bootstrapping over loci . an analysis of molecular variance ( amova ) was carried out for both the pan i locus and the suite of six microsatellite loci in arlequin 3.0 ( excoffier et al . 2005 ) to assess hierarchical partitioning of genetic variance . the genetic relationships among samples were further analysed using principal component analysis and visualized with a multidimensional scaling ( mds ) plot based on a matrix of pairwise fsts . a total of 16 samples , comprising 1471 spawning or near - spawning individuals , were collected from three relatively shallow locations known to be major spawning grounds for icelandic cod ( south west = sw , faxafli bay = fax and the west coast bay , breidafjrdur = bre ; see table 1 for exact details on location ) . otoliths collected during the period 19481996 were recovered from the archived collections at the icelandic marine research institute ( mri ) . otoliths collected prior to 2000 were stored in dry paper envelopes , while the more recent historical samples ( collected in 2000 ) were stored in specially designed plastic storage wells . a contemporary sample ( 2002 ) the choice of sample years was based on the availability of individuals ( sample size ) stratified by years of known high and low stock size . biological information , i.e. total length , age , sex and maturity status , for all individuals used in the genetic analyses was retrieved directly from the mri database . individuals were pooled into 10-year cohort classes based on their year of birth ( 19311940 through to 19912000 ) . overview of icelandic cod samples used in this study : year , date and subarea of sampling ( see note for corresponding coordinates ) , sampling depth ( meters ; entries with asterisks show depth distributions [ 1090% quantiles ] within the subarea for those samples where actual depth of the catch was not available ) , number of individuals ( n ) , amplification success ( asucc ) , proportion of females ( pfem ) , mean total length ( cm ; tlmean ) , length range ( cm ; tlrange ) , mean age ( years ; amean ) , age range ( years ; arange ) . for all samples , dna was derived from archived otoliths with the exception of 2002 for which dna was obtained from fresh gills note : exact coordinates and mean depth of subareas are as follows : subarea south west ( sw ) : 63.564n , 2021w ; subarea faxafli bay ( fax ) : 6464.5n , 2223w ; subarea west coast bay , breiafjrur ( bre ) : 6565.5n , 2324w . dna was extracted from the otolith surface using a chelex / proteinase k protocol ( estoup et al . the dna was preserved at 4c during this study and subsequently moved to a 80c freezer for long - term storage . primers specially designed to amplify a short fragment ( 142 bp ) of the pan i locus from dna that has experienced considerable degradation ( nielsen et al . 2007 ; forward : 5-ggcaaatgaaacccagaaaa , rev : 5-atgacacttgtggcaagcag ) were used for the polymerase chain reaction ( pcr ) . pcr was performed in a 17 l volume containing 3 l of dna product , 1.7 l 10 buffer , 0.51 l of 1.5 mm mgcl2 , 1.7 l of 2.5 mm dntp , 0.25 l of each 10 m primer solution , 0.25 l of 20 mg / ml bsa ( fermentas ) and 0.5 units of dynazyme polymerase ( finnzymes oy , espoo , finland ) . touchdown procedures as follows : initial denaturation step of 2 min at 95c followed by 10 cycles of 30 s at 94c , 45 s of annealing temperature that decreases in each cycle by 0.5c until 55c was reached and 30 s of 70c . this was followed by 25 cycles of 30 s at 94c , 50 s at 55c and 30 s at 70c . a final elongation step of 5 min at 72c was performed . immediately after the pcr , a restriction analysis was carried out on the pcr product ( 17 l ) using 18 units of the enzyme drai ( fermentas ) and accompanying buffer ( 2 l ) . drai cuts at the diagnostic restriction sites in the pcr product , discriminating between the pan i and the pan i alleles . as expected , the digestion pattern was similar to that described by nielsen et al . ( 2007 ) : a single band at 142 bp for the pan i homozygote , two bands of 40 and 102 for the pan i homozygote and all three bands for the heterozygotes . we also analysed genetic variation at six microsatellite loci assumed to be neutral using a subset of those individuals from the pan analysis : gmo2 ( brooker et al . pcr were performed in 10 l volumes containing 2 l of dna , 1 l of 10 buffer , 1 l of 2.5 mm dntp , 0.20.4 units of dynazyme dna polymerase ( finnzymes ) and 12 m of each primer . pcr were performed on a geneamp2700 thermal block using the touchdown procedure described earlier . pcr products were multiplexed and detected on an abi - automatic sequencer ( abi 377 ; applied biosystem ) using genescan 3.1.2 ( biosystems 2000 ) and scored using genemapper 3.7 ( biosystems 2004 ) . the occurrence of genotypic errors resulting from technical artefacts ( null alleles ) or dna quality ( large allele dropouts ) was assessed using the program micro - checker ( van oosterhout et al . samples were processed in a designated area that was decontaminated between procedures using a chlorine solution and uv light . negative controls were run , and comparisons to microsatellite readings were conducted to test for possible contaminations between neighbouring dna wells . changes in the age distribution between sampling years were tested using chi - square tests . in situations of low expected values ( < 5 ) , samples from cod aged 6 years or younger , and eleven years or older , the changes in pan i genotype frequencies between age groups and cohort classes were also tested using chi - square tests . for these tests , individuals aged 5 years or younger , and aged twelve years or older , were pooled , while all intermediate age classes were kept separate . to investigate the long - term consistency of genotypic change by age , we pooled the samples in three time periods . the earliest period consisted of samples collected in 1948 , 1957 , 1959 and 1966 the middle period , characterized by an average fishing mortality of 0.6 , contained samples from 1972 , 1973 , 1976 and 1979 . the latest period comprised samples collected in 1985 , 1996 , 2000 and 2002 and featured several peaks of very high fishing mortality ( > 0.8 ) . we used a multinomial log - linear model ( venables and ripley 2002 ) to explore changes in genotype frequencies with time . the purpose of the model was to describe the long - term changes in genotype frequencies of cohorts 19321999 , after taking into account that some cohorts were only sampled at a younger age and other cohorts only at an older age . in a single model , we fitted the frequencies of pan i genotypes pan i , pan i and pan i as a smooth function of both cohort and age . the smooth functions for cohort and age were natural cubic splines , each with 3 degrees of freedom . the multinomial model ensures that the predicted relative frequencies of the three pan i genotypes sum to one within each cohort - age stratum . potential differences in growth between the pan i genotypes were investigated by analysing length - at - age data , combined for all years . the analyses included cod aged 79 years only since those age classes contained sufficient individuals from all three pan i genotypes in all time periods . to test whether differences in growth of icelandic cod were related to their pan i genotype , mean length was modelled using analysis of covariance ( ancova ) with pan i genotype as the categorical independent variable and age as continuous covariate . for the suite of six microsatellite loci , observed ( ho ) and expected ( he ) heterozygosity were calculated in genetix 4.03 ( belkhir et al . 1999 ) . tests for deviations from hardy weinberg equilibrium ( hwe ) were conducted for both the pan i locus and the suite of six microsatellite loci using the exact test in genepop 3.1 ( raymond and rousset 1995 ) . genetic differentiation between sampling sites and all pairs of populations was estimated with pairwise fst estimates following weir and cockerham ( 1984 ) , and 95% confidence intervals were determined by bootstrapping over loci . an analysis of molecular variance ( amova ) was carried out for both the pan i locus and the suite of six microsatellite loci in arlequin 3.0 ( excoffier et al . the genetic relationships among samples were further analysed using principal component analysis and visualized with a multidimensional scaling ( mds ) plot based on a matrix of pairwise fsts . a hierarchical amova revealed that overall variation at the pan i locus was because of temporal ( fsc = 0.122 , p < 0.001 ) rather than spatial variation ( fct = 0.025 , p = 0.92 ) ( table 2 ) . therefore , in the following analyses , samples were grouped into sample years ( or cohort classes ) independently of sampling location ( and depth ) . results from the analysis of genetic variation at the pan i locus are presented in table 3 . genetic diversity among sample years was highly variable , with observed heterozygosities ranging from 0.153 ( 2002 ) to 0.988 ( 1979 ) , with an average value of 0.617 . genetic diversity within cohort classes was relatively moderate , with observed heterozygosities ranging from 0.395 ( 19912000 cohort class ) to 0.761 ( 19511960 cohort class ) . significant deviations from hwe were found in 50% of the sample years , whereas five of seven cohort classes contained samples that were not in hwe . heterozygote excess was the cause of all of the deviations from hwe ( table 3 ) . hierarchical partitioning of genetic variance at the pan i locus , based on an analysis of molecular variance ( amova ) of samples of icelandic cod ( see table 1 ) observed ( ho ) and expected ( he ) heterozygosity and observed frequencies of pan i allele in icelandic cod samples ( n : sample size ) by cohort class and sample year . bold values indicate significant deviations of fis values from hardy weinberg expectations , after correction for multiple tests as has been previously observed with an independent data set ( marteinsdttir and thorarinsson 1998 ) , the age composition of icelandic cod changed markedly through the study period . the observed shift towards a narrower age distribution resulted from the loss of older age classes and an overall reduction in mean age ( = 851.0 , p < 0.001 ) moreover , our analyses revealed that the pan i locus has been subject to major changes in allele frequencies during the study period , in relation to both age and cohort class . within cohorts , the frequencies of the homozygous genotypes were age dependent ( figs 3 and 4 ) . the pan i genotype was more common among the older spawning cod , while the pan i genotype was most frequently observed among the younger age classes ( = 187 , p < 0.001 ; fig . 3 ) . in fact , no spawning cod younger than 6 years of age carried the pan i genotype . these age - dependent trends in homozygote frequency were still present when the data were split into three different time periods ( fig . a high proportion of heterozygote individuals were found in all age classes ( figs 3 and 4 ) . indeed , as stated earlier , a lack of hwe both within sample years and cohort classes was mainly because of heterozygote excess ( table 3 ) . box and whiskers plot of the age distribution of icelandic cod within each sampling year . age - specific distributions of pan i genotype frequencies ( white : pan i , grey : pan i , black : pan i ) in samples of icelandic cod collected between 1948 and 2002 . the corresponding sample size is listed above each column . observed age - specific frequencies of pan i genotypes ( pan i , pan i and pan i ) for icelandic cod sampled in three time periods : period 1 , 19481966 ; period 2 , 19721979 ; period 3 , 19842002 ( see table 1 for actual years sampled within each period ) . at the inter - cohort level , there was a gradual but strong decline in the frequency of pan i over the study period ( = 199.3 , p < 0.001 ) ( fig . , the pan i genotypes increased in frequency , exceeding 50% in the last 20 years of the study at the expense of the pan i and pan i individuals ( = 85.9 , p < 0.0010 ) ( fig . 5 ) . for those ages represented in our data throughout the study period , predicted genotype frequencies from a multinomial model showed similar temporal trends ( fig . according to the model ( likelihood ratio test versus null model = 276.4 , p < 0.001 ) , the gradual increase in pan i genotype frequency started in the 1960s and was accompanied by decreases in the relative proportions of the two other genotypes . observed pan i genotype frequencies ( white : pan i , grey : pan i , black : pan i ) for icelandic cod from different ( 10 year ) cohort classes . the corresponding sample size is listed above each column . predicted distributions of cohort - specific pan i genotype frequencies for icelandic cod aged 6 , 8 and 10 years , from a multinomial model ( smoothing allowing for three degrees of freedom ) . distinct differences were observed in length - at - age among the pan i genotypes , whereby icelandic cod carrying the pan i genotype had significantly higher length - at - age on average than cod of the pan i and pan i genotypes ( p = 0.048 ) ( fig . average length ( 1 se ) of icelandic cod aged 612 years , sampled in the period 19482002 , and carrying either pan i , pan i or pan i genotype . multilocus genotype information from six microsatellites was obtained from 892 of the individuals utilized in the pan i analyses , representing the same sampling years and sites ( sw , fax , bre ; table 1 ) . genetic diversity in the samples was moderate to high , with observed heterozygosities ranging from 0.594 ( tch22 ) to 0.951 ( tch14 ) . the numbers of alleles ranged from 5 ( tch22 ) to 35 ( tch14 ) . deviations from hwe were detected in 3 of 105 tests after bonferroni correction for multiple tests and were not because of any specific loci or sample . the microsatellite analysis revealed that there was no genetic differentiation ( fst ) among sampling sites ( fst = 0.0004 , p = 0.100 ) . of 105 pairwise ( fst ) comparisons among all samples , none were found to be significantly different from zero and there was an overall fst = 0.0018 ( p = 0.538 ) . this pattern was also evident in a hierarchical amova , which showed that only 0.03% of total genetic variance ( fct = 0.0003 ) could be attributed to variance among sampling locations . the variance between temporal samples was slightly higher ( 0.13% , fsc = 0.0013 ) . this lack of differentiation confirms long - term temporal stability under the assumption of neutrality and a mds analysis ( not shown ) further confirmed the lack of spatial or temporal pattern in the data . our study is the first , to our knowledge , to analyse functional and neutral genetic markers in combination with fisheries data , over a multidecadal time period , in an attempt to explain distinct changes in genetic and phenotypic variation in a fish stock that has been heavily exploited since the 1940s and 50s . by isolating dna from archived cod otoliths , gathered over a 60-year period at spawning sites on the sw coast of iceland , we were able to detect long - term temporal changes in genotype frequencies at the pantophysin ( pan i ) locus , whereas no changes were observed at six microsatellite loci . the significant decline in the pan i genotype , which represented 26% of the sampled population in the 1930s but only 5% in the 1990s , followed changes in the exploitation pattern , which were characterized by increased effort in deeper waters facilitated by larger ships and technological advancements in fishing gear . in the current study , the frequencies of pan i genotypes varied both within and between cohorts . within cohorts , the frequency of the pan i genotype increased with age ( figs 3 and 4 ) . currently , the age - specific distribution of the pan i genotypes is unknown for immature icelandic cod . however , this result strongly indicates that fish of different genotypes mature at different ages . the absence of the pan i genotype among spawning fish aged 35 years is therefore most likely due to cod carrying that genotype not having matured yet and thus not being present on the spawning sites , which provided the data for this study . the gradual increase in the frequency of the pan i genotype from 6 to 9 years of age is thus likely to reflect differences in the rate of maturation . the potential existence of life history variation between cod of different pan i genotypes was also supported by the observation that cod exhibiting the pan i genotype grew at a significantly higher rate than those carrying the pan i and pan i genotypes ( fig . 7 ) . furthermore , our findings complement emerging evidence from tagging studies using data storage tags of two groups of icelandic cod associated with foraging strategy and habitat selection during the feeding season ( plsson and thorsteinsson 2003 ; pampoulie et al . the so - called coastal cod inhabit shallow shelf waters characterized by seasonal temperature trends . in contrast , frontal cod undertake migrations to cold , deep waters ( > 250 m ) where they forage at thermal fronts , making frequent vertical migrations between temperature extremes ( < 0 and > 7c ) . ( 2008 ) revealed that individuals carrying the pan i genotype are most likely to be classified as coastal cod based on their behaviour during feeding migrations , while individuals carrying the pan i genotype most frequently exhibit the migratory behaviour of frontal cod . the heterozygotes were found to exhibit mixed feeding migration strategies . in the present study , we have shown a gradual decline in the frequency of the pan i genotype among spawning cod during the last 60 years . concurrently , the pan i genotype increased in frequency , exceeding 50% in the last 20 years of the study at the expense of the pan i and pan i individuals ( fig . 5 ) . we can not be certain of the primary driving force behind these changes in the proportions of the different genotypes . however , four different hypotheses , three of which can be linked to changes in exploitation patterns , warrant our scrutiny . first , changes in genotypic frequencies may have resulted from changes in stock composition because of the consistent removal of older fish during a period of increased fishing mortality ( marteinsdttir and thorarinsson 1998 ; fig . 2 in this study ) . by the end of the last century , relatively few cod survived to 10 years of age in icelandic waters ( marine research institute 2009 ) . accordingly , the mean age of spawning cod declined from 10 to 6 years during the study period ( table 1 , fig . as previously discussed , the oldest cod in our samples were predominantly of the pan i or pan i genotype , whereas relatively few cod carrying the pan i genotype were found to be aged 9 years or older . therefore , the truncation of the age distribution of icelandic cod , a common feature of commercially exploited fish stocks , is likely to have resulted in the removal of the pan i genotype from the spawning stock at a proportionally faster rate than that of the pan i genotype . secondly , the gradual decline in the frequency of the pan i genotype may also have resulted from the substantial spatial and seasonal changes in fishing effort on icelandic cod that have occurred since wwii . following the termination of fishing by danish seine and bottom trawl within four nautical miles of the coast , the fishery jurisdiction was gradually extended to 200 nautical miles ( see also the introduction ) . at the same time , the introduction of larger vessels resulted in the redirection of fishing effort into deeper offshore waters . this also enabled trawlers to follow the seasonal migration of adult cod , rather than being restricted to inshore breeding grounds during the spawning season ( jnsson 1996 ) . the bathymetric distribution of the pan i genotypes has been recorded for both spawning and migrating cod around iceland ( pampoulie et al . 2009 ) , while this information for juvenile cod is unfortunately scarce . spawning cod sampled in 20022003 in the sw of iceland , the area covered by this study , ranged from being predominantly of the pan i genotype at the shallowest stations to predominantly of the pan i and pan i genotypes at the deeper stations ( pampoulie et al . similar changes in genotypic frequency with depth , at stations all around iceland , were observed by rnason et al . therefore , changes in the exploitation pattern of icelandic cod during the study period are likely to have resulted in relatively greater fishing mortality in deeper water and on the feeding grounds and thus may have led to the disproportionate removal of the pan i genotype from the stock by the commercial fishery . thirdly , we may not be able to fully explain the causes of these long - term temporal changes in genotypic frequency in icelandic cod until our understanding of the population structure further improves . the distinct variation in life history traits , behaviour and pan i genotype frequencies of icelandic cod is persuasive evidence that the current stock designation does not capture the actual geographic scale of population differentiation ( pampoulie et al . accordingly , our microsatellite analysis did not indicate any spatial structure and the long - term stability of the population under the assumption of neutrality could not be rejected . however , the absence of heterogeneity using neutral genetic markers does not contradict the hypothesized existence of different life history components in our data . neutral markers are poorly suited to the detection of changes in genetic diversity because they segregate independently of the selected loci , and in populations with relatively large effective population sizes in particular the level of genetic drift will be low ( conover et al . 2006 ; allendorf and hard 2009 ) . indeed , the population components might experience differential postsettlement selection , likely influenced by environmental factors , but still exhibit a relatively high level of gene flow as a consequence of larval dispersal or mixing on breeding sites , or both . however , if there is some degree of gene flow restriction between the stock components , as a recent study suggests ( t. b. grabowski , b. mcadam , v. thorsteinsson and g. marteinsdttir , unpublished manuscript ) , high effective population sizes and recent separation history are two nonexclusive explanations that could mask underlying stock structure ( case et al . if the icelandic cod stock indeed comprises populations with limited gene flow and/or different selection schedules , then the changes in exploitation patterns described above will have affected the stock complex by removing the pan i genotype at a faster rate than the pan i genotype . furthermore , the different populations may also have been targeted by the fishery in an uneven manner . the overall effects are similar , i.e. they result in long - term changes in genotype frequencies . finally in our fourth hypothesis , we recognize that the genotype distribution at the pan i locus may have changed because of selection by other agents such as those of climatic origin . temperature is the only available long - term proxy for potential environmental changes during this time period . examination of temporal changes in sea surface temperature ( sst ) off the northern coast of iceland ( hanna et al . 2006 ) did not reveal any significant correlations with changes in frequency of the pan i genotypes ( p > 0.05 ) . this is perhaps not surprising as ocean conditions in this area have fluctuated extensively during the study period . between 1925 and 1964 , there was a high inflow of warm atlantic water onto the icelandic shelf resulting in relatively mild temperatures ( malmberg 1986 ; astthorsson et al . the climate shifted dramatically because of increased inflow of icy freshwater from the arctic ( jakobsson 1980 ; malmberg 1986 ; dickson et al . ice years , temperatures dropped far below the long - term average , reaching the lowest recordings in 19681969 ( astthorsson et al . conditions subsequently remained more stable , until the current situation of gradual warming , which started in 1996 ( astthorsson et al . these striking fluctuations in temperature and salinity of icelandic waters thus do not appear responsible for the gradual and consistent changes in the genotype frequencies observed in this study . other studies have also shown that there does not appear to be a direct link between pan i genotype frequencies in cod and water temperature ( nielsen et al . unfortunately , the quantitative investigation of this potential relationship for icelandic cod is limited by the quality and quantity of available environmental data . although the sst time series from northern icelandic waters ( hanna et al . 2006 ) is thought to be relatively representative of the average environmental conditions experienced by the stock in general , icelandic cod are confronted with highly fluctuating environmental conditions throughout their lifespan ( jakobsson and stefnsson 1998 ) ; variability that can probably only be reliably captured through data storage tags . furthermore , the life history stage(s ) at which temperature affects the genotypic distribution of the pan i locus of cod is unknown . in conclusion , although the observed changes in genotype frequencies at the pan i locus could be the result of simultaneous selection by both trends in temperature and fishing , the information that does exist indicates that although there have been persistent changes in environmental conditions in icelandic waters , those changes alone are insufficient to explain the strong selection against the pan i observed during the last 20 years of the study . a recent study has also attempted to explore the effects of exploitation on genetic diversity at the pan i locus in icelandic cod using samples from spawning areas around iceland over a 3-year period ( 20052007 ) ( rnason et al . the authors estimated the fitness across age groups at the inter - cohort level and concluded that , in contrast to our study , the changes in the frequencies of the pan i and pan i genotypes were because of selection against the pan i genotype ( the coastal component ) by high near - shore fishing pressure . first , the changes in pan i homozygote frequencies observed in our historical time series appear concomitant with the known changes that took place in the fishery during that period . it is , in principle , possible for an abrupt shift in the selection regime to have taken place in recent years , and this is the conclusion reached by rnason et al . it is worth noting though that the frequencies of the pan i genotype did not appear to have declined in our contemporary sample ( collected in 2002 ) in comparison with the earlier years . secondly , our study indicates that the genotypic variation observed within cohorts is because of differences in maturation rates , with cod carrying the pan i genotype more likely to mature at a later age . ( 2009 ) also detected distinct changes in pan i genotype frequencies at the intra - cohort level , i.e. the pan i genotype increased in frequency with age of the cod . therefore , the reported change in the frequency of the pan i genotype over the 3-year period in their study may actually have been because of a gradual increase in the number of pan i individuals as they matured and entered the spawning grounds , rather than the intense removal of pan i cod by the fishery . a thorough investigation of maturation rates of cod in relation to pantophysin genotypes would therefore help resolve the conflicting conclusions from these two studies . what is apparent is that historical or temporally replicated data , such as that used in our study , are of the utmost importance when examining changes in the genetic composition of populations that could have been potentially brought about by anthropogenic effects . significant excess of pan i heterozygotes was observed in five of seven cohort classes and 50% of sampling years . heterozygote excess at the pantophysin locus has been reported in other studies of atlantic cod ( jnsdttir et al . 1999 , 2001 ; beacham et al . 2009 ) ; however , the magnitudes in the present study are higher than typically reported . the possibility of genotyping errors because of incomplete enzyme digestion , leading to inflated heterozygote scores ( this would not affect pan i homozygotes ) was thus examined . however , repetitions of samples ( ca . 30% ) showed consistent results thus rendering the hypothesis of mis - scoring because of partial digestion unlikely . another explanation for the heterozygote excess observed in this study is that heterozygotes have a selective advantage i.e. in a highly fluctuating environment , such as that inhabited by icelandic cod , preservation of both alleles could be beneficial both in the short and in the longer term . the fact that polymorphisms have been maintained at the pan i locus for at least two million years ( pogson and mesa 2004 ) indicates there are ongoing natural selection processes influencing the genotypic distribution ( canino et al . however , heterozygotes do not appear to have an advantage in growth or longevity . indeed , even if a fluctuating environment can provide a favourable basis for protected polymorphisms , the conditions required for this are often quite restrictive ( hedrick 2006 ) . the strength of selection needed to explain such departures from hwe would probably thus be unrealistically elevated . significant heterozygote excess at the pan i locus has also been attributed to the confounding effect of sexes ( karlsson and mork 2003 ) ; however , this was not supported by our data . we therefore propose another explanation : if heterozygotes have maturation rates that are similar to those of pan i homozygotes , i.e. allele a exhibits some dominance ( fig . 3 ) , the recruitment of pan i and pan i cod to the spawning grounds would occur before the pan i homozygotes and would thus generate excesses of heterozygotes . we therefore find it likely that the observed heterozygote excess represents a dynamical system influenced by behavioural differences in the stock ( rnason et al . , this hypothesis could be examined through future investigations of maturation rates associated with the pan i genotypes . in conclusion , decades of high fishing pressure have resulted in evident changes in the age composition of icelandic cod ( marteinsdttir and thorarinsson 1998 ; this paper ) and are also likely to have altered the maturation schedule of this important fish stock ( pardoe et al . , we also show that commercial fishing has likely led to loss of adaptive variation at the pantophysin locus over a period of six decades . the analysis of historical genetic material from archived otolith collections , and more specifically non - neutral markers such as the pan i locus , has thus proved their worth in the assessment of the long - term effects of exploitation on commercial fish stocks such as atlantic cod . our results suggest that fisheries can shape the genetic composition of a fish population over a relatively short time period , thus supporting the calls for an evolutionary dimension to fisheries management ( e.g. conover 2000 ; law 2000 ; jrgensen et al . 2007 ; kuparinen and meril 2007 ) , ideally through the establishment of genetic monitoring programs based on an examination of functional as well as neutral markers ( bradbury et al . 2010 ) and incorporation of genetic perspectives ( andr et al . 2010 ) into management objectives that are concerned with the long - term sustainability of harvested populations .

the intense fishing mortality imposed on atlantic cod in icelandic waters during recent decades has resulted in marked changes in stock abundance , as well as in age and size composition . using a molecular marker known to be under selection ( pan i ) along with a suite of six neutral microsatellite loci , we analysed an archived data set and revealed evidence of distinct temporal changes in the frequencies of genotypes at the pan i locus among spawning icelandic cod , collected between 1948 and 2002 , a period characterized by high fishing pressure . concurrently , temporal stability in the composition of the microsatellite loci was established within the same data set . the frequency of the pan ibb genotype decreased over a period of six decades , concomitant with considerable spatial and technical changes in fishing effort that resulted in the disappearance of older individuals from the fishable stock . consequently , these changes have likely led to a change in the genotype frequencies at this locus in the spawning stock of icelandic cod . the study highlights the value of molecular genetic approaches that combine functional and neutral markers examined in the same set of individuals for investigations of the selective effects of harvesting and reiterates the need for an evolutionary dimension to fisheries management .

Introduction Material and methods Sampling DNA extraction and analysis Statistical analyses Results Discussion

2008 ) , but unambiguous genetic evidence of contemporary evolution in marine fish populations has proven elusive because of the limited availability of adaptive variation estimates , as well as historical molecular data sets ( conover et al . indeed , because of the large effective population sizes of exploited marine fishes , neutral markers are unlikely to exhibit changes in a signal over a relatively short time period , while loci under selection might readily respond to changes in selection regime . like most cod ( gadus morhua ) stocks in the north atlantic , icelandic cod has experienced a drastic reduction in abundance and spawning stock biomass ( ssb ) during the last few decades . estimated total spawning stock biomass ( histogram bars ) and average fishing mortality of age groups 510 ( line ) of cod in icelandic waters during the period 19482002 ( marine research institute 2009 ) . since wwii there have been substantial spatial and seasonal changes in the exploitation pattern of icelandic cod . this , combined with the stricter inshore fishing regulations , improvements in fishing technology , and the introduction of larger vessels , resulted in the redirection of fishing effort into deeper offshore waters . alongside the declines in stock abundance , there have been notable alterations in the life history of icelandic cod , whereby far fewer fish now survive beyond 7 years of age , resulting in severely truncated age and size distributions ( schopka 1994 ; marteinsdttir and thorarinsson 1998 ) . a recent study , which estimated pmrns for 36 cohorts of icelandic cod , found evidence that a shift towards maturation at smaller sizes and younger ages has occurred independently of changes in growth , condition and temperature ( pardoe et al . this change in the maturation schedule of icelandic cod , along with the loss of older and larger repeat spawners , raises concerns for the stock 's reproductive potential , and consequently its ability to withstand harvesting and future environmental change ( law 2007 ; rnason et al . in this study , we used archived otoliths , collected between 1948 and 2002 , to jointly examine phenotypic and genetic data . our goal was to examine historical trends in the genetic composition of the icelandic cod stock using both selected and neutral markers . temporal stability in the allele frequency distribution of neutral markers would reflect stability in population structure and minimal drift , as is expected when effective population sizes are large . we examined polymorphism at the pantophysin ( pan i ) locus ( pogson et al . 1995 ; pogson 2001 ) , a genetic marker known to be under selection or closely linked to a gene under selection , as well as a suite of six microsatellite loci assumed to be neutral . the two main allele classes within the pan i locus , pan i and pan i , are differentiated by a dra i restriction site ( pogson 2001 ) . we found clear evidence that major changes in genetic variation at the pan i locus of icelandic cod have occurred alongside those in fishing patterns and age composition of the stock , whereas no temporal changes were detected with neutral genetic markers . overview of icelandic cod samples used in this study : year , date and subarea of sampling ( see note for corresponding coordinates ) , sampling depth ( meters ; entries with asterisks show depth distributions [ 1090% quantiles ] within the subarea for those samples where actual depth of the catch was not available ) , number of individuals ( n ) , amplification success ( asucc ) , proportion of females ( pfem ) , mean total length ( cm ; tlmean ) , length range ( cm ; tlrange ) , mean age ( years ; amean ) , age range ( years ; arange ) . primers specially designed to amplify a short fragment ( 142 bp ) of the pan i locus from dna that has experienced considerable degradation ( nielsen et al . drai cuts at the diagnostic restriction sites in the pcr product , discriminating between the pan i and the pan i alleles . we also analysed genetic variation at six microsatellite loci assumed to be neutral using a subset of those individuals from the pan analysis : gmo2 ( brooker et al . the purpose of the model was to describe the long - term changes in genotype frequencies of cohorts 19321999 , after taking into account that some cohorts were only sampled at a younger age and other cohorts only at an older age . in a single model , we fitted the frequencies of pan i genotypes pan i , pan i and pan i as a smooth function of both cohort and age . for the suite of six microsatellite loci , observed ( ho ) and expected ( he ) heterozygosity were calculated in genetix 4.03 ( belkhir et al . tests for deviations from hardy weinberg equilibrium ( hwe ) were conducted for both the pan i locus and the suite of six microsatellite loci using the exact test in genepop 3.1 ( raymond and rousset 1995 ) . an analysis of molecular variance ( amova ) was carried out for both the pan i locus and the suite of six microsatellite loci in arlequin 3.0 ( excoffier et al . a total of 16 samples , comprising 1471 spawning or near - spawning individuals , were collected from three relatively shallow locations known to be major spawning grounds for icelandic cod ( south west = sw , faxafli bay = fax and the west coast bay , breidafjrdur = bre ; see table 1 for exact details on location ) . overview of icelandic cod samples used in this study : year , date and subarea of sampling ( see note for corresponding coordinates ) , sampling depth ( meters ; entries with asterisks show depth distributions [ 1090% quantiles ] within the subarea for those samples where actual depth of the catch was not available ) , number of individuals ( n ) , amplification success ( asucc ) , proportion of females ( pfem ) , mean total length ( cm ; tlmean ) , length range ( cm ; tlrange ) , mean age ( years ; amean ) , age range ( years ; arange ) . primers specially designed to amplify a short fragment ( 142 bp ) of the pan i locus from dna that has experienced considerable degradation ( nielsen et al . drai cuts at the diagnostic restriction sites in the pcr product , discriminating between the pan i and the pan i alleles . we also analysed genetic variation at six microsatellite loci assumed to be neutral using a subset of those individuals from the pan analysis : gmo2 ( brooker et al . the purpose of the model was to describe the long - term changes in genotype frequencies of cohorts 19321999 , after taking into account that some cohorts were only sampled at a younger age and other cohorts only at an older age . in a single model , we fitted the frequencies of pan i genotypes pan i , pan i and pan i as a smooth function of both cohort and age . for the suite of six microsatellite loci , observed ( ho ) and expected ( he ) heterozygosity were calculated in genetix 4.03 ( belkhir et al . tests for deviations from hardy weinberg equilibrium ( hwe ) were conducted for both the pan i locus and the suite of six microsatellite loci using the exact test in genepop 3.1 ( raymond and rousset 1995 ) . an analysis of molecular variance ( amova ) was carried out for both the pan i locus and the suite of six microsatellite loci in arlequin 3.0 ( excoffier et al . a hierarchical amova revealed that overall variation at the pan i locus was because of temporal ( fsc = 0.122 , p < 0.001 ) rather than spatial variation ( fct = 0.025 , p = 0.92 ) ( table 2 ) . results from the analysis of genetic variation at the pan i locus are presented in table 3 . hierarchical partitioning of genetic variance at the pan i locus , based on an analysis of molecular variance ( amova ) of samples of icelandic cod ( see table 1 ) observed ( ho ) and expected ( he ) heterozygosity and observed frequencies of pan i allele in icelandic cod samples ( n : sample size ) by cohort class and sample year . bold values indicate significant deviations of fis values from hardy weinberg expectations , after correction for multiple tests as has been previously observed with an independent data set ( marteinsdttir and thorarinsson 1998 ) , the age composition of icelandic cod changed markedly through the study period . the observed shift towards a narrower age distribution resulted from the loss of older age classes and an overall reduction in mean age ( = 851.0 , p < 0.001 ) moreover , our analyses revealed that the pan i locus has been subject to major changes in allele frequencies during the study period , in relation to both age and cohort class . within cohorts , the frequencies of the homozygous genotypes were age dependent ( figs 3 and 4 ) . age - specific distributions of pan i genotype frequencies ( white : pan i , grey : pan i , black : pan i ) in samples of icelandic cod collected between 1948 and 2002 . observed age - specific frequencies of pan i genotypes ( pan i , pan i and pan i ) for icelandic cod sampled in three time periods : period 1 , 19481966 ; period 2 , 19721979 ; period 3 , 19842002 ( see table 1 for actual years sampled within each period ) . at the inter - cohort level , there was a gradual but strong decline in the frequency of pan i over the study period ( = 199.3 , p < 0.001 ) ( fig . , the pan i genotypes increased in frequency , exceeding 50% in the last 20 years of the study at the expense of the pan i and pan i individuals ( = 85.9 , p < 0.0010 ) ( fig . observed pan i genotype frequencies ( white : pan i , grey : pan i , black : pan i ) for icelandic cod from different ( 10 year ) cohort classes . distinct differences were observed in length - at - age among the pan i genotypes , whereby icelandic cod carrying the pan i genotype had significantly higher length - at - age on average than cod of the pan i and pan i genotypes ( p = 0.048 ) ( fig . average length ( 1 se ) of icelandic cod aged 612 years , sampled in the period 19482002 , and carrying either pan i , pan i or pan i genotype . multilocus genotype information from six microsatellites was obtained from 892 of the individuals utilized in the pan i analyses , representing the same sampling years and sites ( sw , fax , bre ; table 1 ) . our study is the first , to our knowledge , to analyse functional and neutral genetic markers in combination with fisheries data , over a multidecadal time period , in an attempt to explain distinct changes in genetic and phenotypic variation in a fish stock that has been heavily exploited since the 1940s and 50s . by isolating dna from archived cod otoliths , gathered over a 60-year period at spawning sites on the sw coast of iceland , we were able to detect long - term temporal changes in genotype frequencies at the pantophysin ( pan i ) locus , whereas no changes were observed at six microsatellite loci . the significant decline in the pan i genotype , which represented 26% of the sampled population in the 1930s but only 5% in the 1990s , followed changes in the exploitation pattern , which were characterized by increased effort in deeper waters facilitated by larger ships and technological advancements in fishing gear . in the current study , the frequencies of pan i genotypes varied both within and between cohorts . within cohorts , the frequency of the pan i genotype increased with age ( figs 3 and 4 ) . currently , the age - specific distribution of the pan i genotypes is unknown for immature icelandic cod . the absence of the pan i genotype among spawning fish aged 35 years is therefore most likely due to cod carrying that genotype not having matured yet and thus not being present on the spawning sites , which provided the data for this study . the gradual increase in the frequency of the pan i genotype from 6 to 9 years of age is thus likely to reflect differences in the rate of maturation . in the present study , we have shown a gradual decline in the frequency of the pan i genotype among spawning cod during the last 60 years . concurrently , the pan i genotype increased in frequency , exceeding 50% in the last 20 years of the study at the expense of the pan i and pan i individuals ( fig . we can not be certain of the primary driving force behind these changes in the proportions of the different genotypes . first , changes in genotypic frequencies may have resulted from changes in stock composition because of the consistent removal of older fish during a period of increased fishing mortality ( marteinsdttir and thorarinsson 1998 ; fig . as previously discussed , the oldest cod in our samples were predominantly of the pan i or pan i genotype , whereas relatively few cod carrying the pan i genotype were found to be aged 9 years or older . therefore , the truncation of the age distribution of icelandic cod , a common feature of commercially exploited fish stocks , is likely to have resulted in the removal of the pan i genotype from the spawning stock at a proportionally faster rate than that of the pan i genotype . secondly , the gradual decline in the frequency of the pan i genotype may also have resulted from the substantial spatial and seasonal changes in fishing effort on icelandic cod that have occurred since wwii . at the same time , the introduction of larger vessels resulted in the redirection of fishing effort into deeper offshore waters . spawning cod sampled in 20022003 in the sw of iceland , the area covered by this study , ranged from being predominantly of the pan i genotype at the shallowest stations to predominantly of the pan i and pan i genotypes at the deeper stations ( pampoulie et al . therefore , changes in the exploitation pattern of icelandic cod during the study period are likely to have resulted in relatively greater fishing mortality in deeper water and on the feeding grounds and thus may have led to the disproportionate removal of the pan i genotype from the stock by the commercial fishery . thirdly , we may not be able to fully explain the causes of these long - term temporal changes in genotypic frequency in icelandic cod until our understanding of the population structure further improves . the distinct variation in life history traits , behaviour and pan i genotype frequencies of icelandic cod is persuasive evidence that the current stock designation does not capture the actual geographic scale of population differentiation ( pampoulie et al . neutral markers are poorly suited to the detection of changes in genetic diversity because they segregate independently of the selected loci , and in populations with relatively large effective population sizes in particular the level of genetic drift will be low ( conover et al . if the icelandic cod stock indeed comprises populations with limited gene flow and/or different selection schedules , then the changes in exploitation patterns described above will have affected the stock complex by removing the pan i genotype at a faster rate than the pan i genotype . finally in our fourth hypothesis , we recognize that the genotype distribution at the pan i locus may have changed because of selection by other agents such as those of climatic origin . 2006 ) did not reveal any significant correlations with changes in frequency of the pan i genotypes ( p > 0.05 ) . these striking fluctuations in temperature and salinity of icelandic waters thus do not appear responsible for the gradual and consistent changes in the genotype frequencies observed in this study . other studies have also shown that there does not appear to be a direct link between pan i genotype frequencies in cod and water temperature ( nielsen et al . furthermore , the life history stage(s ) at which temperature affects the genotypic distribution of the pan i locus of cod is unknown . in conclusion , although the observed changes in genotype frequencies at the pan i locus could be the result of simultaneous selection by both trends in temperature and fishing , the information that does exist indicates that although there have been persistent changes in environmental conditions in icelandic waters , those changes alone are insufficient to explain the strong selection against the pan i observed during the last 20 years of the study . a recent study has also attempted to explore the effects of exploitation on genetic diversity at the pan i locus in icelandic cod using samples from spawning areas around iceland over a 3-year period ( 20052007 ) ( rnason et al . the authors estimated the fitness across age groups at the inter - cohort level and concluded that , in contrast to our study , the changes in the frequencies of the pan i and pan i genotypes were because of selection against the pan i genotype ( the coastal component ) by high near - shore fishing pressure . first , the changes in pan i homozygote frequencies observed in our historical time series appear concomitant with the known changes that took place in the fishery during that period . it is worth noting though that the frequencies of the pan i genotype did not appear to have declined in our contemporary sample ( collected in 2002 ) in comparison with the earlier years . ( 2009 ) also detected distinct changes in pan i genotype frequencies at the intra - cohort level , i.e. therefore , the reported change in the frequency of the pan i genotype over the 3-year period in their study may actually have been because of a gradual increase in the number of pan i individuals as they matured and entered the spawning grounds , rather than the intense removal of pan i cod by the fishery . what is apparent is that historical or temporally replicated data , such as that used in our study , are of the utmost importance when examining changes in the genetic composition of populations that could have been potentially brought about by anthropogenic effects . in a highly fluctuating environment , such as that inhabited by icelandic cod , preservation of both alleles could be beneficial both in the short and in the longer term . the fact that polymorphisms have been maintained at the pan i locus for at least two million years ( pogson and mesa 2004 ) indicates there are ongoing natural selection processes influencing the genotypic distribution ( canino et al . significant heterozygote excess at the pan i locus has also been attributed to the confounding effect of sexes ( karlsson and mork 2003 ) ; however , this was not supported by our data . 3 ) , the recruitment of pan i and pan i cod to the spawning grounds would occur before the pan i homozygotes and would thus generate excesses of heterozygotes . , this hypothesis could be examined through future investigations of maturation rates associated with the pan i genotypes . in conclusion , decades of high fishing pressure have resulted in evident changes in the age composition of icelandic cod ( marteinsdttir and thorarinsson 1998 ; this paper ) and are also likely to have altered the maturation schedule of this important fish stock ( pardoe et al . , we also show that commercial fishing has likely led to loss of adaptive variation at the pantophysin locus over a period of six decades . the analysis of historical genetic material from archived otolith collections , and more specifically non - neutral markers such as the pan i locus , has thus proved their worth in the assessment of the long - term effects of exploitation on commercial fish stocks such as atlantic cod . our results suggest that fisheries can shape the genetic composition of a fish population over a relatively short time period , thus supporting the calls for an evolutionary dimension to fisheries management ( e.g.

neuronal gamma - band synchronization in visual cortex has been associated with several important functions , particularly perceptual grouping and selection ( eckhorn et al . 1988 ; gray et al . 1989 ; kreiter and singer 1996 ; livingstone 1996 ; fries et al . 2000 ) , attentional stimulus selection ( fries , reynolds , et al . 2001 ; bichot et al . 2005 ; taylor et al . 2005 ; bauer et al . 2006 ; womelsdorf et al . 2006 ; fries et al . 2008 ; buffalo et al . 2011 ; bosman et al . 2012 ; grothe et al . 2012 ) , and efficient stimulus representation and signaling ( womelsdorf et al . 2007 ; all of these studies used artificial stimuli , like bars , gratings , letter - like symbols , or bezier curves , always on uniform backgrounds . if gamma - band synchronization plays a role in stimulus processing , perceptual grouping , or selective attention during natural viewing , it needs to be present during free viewing of natural images . so far , this has not been demonstrated , but to the contrary , several studies strongly suggest that free viewing of natural images eliminates gamma - band activity . a previous study used an interesting approach when aiming to emulate natural viewing ( kayser et al . the authors first mounted a camera onto the head of a cat and recorded movies while the cat roamed through nature . subsequently , they presented these movies to an awake head - fixed cat and recorded neuronal activity from primary visual cortex . they report that such movies primarily induce broad - band local field potential ( lfp ) power increases between 100 and 200 hz , which differ strongly from band - limited gamma ( 4080 hz ) power increases that they find for grating stimuli , and rather resemble power changes that they observe when presenting pink - noise stimuli . another recent study investigated single units and lfp in area v1 of cebus monkeys freely viewing natural images ( ito et al . these authors focus on saccade - related modulations of coincident spiking and of lfp locking . yet , as an example , they also show a time 1c ) and , while it reveals alpha- and beta - band modulations , there are no clear gamma - band peaks . the 2 panels are rendered from photographs taken during the electrode implantation surgeries , after placement of the ecog grid onto the brain , before closure of the dura . lus : lunate sulcus ; sts : superior temporal sulcus ; as : arcuate sulcus ; cs : central sulcus ; ips : intraparietal sulcus . large blue ( red ) dots indicate the positions of electrodes assigned to area v1 ( v4 ) . the 2 panels are rendered from photographs taken during the electrode implantation surgeries , after placement of the ecog grid onto the brain , before closure of the dura . lus : lunate sulcus ; sts : superior temporal sulcus ; as : arcuate sulcus ; cs : central sulcus ; ips : intraparietal sulcus . other studies demonstrated that gamma - band activity is strongly reduced for grating stimuli of reduced size ( gieselmann and thiele 2008 ) or for 2 gratings superimposed to form a plaid ( lima et al . natural images often contain small and/or superimposed components and might , therefore , induce only marginal gamma - band activity . finally , the frequency of gamma - band activity can shift by at least 25 hz upon changes in stimulus properties like size ( gieselmann and thiele 2008 ) or contrast ( ray and maunsell 2010 ; roberts et al . therefore , natural images with their wide range of object sizes and contrasts might lead to local gamma - band activities with a wide range of frequencies , which might not summate effectively and thereby lead to merely weak and broad - band power increases . since an absence of gamma - band activity during free viewing of natural images we recorded lfp from electrocorticographic ( ecog ) grid electrodes covering areas v1 , v4 , and several additional areas of the left hemisphere in 2 macaque monkeys freely viewing natural images . all animal procedures were approved by the ethics committee of the radboud university , nijmegen , netherlands . two adult male macaque monkeys were used in this study and we will refer to them as monkey p and monkey a. monkey p in this study is the same as monkey p in bosman et al . stimuli were presented on a cathode ray tube ( crt ) screen refreshing at 120 hz noninterlaced and positioned such that 32 pixels corresponded to 1 of visual angle ( ) . for the data reported in this study , monkeys were required to fixate for 0.63 s on a fixation point ( 0.12 by 0.12 black square ) centered on a gray background , after which a natural image was presented , which was again centered on the background screen . grayscale images were shown for 3.56 s ( flat random distribution ) , and color images for 1.5 s. once the image had appeared on the screen , the monkey could view it freely . if the monkey kept its gaze on the image as long as it was presented , it was given a juice reward after stimulus offset . because this task was very easy for the monkeys , almost every trial was rewarded . we used 49 grayscale images and 16 color images , with grayscale and color images presented in separate sessions . grayscale images subtended 16-by-16 , and color images 18.5-by-18.5. each grayscale image was presented for an average of 15 trials , and each color image for an average of 22 trials . eye position was recorded with an infrared camera system ( thomas recording et-49b system ) at a sampling rate of 230 hz . the monkeys were implanted with ecog grid electrodes , consisting of 252 subdural electrodes distributed across several superficial areas ( rubehn et al . 2012 ) . unless stated otherwise , we selected electrodes over v1 and v4 that were strongly driven by stimuli within the central 4 of eccentricity . due to placement of the ecog grid onto the dorsal parts of v1 and v4 in the left hemisphere , correspondingly , we accepted analysis epochs when the gaze of the monkey was at least 4 away from the lower and the right border of the natural image for at least 90% of the epoch duration . this ensured that the responses of the recorded sites were due to the natural image rather than the screen background . in monkey p ( monkey a ) , we used 43 ( 42 ) electrodes on v1 and 16 ( 14 ) on v4 . the assignment of electrodes to visual areas was based on intraoperative photographs and brain atlases , and used primarily sulcal landmarks . for most of the electrodes , , some of the most anterior electrodes assigned to v1 might as well be over v2 , and the most lateral electrodes assigned to v4 might as well be over the temporal - occipital area ( teo ) . electrophysiological signals were impedance buffered and amplified 20 times through eight 32-channel headstages ( plexon headstage 32 v - g20 ) . they were then low - pass filtered at 8 khz and digitized at roughly 32 khz ( neuralynx digital lynx 256 channel system ) . results from those recordings have been published along with more information on the typical receptive fields obtained with the ecog electrodes ( bosman et al . all analyses were done in matlab ( the mathworks ) , and using the fieldtrip open source matlab toolbox ( http://fieldtrip.fcdonders.nl/ ) ( oostenveld et al . signals were low - pass filtered at 250 hz and downsampled to 1 khz . during recordings , the signal from each electrode was differentiated against a common reference and we refer to this signal as the unipolar lfp . offline , signals from pairs of immediately neighboring electrodes were subtracted from each other to remove the common recording reference , and we refer to this bipolar derivation as a site and to the resulting signal as a bipolar or locally differentiated lfp or just as lfp . power - line artifacts were removed by subtracting the discrete fourier transforms at 50 and 100 hz . we performed frequency - resolved , that is , spectral , analyses of lfp power . for figures 24 , we limited data selection and processing to the absolute minimum . in figure 2b , the analysis covered 20140 hz in steps of 2 hz , using for each frequency an epoch length of 4 cycles . figure 2c is based on 3 slightly overlapping 0.5 s epochs , multitapered with 8 tapers ( mitra and pesaran 1999 ) to achieve an 8-hz spectral smoothing . figures 3 and 4 are based on all nonoverlapping 0.5 s epochs from 0.3 s after stimulus onset until stimulus offset , during which the receptive fields ( rfs ) remained on the image ( see above for details ) , compared with a 0.5-s prestimulus baseline . we aimed at focusing on the effect of visual processing during free viewing , rather than the effects of saccades . therefore , we selected all 0.25 s epochs that started 0.125 s after a saccade and did not include the following saccade . those epochs were hanning tapered . saccades were defined as follows : the vertical and horizontal eye position traces were low - pass filtered at 50 hz , differentiated to obtain vertical and horizontal eye velocity , and combined to obtain overall eye speed . saccades following other saccades within < 20 ms were discarded . in figure 6 , we investigated the dynamics of spectral power around the time of a saccade . for this analysis , we selected epochs from 0.2 s before saccades until 0.3 s after the saccade , during which the rfs were on the image ( see above for details ) . the subsequent time frequency analysis was the same as in figure 2e , that is , using 4 cycles per frequency , hanning tapered . for figure 6 , we selected those v1 and v4 sites that showed the top third ( v1 ) and top half ( v4 ) gamma - band power increases during natural viewing . the gamma - frequency band was defined as 5080 hz in both monkeys . where power during free viewing of natural images was normalized by power during the prestimulus baseline , the respective power values were first averaged across trials , separately for the 2 epochs , before normalization . figure 2.gamma-band oscillation in the raw lfp of one example site during one example visual exploration . ( a ) raw unipolar lfp trace during one visual exploration of the photograph of 2 oranges . the insets above the lfp show the stimulus presented to the monkey at the respective time point , and superimposed the eye position trace around that time point in blue , and the eye position trace during this exploration so far in gray . the fixation point is shown as a cross to make it visible behind the eye trace , while it was actually a small green square . ( b ) time frequency analysis of absolute power of the same example unipolar lfp . ( c ) power change spectrum of the same unipolar lfp during visual exploration versus prestimulus baseline . ( d f ) same as ( a c ) for an example locally bipolar lfp . each panel shows the natural image presented to the animal and the corresponding spectra ( red : monkey p , blue : monkey a ) of power changes in percent , comparing free viewing of the respective image with a prestimulus baseline , averaged over all v1 sites ( shaded regions indicate 1 standard error of the mean , sem ) . the spectra were scaled such that the lower end of the y - axis corresponds to zero change and the upper end corresponds to the percent change value that is indicated in the upper - left corner of the respective panel . ( a ) histograms of changes in peak gamma - band power across all grayscale images , separately for monkey p ( red ) and monkey a ( blue ) . ( c ) histograms of the negative decadic logarithm of the p - values derived from 2-sided paired t - tests comparing gamma - band power ( 5080 hz ) between natural viewing and prestimulus fixation baseline , across trials . color coding as in a and b. ( d ) same as in ( c ) , but for the color images . vertical lines indicate significance thresholds of * p = 0.05 and * * * p = 0.001 . figure 5.power change spectra averaged across all natural images for 4 example sites in both monkeys . ( a d ) spectra of power change ( 1 sem ) during free viewing , when compared with baseline , averaged across all natural images , for the example sites indicated in ( e ) . ( f ) topographical distribution of the corresponding t - values for the comparison of free viewing of natural images versus baseline . ( g l ) same analyses as in ( a f ) , but for monkey a. the gamma - band was defined as 5080 hz in both monkeys . all analyses shown in this figure use fixation periods in between saccades ( see methods for details ) . lus : lunate sulcus ; sts : superior temporal sulcus ; as : arcuate sulcus ; cs : central sulcus ; ips : intraparietal sulcus . frequency analysis of power ( expressed as percent change from prestimulus baseline ) in area v1 around the time of a saccade of monkey p. the saccade is indicated by the vertical black line at time zero ( moment of peak saccade velocity ) . ( c ) saccade - aligned erp ( averaged after single - trial rectification ) in area v1 . frequency analysis of the power of the erp ( expressed as percent of the prestimulus baseline ) . ( g l ) same as ( a f ) , but for monkey a. gamma - band oscillation in the raw lfp of one example site during one example visual exploration . ( a ) raw unipolar lfp trace during one visual exploration of the photograph of 2 oranges . the insets above the lfp show the stimulus presented to the monkey at the respective time point , and superimposed the eye position trace around that time point in blue , and the eye position trace during this exploration so far in gray . the fixation point is shown as a cross to make it visible behind the eye trace , while it was actually a small green square . ( b ) time frequency analysis of absolute power of the same example unipolar lfp . ( c ) power change spectrum of the same unipolar lfp during visual exploration versus prestimulus baseline . ( d f ) same as ( a c ) for an example locally bipolar lfp . each panel shows the natural image presented to the animal and the corresponding spectra ( red : monkey p , blue : monkey a ) of power changes in percent , comparing free viewing of the respective image with a prestimulus baseline , averaged over all v1 sites ( shaded regions indicate 1 standard error of the mean , sem ) . the spectra were scaled such that the lower end of the y - axis corresponds to zero change and the upper end corresponds to the percent change value that is indicated in the upper - left corner of the respective panel . ( a ) histograms of changes in peak gamma - band power across all grayscale images , separately for monkey p ( red ) and monkey a ( blue ) . ( c ) histograms of the negative decadic logarithm of the p - values derived from 2-sided paired t - tests comparing gamma - band power ( 5080 hz ) between natural viewing and prestimulus fixation baseline , across trials . color coding as in a and b. ( d ) same as in ( c ) , but for the color images . vertical lines indicate significance thresholds of * p = 0.05 and * * * p = 0.001 . power change spectra averaged across all natural images for 4 example sites in both monkeys . ( a d ) spectra of power change ( 1 sem ) during free viewing , when compared with baseline , averaged across all natural images , for the example sites indicated in ( e ) . ( f ) topographical distribution of the corresponding t - values for the comparison of free viewing of natural images versus baseline . ( g l ) same analyses as in ( a f ) , but for monkey a. the gamma - band was defined as 5080 hz in both monkeys . all analyses shown in this figure use fixation periods in between saccades ( see methods for details ) . lus : lunate sulcus ; sts : superior temporal sulcus ; as : arcuate sulcus ; cs : central sulcus ; ips : intraparietal sulcus . ( a ) time frequency analysis of power ( expressed as percent change from prestimulus baseline ) in area v1 around the time of a saccade of monkey p. the saccade is indicated by the vertical black line at time zero ( moment of peak saccade velocity ) . ( c ) saccade - aligned erp ( averaged after single - trial rectification ) in area v1 . ( e ) time frequency analysis of the power of the erp ( expressed as percent of the prestimulus baseline ) . ( g l ) same as ( a f ) , but for monkey a. all animal procedures were approved by the ethics committee of the radboud university , nijmegen , netherlands . two adult male macaque monkeys were used in this study and we will refer to them as monkey p and monkey a. monkey p in this study is the same as monkey p in bosman et al . stimuli were presented on a cathode ray tube ( crt ) screen refreshing at 120 hz noninterlaced and positioned such that 32 pixels corresponded to 1 of visual angle ( ) . for the data reported in this study , monkeys were required to fixate for 0.63 s on a fixation point ( 0.12 by 0.12 black square ) centered on a gray background , after which a natural image was presented , which was again centered on the background screen . grayscale images were shown for 3.56 s ( flat random distribution ) , and color images for 1.5 s. once the image had appeared on the screen , the monkey could view it freely . if the monkey kept its gaze on the image as long as it was presented , it was given a juice reward after stimulus offset . because this task was very easy for the monkeys , almost every trial was rewarded . we used 49 grayscale images and 16 color images , with grayscale and color images presented in separate sessions . grayscale images subtended 16-by-16 , and color images 18.5-by-18.5. each grayscale image was presented for an average of 15 trials , and each color image for an average of 22 trials . eye position was recorded with an infrared camera system ( thomas recording et-49b system ) at a sampling rate of 230 hz . the monkeys were implanted with ecog grid electrodes , consisting of 252 subdural electrodes distributed across several superficial areas ( rubehn et al . 2009 ; bosman et al . 2012 ) . unless stated otherwise , we selected electrodes over v1 and v4 that were strongly driven by stimuli within the central 4 of eccentricity . due to placement of the ecog grid onto the dorsal parts of v1 and v4 in the left hemisphere , receptive fields were in the lower right visual quadrant . correspondingly , we accepted analysis epochs when the gaze of the monkey was at least 4 away from the lower and the right border of the natural image for at least 90% of the epoch duration . this ensured that the responses of the recorded sites were due to the natural image rather than the screen background . in monkey p ( monkey a ) , we used 43 ( 42 ) electrodes on v1 and 16 ( 14 ) on v4 . the assignment of electrodes to visual areas was based on intraoperative photographs and brain atlases , and used primarily sulcal landmarks . for most of the electrodes , , some of the most anterior electrodes assigned to v1 might as well be over v2 , and the most lateral electrodes assigned to v4 might as well be over the temporal - occipital area ( teo ) . electrophysiological signals were impedance buffered and amplified 20 times through eight 32-channel headstages ( plexon headstage 32 v - g20 ) . they were then low - pass filtered at 8 khz and digitized at roughly 32 khz ( neuralynx digital lynx 256 channel system ) . results from those recordings have been published along with more information on the typical receptive fields obtained with the ecog electrodes ( bosman et al . all analyses were done in matlab ( the mathworks ) , and using the fieldtrip open source matlab toolbox ( http://fieldtrip.fcdonders.nl/ ) ( oostenveld et al . signals were low - pass filtered at 250 hz and downsampled to 1 khz . during recordings , the signal from each electrode was differentiated against a common reference and we refer to this signal as the unipolar lfp . offline , signals from pairs of immediately neighboring electrodes were subtracted from each other to remove the common recording reference , and we refer to this bipolar derivation as a site and to the resulting signal as a bipolar or locally differentiated lfp or just as lfp . power - line artifacts were removed by subtracting the discrete fourier transforms at 50 and 100 hz . we performed frequency - resolved , that is , spectral , analyses of lfp power . for figures 24 , we limited data selection and processing to the absolute minimum . in figure 2b , the analysis covered 20140 hz in steps of 2 hz , using for each frequency an epoch length of 4 cycles . figure 2c is based on 3 slightly overlapping 0.5 s epochs , multitapered with 8 tapers ( mitra and pesaran 1999 ) to achieve an 8-hz spectral smoothing . figures 3 and 4 are based on all nonoverlapping 0.5 s epochs from 0.3 s after stimulus onset until stimulus offset , during which the receptive fields ( rfs ) remained on the image ( see above for details ) , compared with a 0.5-s prestimulus baseline . these epochs were hanning tapered and fourier transformed . for figure 5 , we aimed at focusing on the effect of visual processing during free viewing , rather than the effects of saccades . therefore , we selected all 0.25 s epochs that started 0.125 s after a saccade and did not include the following saccade . saccades were defined as follows : the vertical and horizontal eye position traces were low - pass filtered at 50 hz , differentiated to obtain vertical and horizontal eye velocity , and combined to obtain overall eye speed . , we investigated the dynamics of spectral power around the time of a saccade . for this analysis , we selected epochs from 0.2 s before saccades until 0.3 s after the saccade , during which the rfs were on the image ( see above for details ) . the subsequent time frequency analysis was the same as in figure 2e , that is , using 4 cycles per frequency , hanning tapered . for figure 6 , we selected those v1 and v4 sites that showed the top third ( v1 ) and top half ( v4 ) gamma - band power increases during natural viewing . the gamma - frequency band was defined as 5080 hz in both monkeys . where power during free viewing of natural images was normalized by power during the prestimulus baseline , the respective power values were first averaged across trials , separately for the 2 epochs , before normalization . figure 2.gamma-band oscillation in the raw lfp of one example site during one example visual exploration . ( a ) raw unipolar lfp trace during one visual exploration of the photograph of 2 oranges . the insets above the lfp show the stimulus presented to the monkey at the respective time point , and superimposed the eye position trace around that time point in blue , and the eye position trace during this exploration so far in gray . the fixation point is shown as a cross to make it visible behind the eye trace , while it was actually a small green square . ( b ) time frequency analysis of absolute power of the same example unipolar lfp . ( c ) power change spectrum of the same unipolar lfp during visual exploration versus prestimulus baseline . ( d f ) same as ( a c ) for an example locally bipolar lfp . each panel shows the natural image presented to the animal and the corresponding spectra ( red : monkey p , blue : monkey a ) of power changes in percent , comparing free viewing of the respective image with a prestimulus baseline , averaged over all v1 sites ( shaded regions indicate 1 standard error of the mean , sem ) . the spectra were scaled such that the lower end of the y - axis corresponds to zero change and the upper end corresponds to the percent change value that is indicated in the upper - left corner of the respective panel . ( a ) histograms of changes in peak gamma - band power across all grayscale images , separately for monkey p ( red ) and monkey a ( blue ) . ( c ) histograms of the negative decadic logarithm of the p - values derived from 2-sided paired t - tests comparing gamma - band power ( 5080 hz ) between natural viewing and prestimulus fixation baseline , across trials . color coding as in a and b. ( d ) same as in ( c ) , but for the color images . vertical lines indicate significance thresholds of * p = 0.05 and * * * p = 0.001 . figure 5.power change spectra averaged across all natural images for 4 example sites in both monkeys . ( a d ) spectra of power change ( 1 sem ) during free viewing , when compared with baseline , averaged across all natural images , for the example sites indicated in ( e ) . ( f ) topographical distribution of the corresponding t - values for the comparison of free viewing of natural images versus baseline . ( g l ) same analyses as in ( a f ) , but for monkey a. the gamma - band was defined as 5080 hz in both monkeys . all analyses shown in this figure use fixation periods in between saccades ( see methods for details ) . lus : lunate sulcus ; sts : superior temporal sulcus ; as : arcuate sulcus ; cs : central sulcus ; ips : intraparietal sulcus . frequency analysis of power ( expressed as percent change from prestimulus baseline ) in area v1 around the time of a saccade of monkey p. the saccade is indicated by the vertical black line at time zero ( moment of peak saccade velocity ) . ( c ) saccade - aligned erp ( averaged after single - trial rectification ) in area v1 . ( e ) time frequency analysis of the power of the erp ( expressed as percent of the prestimulus baseline ) . ( g l ) same as ( a f ) , but for monkey a. gamma - band oscillation in the raw lfp of one example site during one example visual exploration . ( a ) raw unipolar lfp trace during one visual exploration of the photograph of 2 oranges . the insets above the lfp show the stimulus presented to the monkey at the respective time point , and superimposed the eye position trace around that time point in blue , and the eye position trace during this exploration so far in gray . the fixation point is shown as a cross to make it visible behind the eye trace , while it was actually a small green square . ( b ) time frequency analysis of absolute power of the same example unipolar lfp . ( c ) power change spectrum of the same unipolar lfp during visual exploration versus prestimulus baseline . ( d f ) same as ( a c ) for an example locally bipolar lfp . each panel shows the natural image presented to the animal and the corresponding spectra ( red : monkey p , blue : monkey a ) of power changes in percent , comparing free viewing of the respective image with a prestimulus baseline , averaged over all v1 sites ( shaded regions indicate 1 standard error of the mean , sem ) . the spectra were scaled such that the lower end of the y - axis corresponds to zero change and the upper end corresponds to the percent change value that is indicated in the upper - left corner of the respective panel . ( a ) histograms of changes in peak gamma - band power across all grayscale images , separately for monkey p ( red ) and monkey a ( blue ) . ( c ) histograms of the negative decadic logarithm of the p - values derived from 2-sided paired t - tests comparing gamma - band power ( 5080 hz ) between natural viewing and prestimulus fixation baseline , across trials . color coding as in a and b. ( d ) same as in ( c ) , but for the color images . vertical lines indicate significance thresholds of * p = 0.05 and * * * p = 0.001 . power change spectra averaged across all natural images for 4 example sites in both monkeys . ( a d ) spectra of power change ( 1 sem ) during free viewing , when compared with baseline , averaged across all natural images , for the example sites indicated in ( e ) . ( f ) topographical distribution of the corresponding t - values for the comparison of free viewing of natural images versus baseline . ( g l ) same analyses as in ( a f ) , but for monkey a. the gamma - band was defined as 5080 hz in both monkeys . all analyses shown in this figure use fixation periods in between saccades ( see methods for details ) . lus : lunate sulcus ; sts : superior temporal sulcus ; as : arcuate sulcus ; cs : central sulcus ; ips : intraparietal sulcus . ( a ) time frequency analysis of power ( expressed as percent change from prestimulus baseline ) in area v1 around the time of a saccade of monkey p. the saccade is indicated by the vertical black line at time zero ( moment of peak saccade velocity ) . ( c ) saccade - aligned erp ( averaged after single - trial rectification ) in area v1 . frequency analysis of the power of the erp ( expressed as percent of the prestimulus baseline ) . ( g l ) same as ( a f ) , but for monkey a. figure 2a shows an example raw , unipolar lfp trace of one electrode in area v1 recorded while monkey p explored a photograph of 2 oranges . the stimulus replaced the fixation point at time zero and , within < 100 ms , the lfp was dominated by strongly rhythmic gamma - band oscillations . these lfp data were only filtered broadly , between 1 and 250 hz . the time frequency analysis of absolute power ( fig . 2b ) shows that the gamma - band activity was band - limited with a peak between 50 and 80 hz and largely sustained , with interruptions after saccades , as will be investigated in more detail later . figure 2c shows the change in spectral power during natural viewing when compared with the baseline , documenting a prominent gamma - band peak . figure 2d f shows the same example trial for a locally differentiated ( i.e. , bipolar ) lfp , demonstrating that the gamma - band response in this signal was very similar to the unipolar lfp . among all images , the oranges induced the strongest gamma - band oscillations . yet , clear gamma - band peaks could be seen in the power change spectra for all of the images and across most of the sites in v1 and v4 . to document that gamma - band activity during free viewing of natural images was robust , we show 1 ) spectra averaged over v1 sites , separately for 24 example images ( fig . 3 ) , 2 ) distributions of gamma power changes averaged over v1 sites and , corresponding p - values , separately for all images ( fig . 4 ) , 3 ) spectra averaged over all images , separately for example v1 , v4 , and nonvisual sites ( fig . 5 ) , 4 ) topographies of gamma power changes ( and corresponding t - values ) averaged over all images , characterizing gamma activity for each site across the ecog grid ( fig . figure 3 shows the power change spectra averaged over all sites in area v1 , separately for the 2 monkeys ( monkey p : red and monkey a : blue ) , for 12 grayscale and 12 color images . figure 4 documents the corresponding peak gamma power changes across all natural images and demonstrates that each image individually induced a highly significant gamma power enhancement . also , for each image , power enhancements during natural viewing compared with prestimulus baseline ( i.e. , log10[power during natural viewing / power during prestimulus baseline ] ) were significantly stronger in the gamma - frequency band ( 5080 hz ) when compared with a 150- to 200-hz band ( largest p - value was 0.0007 , paired t - test across trials ) , indicating that gamma power enhancements are most likely not due to broad - band power enhancements as have been found in other ecog recordings ( miller et al . figure 5a shows , for an example v1 site in monkey p , the power change spectrum averaged over all images . figure 5b shows a very similar gamma - band peak in an example site from area v4 . in contrast , 2 example sites in parietal and frontal cortex did not show enhanced gamma - band activity during natural viewing ( fig . figure 5e shows gamma - band power changes , and figure 5f the corresponding t - values [ comparing log(power ) between natural viewing and baseline ] across all sites in the ecog grid . the topographies reveal enhanced gamma - band power primarily in early - to - intermediate visual cortex covered by the subdural grid , that is , areas v1 , v2 , v4 , and teo . notably , outside the visual areas , natural viewing slightly reduced gamma - band power when compared with baseline . l confirms these results in monkey a. figure 2b had suggested that saccades interrupt ongoing gamma - band oscillations . to investigate this further , figure 6 shows time frequency analyses aligned to saccades . in both monkeys and in both areas v1 and v4 , the sustained presaccade gamma activity with peak power at frequencies of > 50 hz was , after the saccade , replaced by gamma with peak power at a frequency that slid downwards until a frequency of approximately 3040 hz was reached ( fig . this 30- to 40-hz activity peaked at a postsaccadic latency of 76 ms in v1 and 89 ms in v4 ( table 1 , upper part ) . the postsaccadic 30- to 40-hz activity co - occurred in time with a peak in the event - related potential ( erp ; fig . 6c , d , i , j ; table 1 , middle part ) . the time frequency analysis of the erp ( fig . 6e , f , k , l ; table 1 , lower part ) showed that the saccade - aligned erp accounted for only a small fraction of the 30- to 40-hz activity . table 1peak latencies of postsaccadic transientsv1 peak latency ( ms)v4 peak latency ( ms)tfr of lfp monkey p7888 monkey a7489 average76 388.5 1erp monkey p7295 monkey a7988 average75.5 591.5 1tfr of erp monkey p7099 monkey a7488 average72 393.5 8the upper part of the table gives the peak latencies of the postsaccadic transient in 3040 hz lfp power , separately for v1 and v4 , and for the 2 monkeys as well as their mean . the bottom part gives the peak latencies of the postsaccadic 30- to 40-hz power of the erp . peak latencies of postsaccadic transients the upper part of the table gives the peak latencies of the postsaccadic transient in 3040 hz lfp power , separately for v1 and v4 , and for the 2 monkeys as well as their mean . the bottom part gives the peak latencies of the postsaccadic 30- to 40-hz power of the erp . we found strong , sustained , and band - limited gamma - band activity in the visual cortex of 2 macaque monkeys during free viewing of natural images . in area v1 , this gamma - band activity was present for all natural images tested . enhanced gamma - band activity during natural viewing occurred over most of the recorded visual cortex , while it was largely absent over other regions . after saccades , the sustained gamma - band activity of 5080 hz was replaced by gamma - band activity with a peak frequency that slid downwards until a frequency of approximately 3040 hz was reached at around 80 ms postsaccade . the strongest gamma - band increases occurred in both monkeys for the orange and the banana photograph . thus , the particularly strong gamma - band activity might be due to the familiarity and/or the appetitive character of these stimuli . we found gamma - band activity during natural viewing with lfp recordings from an ecog grid , rather than , for example , with single - unit spike recordings . while this does not allow us to demonstrate gamma - band synchronization in spike trains directly , it is very likely that also the spikes showed gamma - band rhythms . the lfp recordings reflect mainly synaptic currents and the resulting membrane potential fluctuations ( mitzdorf 1985 ) . these synaptic activities , in turn , result from spiking activity , mostly from nearby cortical neurons . simultaneous recordings of spikes and lfps have documented that gamma - band power enhancements are typically accompanied by gamma - band , spike - field coherence ( fries , reynolds , et al . 2001 ; fries , schrder , et al . a previous study had investigated visual cortical responses to natural scenes , using recordings from awake cat area 18 , while the animals were presented with natural movies ( kayser et al . these movies induced activity that did not differ significantly from activity induced by visual pink noise , but it did differ strongly from gamma - band activity induced by gratings . we think that the discrepancy to our study is due to their use of a particular type of movies . as stated in that study , these [ movies ] were recorded from a camera mounted to a cat 's head while the animal was exploring different local environments such as forests and meadows [ ] . the motion of the cat 's head and the mounted camera resulted in strong and frequent movements of the entire image . it has been shown previously that such strong image motion transients lead to stimulus - locked lfp components , which replace the stimulus - induced , gamma - band response ( kruse and eckhorn 1996 ) . kruse and eckhorn had also investigated activity in cat primary visual cortex . when they presented smoothly moving stimuli , this induced gamma - band oscillations . in contrast , when they superimposed onto the smooth stimulus movement dynamically changing accelerations , gamma - band oscillations were replaced by stimulus - locked response components . thus , when visual stimuli are static or moving smoothly , visual cortex generates neuronal synchronization rhythmically in the gamma - frequency band ( friedman - hill et al . 2000 ) ; when the visual input entails unpredictable transients , neurons temporarily synchronize to these stimulus transients ( kruse and eckhorn 1996 ) . since synchronization is most likely crucial for the successful communication to postsynaptic targets ( fries 2009 ) , the intrinsically generated neuronal gamma - band synchronization to stimuli without transients is likely subserving their continued signaling , and the externally triggered neuronal synchronization to stimulus transients likely subserves the signaling of salient events in the visually observed world . typically , such salient events are the appearance or the motion onset of an object within the visual field . rarely is there a motion transient in our entire visual input due to motion of the entire visual world around us . in contrast , in the movies of kayser et al . , the motion transients regularly affected the entire visual field , as they were generated by movements of the camera mounted on the head of a cat , and they occurred very frequently if not continuously . we note that , during natural viewing , motion transients of the entire visual field do occur , mostly due to saccades . yet , those transients are generated by the visual system itself and are therefore dealt with in a different way , as is obvious from our perceptual experience . visual motion transients due to our own eye movements ( or body movements ) are , in healthy subjects , not experienced as movements of the external world . in contrast , the motion transients in the movies of kayser et al . are experienced as strong and frequent movements of the entire external world . another previous study used actual free viewing of natural images , like in our study , and recorded neuronal activity from area 17 of cebus monkeys ( ito et al . this study finds that lfp modulations in the beta frequency range are initiated in v1 with the beginning of saccades , and that this signal appears to modulate the timing of the onset of visually evoked spiking activity during fixations , leading to a locking of these first spikes to a specific phase of the lfp modulation . ( 2009 ) , particularly with regard to the presence of gamma - band activity in the bosman et al . they suggest that the absence of gamma in their study might be a genuine consequence of the free viewing of natural images , whereas the presence of gamma in the bosman et al . study might be due to stimulation with high contrast gratings and to fixation with attention to a peripheral target . our current study shows that , also during free viewing of natural images , strong gamma - band activity is present in early and intermediate visual cortices . it could be argued that one reason for this discrepancy is our use of rhesus monkeys , while ito et al . however , we think that this species difference is very unlikely to cause the difference , because gamma - band activity has been described across many species , including mouse ( nase et al . 2003 ) and human ( hoogenboom et al . 2006 , 2010 ) , that is , species separated much farther than cebus and rhesus monkeys . rather , the predominance of beta - band ( 1316 hz ) activity in the ito et al . the paper does not specify the laminar origin of the signals , yet another recent study in monkey area v1 demonstrated stimulus - induced 6- to 16-hz activity to predominate in deep layers ( buffalo et al . the ecog signal most likely results from the currents flowing in the tissue under the ecog electrode . while this signal does not allow to attribute the recorded neuronal currents to particular layers , it is certainly reflecting also the superficial layers , where gamma - band activity is particularly strong ( maier et al . the results described here suggest that gamma - band synchronization is present generally in the visual cortex during natural viewing . this is of great importance for proposals suggesting functional roles for gamma - band synchronization ( singer and gray 1995 ; engel et al . our results suggest that those proposals do not only apply to artificial laboratory conditions , but that they apply also to natural viewing . future studies will need to test those proposals directly under natural viewing conditions . combining natural viewing with proper operationalization of the investigated cognitive functions and this work was supported by grants from the european science foundation ( european young investigator award to p.f . ) , the european union ( health - f2 - 2008 - 200728 to p.f . ) , the loewe program ( neuronale koordination forschungsschwerpunkt frankfurt to p.f . ) , the netherlands organization for scientific research ( vici grant 453 - 04 - 002 to p.d.w . and veni grant 451 - 09 - 025 to m.r . ) , and the smart mix program of the netherlands ministry of economic affairs and the netherlands ministry of education , culture , and science ( braingain to r.o . and p.f . ) . funding to pay the open access publication charges for this article was provided by the ernst strngmann institute ( esi ) for neuroscience in cooperation with max planck society .

gamma - band activity in visual cortex has been implicated in several cognitive operations , like perceptual grouping and attentional selection . so far , it has been studied primarily under well - controlled visual fixation conditions and using well - controlled stimuli , like isolated bars or patches of grating . if gamma - band activity is to subserve its purported functions outside of the laboratory , it should be present during natural viewing conditions . we recorded neuronal activity with a 252-channel electrocorticographic ( ecog ) grid covering large parts of the left hemisphere of 2 macaque monkeys , while they freely viewed natural images . we found that natural viewing led to pronounced gamma - band activity in the visual cortex . in area v1 , gamma - band activity during natural viewing showed a clear spectral peak indicative of oscillatory activity between 50 and 80 hz and was highly significant for each of 65 natural images . across the ecog grid , gamma - band activity during natural viewing was present over most of the recorded visual cortex and absent over most remaining cortex . after saccades , the gamma peak frequency slid down to 3040 hz at around 80 ms postsaccade , after which the sustained 50- to 80-hz gamma - band activity resumed . we propose that gamma - band activity plays an important role during natural viewing .

Introduction Materials and Methods Stimuli and Behavioral Paradigm Data Acquisition Data Analysis and Statistical Testing Results Discussion Funding

neuronal gamma - band synchronization in visual cortex has been associated with several important functions , particularly perceptual grouping and selection ( eckhorn et al . if gamma - band synchronization plays a role in stimulus processing , perceptual grouping , or selective attention during natural viewing , it needs to be present during free viewing of natural images . so far , this has not been demonstrated , but to the contrary , several studies strongly suggest that free viewing of natural images eliminates gamma - band activity . subsequently , they presented these movies to an awake head - fixed cat and recorded neuronal activity from primary visual cortex . they report that such movies primarily induce broad - band local field potential ( lfp ) power increases between 100 and 200 hz , which differ strongly from band - limited gamma ( 4080 hz ) power increases that they find for grating stimuli , and rather resemble power changes that they observe when presenting pink - noise stimuli . another recent study investigated single units and lfp in area v1 of cebus monkeys freely viewing natural images ( ito et al . yet , as an example , they also show a time 1c ) and , while it reveals alpha- and beta - band modulations , there are no clear gamma - band peaks . the 2 panels are rendered from photographs taken during the electrode implantation surgeries , after placement of the ecog grid onto the brain , before closure of the dura . the 2 panels are rendered from photographs taken during the electrode implantation surgeries , after placement of the ecog grid onto the brain , before closure of the dura . other studies demonstrated that gamma - band activity is strongly reduced for grating stimuli of reduced size ( gieselmann and thiele 2008 ) or for 2 gratings superimposed to form a plaid ( lima et al . natural images often contain small and/or superimposed components and might , therefore , induce only marginal gamma - band activity . finally , the frequency of gamma - band activity can shift by at least 25 hz upon changes in stimulus properties like size ( gieselmann and thiele 2008 ) or contrast ( ray and maunsell 2010 ; roberts et al . therefore , natural images with their wide range of object sizes and contrasts might lead to local gamma - band activities with a wide range of frequencies , which might not summate effectively and thereby lead to merely weak and broad - band power increases . since an absence of gamma - band activity during free viewing of natural images we recorded lfp from electrocorticographic ( ecog ) grid electrodes covering areas v1 , v4 , and several additional areas of the left hemisphere in 2 macaque monkeys freely viewing natural images . for the data reported in this study , monkeys were required to fixate for 0.63 s on a fixation point ( 0.12 by 0.12 black square ) centered on a gray background , after which a natural image was presented , which was again centered on the background screen . due to placement of the ecog grid onto the dorsal parts of v1 and v4 in the left hemisphere , correspondingly , we accepted analysis epochs when the gaze of the monkey was at least 4 away from the lower and the right border of the natural image for at least 90% of the epoch duration . for most of the electrodes , , some of the most anterior electrodes assigned to v1 might as well be over v2 , and the most lateral electrodes assigned to v4 might as well be over the temporal - occipital area ( teo ) . in figure 2b , the analysis covered 20140 hz in steps of 2 hz , using for each frequency an epoch length of 4 cycles . figures 3 and 4 are based on all nonoverlapping 0.5 s epochs from 0.3 s after stimulus onset until stimulus offset , during which the receptive fields ( rfs ) remained on the image ( see above for details ) , compared with a 0.5-s prestimulus baseline . for figure 6 , we selected those v1 and v4 sites that showed the top third ( v1 ) and top half ( v4 ) gamma - band power increases during natural viewing . the gamma - frequency band was defined as 5080 hz in both monkeys . each panel shows the natural image presented to the animal and the corresponding spectra ( red : monkey p , blue : monkey a ) of power changes in percent , comparing free viewing of the respective image with a prestimulus baseline , averaged over all v1 sites ( shaded regions indicate 1 standard error of the mean , sem ) . the spectra were scaled such that the lower end of the y - axis corresponds to zero change and the upper end corresponds to the percent change value that is indicated in the upper - left corner of the respective panel . ( c ) histograms of the negative decadic logarithm of the p - values derived from 2-sided paired t - tests comparing gamma - band power ( 5080 hz ) between natural viewing and prestimulus fixation baseline , across trials . ( g l ) same analyses as in ( a f ) , but for monkey a. the gamma - band was defined as 5080 hz in both monkeys . frequency analysis of power ( expressed as percent change from prestimulus baseline ) in area v1 around the time of a saccade of monkey p. the saccade is indicated by the vertical black line at time zero ( moment of peak saccade velocity ) . ( c ) saccade - aligned erp ( averaged after single - trial rectification ) in area v1 . ( g l ) same as ( a f ) , but for monkey a. gamma - band oscillation in the raw lfp of one example site during one example visual exploration . ( a ) raw unipolar lfp trace during one visual exploration of the photograph of 2 oranges . each panel shows the natural image presented to the animal and the corresponding spectra ( red : monkey p , blue : monkey a ) of power changes in percent , comparing free viewing of the respective image with a prestimulus baseline , averaged over all v1 sites ( shaded regions indicate 1 standard error of the mean , sem ) . ( a ) histograms of changes in peak gamma - band power across all grayscale images , separately for monkey p ( red ) and monkey a ( blue ) . ( c ) histograms of the negative decadic logarithm of the p - values derived from 2-sided paired t - tests comparing gamma - band power ( 5080 hz ) between natural viewing and prestimulus fixation baseline , across trials . ( f ) topographical distribution of the corresponding t - values for the comparison of free viewing of natural images versus baseline . ( g l ) same analyses as in ( a f ) , but for monkey a. the gamma - band was defined as 5080 hz in both monkeys . ( a ) time frequency analysis of power ( expressed as percent change from prestimulus baseline ) in area v1 around the time of a saccade of monkey p. the saccade is indicated by the vertical black line at time zero ( moment of peak saccade velocity ) . due to placement of the ecog grid onto the dorsal parts of v1 and v4 in the left hemisphere , receptive fields were in the lower right visual quadrant . this ensured that the responses of the recorded sites were due to the natural image rather than the screen background . for most of the electrodes , , some of the most anterior electrodes assigned to v1 might as well be over v2 , and the most lateral electrodes assigned to v4 might as well be over the temporal - occipital area ( teo ) . in figure 2b , the analysis covered 20140 hz in steps of 2 hz , using for each frequency an epoch length of 4 cycles . for figure 6 , we selected those v1 and v4 sites that showed the top third ( v1 ) and top half ( v4 ) gamma - band power increases during natural viewing . the gamma - frequency band was defined as 5080 hz in both monkeys . where power during free viewing of natural images was normalized by power during the prestimulus baseline , the respective power values were first averaged across trials , separately for the 2 epochs , before normalization . ( a ) raw unipolar lfp trace during one visual exploration of the photograph of 2 oranges . the spectra were scaled such that the lower end of the y - axis corresponds to zero change and the upper end corresponds to the percent change value that is indicated in the upper - left corner of the respective panel . ( a ) histograms of changes in peak gamma - band power across all grayscale images , separately for monkey p ( red ) and monkey a ( blue ) . ( c ) histograms of the negative decadic logarithm of the p - values derived from 2-sided paired t - tests comparing gamma - band power ( 5080 hz ) between natural viewing and prestimulus fixation baseline , across trials . ( f ) topographical distribution of the corresponding t - values for the comparison of free viewing of natural images versus baseline . ( g l ) same analyses as in ( a f ) , but for monkey a. the gamma - band was defined as 5080 hz in both monkeys . frequency analysis of power ( expressed as percent change from prestimulus baseline ) in area v1 around the time of a saccade of monkey p. the saccade is indicated by the vertical black line at time zero ( moment of peak saccade velocity ) . ( c ) saccade - aligned erp ( averaged after single - trial rectification ) in area v1 . ( g l ) same as ( a f ) , but for monkey a. gamma - band oscillation in the raw lfp of one example site during one example visual exploration . ( a ) raw unipolar lfp trace during one visual exploration of the photograph of 2 oranges . each panel shows the natural image presented to the animal and the corresponding spectra ( red : monkey p , blue : monkey a ) of power changes in percent , comparing free viewing of the respective image with a prestimulus baseline , averaged over all v1 sites ( shaded regions indicate 1 standard error of the mean , sem ) . the spectra were scaled such that the lower end of the y - axis corresponds to zero change and the upper end corresponds to the percent change value that is indicated in the upper - left corner of the respective panel . ( c ) histograms of the negative decadic logarithm of the p - values derived from 2-sided paired t - tests comparing gamma - band power ( 5080 hz ) between natural viewing and prestimulus fixation baseline , across trials . ( g l ) same analyses as in ( a f ) , but for monkey a. the gamma - band was defined as 5080 hz in both monkeys . ( a ) time frequency analysis of power ( expressed as percent change from prestimulus baseline ) in area v1 around the time of a saccade of monkey p. the saccade is indicated by the vertical black line at time zero ( moment of peak saccade velocity ) . ( c ) saccade - aligned erp ( averaged after single - trial rectification ) in area v1 . ( g l ) same as ( a f ) , but for monkey a. figure 2a shows an example raw , unipolar lfp trace of one electrode in area v1 recorded while monkey p explored a photograph of 2 oranges . the stimulus replaced the fixation point at time zero and , within < 100 ms , the lfp was dominated by strongly rhythmic gamma - band oscillations . 2b ) shows that the gamma - band activity was band - limited with a peak between 50 and 80 hz and largely sustained , with interruptions after saccades , as will be investigated in more detail later . figure 2c shows the change in spectral power during natural viewing when compared with the baseline , documenting a prominent gamma - band peak . , bipolar ) lfp , demonstrating that the gamma - band response in this signal was very similar to the unipolar lfp . among all images , the oranges induced the strongest gamma - band oscillations . yet , clear gamma - band peaks could be seen in the power change spectra for all of the images and across most of the sites in v1 and v4 . to document that gamma - band activity during free viewing of natural images was robust , we show 1 ) spectra averaged over v1 sites , separately for 24 example images ( fig . 5 ) , 4 ) topographies of gamma power changes ( and corresponding t - values ) averaged over all images , characterizing gamma activity for each site across the ecog grid ( fig . figure 3 shows the power change spectra averaged over all sites in area v1 , separately for the 2 monkeys ( monkey p : red and monkey a : blue ) , for 12 grayscale and 12 color images . figure 4 documents the corresponding peak gamma power changes across all natural images and demonstrates that each image individually induced a highly significant gamma power enhancement . also , for each image , power enhancements during natural viewing compared with prestimulus baseline ( i.e. , log10[power during natural viewing / power during prestimulus baseline ] ) were significantly stronger in the gamma - frequency band ( 5080 hz ) when compared with a 150- to 200-hz band ( largest p - value was 0.0007 , paired t - test across trials ) , indicating that gamma power enhancements are most likely not due to broad - band power enhancements as have been found in other ecog recordings ( miller et al . in contrast , 2 example sites in parietal and frontal cortex did not show enhanced gamma - band activity during natural viewing ( fig . figure 5e shows gamma - band power changes , and figure 5f the corresponding t - values [ comparing log(power ) between natural viewing and baseline ] across all sites in the ecog grid . the topographies reveal enhanced gamma - band power primarily in early - to - intermediate visual cortex covered by the subdural grid , that is , areas v1 , v2 , v4 , and teo . notably , outside the visual areas , natural viewing slightly reduced gamma - band power when compared with baseline . in both monkeys and in both areas v1 and v4 , the sustained presaccade gamma activity with peak power at frequencies of > 50 hz was , after the saccade , replaced by gamma with peak power at a frequency that slid downwards until a frequency of approximately 3040 hz was reached ( fig . the postsaccadic 30- to 40-hz activity co - occurred in time with a peak in the event - related potential ( erp ; fig . peak latencies of postsaccadic transients the upper part of the table gives the peak latencies of the postsaccadic transient in 3040 hz lfp power , separately for v1 and v4 , and for the 2 monkeys as well as their mean . we found strong , sustained , and band - limited gamma - band activity in the visual cortex of 2 macaque monkeys during free viewing of natural images . in area v1 , this gamma - band activity was present for all natural images tested . enhanced gamma - band activity during natural viewing occurred over most of the recorded visual cortex , while it was largely absent over other regions . after saccades , the sustained gamma - band activity of 5080 hz was replaced by gamma - band activity with a peak frequency that slid downwards until a frequency of approximately 3040 hz was reached at around 80 ms postsaccade . the strongest gamma - band increases occurred in both monkeys for the orange and the banana photograph . thus , the particularly strong gamma - band activity might be due to the familiarity and/or the appetitive character of these stimuli . we found gamma - band activity during natural viewing with lfp recordings from an ecog grid , rather than , for example , with single - unit spike recordings . while this does not allow us to demonstrate gamma - band synchronization in spike trains directly , it is very likely that also the spikes showed gamma - band rhythms . simultaneous recordings of spikes and lfps have documented that gamma - band power enhancements are typically accompanied by gamma - band , spike - field coherence ( fries , reynolds , et al . these movies induced activity that did not differ significantly from activity induced by visual pink noise , but it did differ strongly from gamma - band activity induced by gratings . it has been shown previously that such strong image motion transients lead to stimulus - locked lfp components , which replace the stimulus - induced , gamma - band response ( kruse and eckhorn 1996 ) . when they presented smoothly moving stimuli , this induced gamma - band oscillations . in contrast , when they superimposed onto the smooth stimulus movement dynamically changing accelerations , gamma - band oscillations were replaced by stimulus - locked response components . thus , when visual stimuli are static or moving smoothly , visual cortex generates neuronal synchronization rhythmically in the gamma - frequency band ( friedman - hill et al . since synchronization is most likely crucial for the successful communication to postsynaptic targets ( fries 2009 ) , the intrinsically generated neuronal gamma - band synchronization to stimuli without transients is likely subserving their continued signaling , and the externally triggered neuronal synchronization to stimulus transients likely subserves the signaling of salient events in the visually observed world . , the motion transients regularly affected the entire visual field , as they were generated by movements of the camera mounted on the head of a cat , and they occurred very frequently if not continuously . we note that , during natural viewing , motion transients of the entire visual field do occur , mostly due to saccades . in contrast , the motion transients in the movies of kayser et al . another previous study used actual free viewing of natural images , like in our study , and recorded neuronal activity from area 17 of cebus monkeys ( ito et al . this study finds that lfp modulations in the beta frequency range are initiated in v1 with the beginning of saccades , and that this signal appears to modulate the timing of the onset of visually evoked spiking activity during fixations , leading to a locking of these first spikes to a specific phase of the lfp modulation . ( 2009 ) , particularly with regard to the presence of gamma - band activity in the bosman et al . they suggest that the absence of gamma in their study might be a genuine consequence of the free viewing of natural images , whereas the presence of gamma in the bosman et al . our current study shows that , also during free viewing of natural images , strong gamma - band activity is present in early and intermediate visual cortices . it could be argued that one reason for this discrepancy is our use of rhesus monkeys , while ito et al . however , we think that this species difference is very unlikely to cause the difference , because gamma - band activity has been described across many species , including mouse ( nase et al . rather , the predominance of beta - band ( 1316 hz ) activity in the ito et al . the paper does not specify the laminar origin of the signals , yet another recent study in monkey area v1 demonstrated stimulus - induced 6- to 16-hz activity to predominate in deep layers ( buffalo et al . while this signal does not allow to attribute the recorded neuronal currents to particular layers , it is certainly reflecting also the superficial layers , where gamma - band activity is particularly strong ( maier et al . the results described here suggest that gamma - band synchronization is present generally in the visual cortex during natural viewing . future studies will need to test those proposals directly under natural viewing conditions . combining natural viewing with proper operationalization of the investigated cognitive functions and this work was supported by grants from the european science foundation ( european young investigator award to p.f . )

gaseous microemboli are a concern during surgery with cardiopulmonary bypass as potential cause of neurocognitive deficit since these microbubbles were verified by ultrasonic microbubble counters operating with blood flow rates up to 6 l / min [ 1 , 2 ] . in haemodialysis ( hd ) with much lower blood flow rates ( up to 0.6 l / min ) , microbubbles have also been a concern , but proper measurement was difficult . microemboli were detected in the subclavian vein downstream from the arteriovenous fistula of hd patients a decade ago [ 3 , 4 ] . it was speculated that microbubbles lodge in the capillary bed of various organs , mainly the lungs , and that they obstruct the blood flow in the capillaries , causing tissue ischaemia and micro - infarct , followed by inflammatory reaction , complement activation and tissue damage . patients who undergo surgery with cardiopulmonary bypass are usually treated only once , whereas hd patients are repeatedly treated , in total > 600 h each year . any infusion of microbubbles during hd may become significant for the patient 's health due to the cumulative exposure time . when it became technically possible to measure microbubbles in hd , it was demonstrated that they may pass the venous bubble catcher and the safety system against air infusion without triggering an alarm , in vitro and during treatments . they observed a significantly higher number of patients with elevated embolic counts on the arterial side than would be expected if an open foramen ovale was the only option , and concluded that a considerable number of microemboli seem to pass the lung . two years later , a case report of a chronic hd patient was published : he suffered from cardiac arrest and died . the autopsy revealed a closed foramen ovale ; the microscopic investigation verified microembolies of air that were surrounded by fibrin in the lungs , the heart and the brain . the investigators concluded that microbubbles were present before death , passed the cardiopulmonary circulation , entered the left heart and were distributed throughout the body . autopsies of five patients ( died either during or up to 2.3 days after the hd session ) revealed pulmonary fibrosis and pulmonary microemboli ( fibrin around air bubbles ) . a study by british investigators confirmed microemboli in the arteriovenous fistulae of hd patients during their treatment , but found no evidence of microembolization in the middle cerebral artery . microbubbles were determined at the site of the arteriovenous access for different blood levels in the venous bubble catcher in a randomized , double - blinded study : the lowest number of microbubbles was observed during high blood levels , confirmed in a second study . recently , an australian group published a computational analysis of microbubbles in hd : most bubbles with a diameter of < 50 m , and many bubbles in the range of 50200 m pass through the venous bubble catcher . it seems that microbubbles generally occur in hd treatments . up to now , the available information on the potentially harmful microbubbles in hd is not consistent . the aim of our investigation was to detect possible sources of the generated microbubbles and to increase the knowledge on microbubble formation . the study was designed as purely observational , non - interventional investigation in a single centre , without documentation of patient data . twenty - nine treatments of 21 patients were observed , covering a broad range of patient and treatment conditions ( types of blood access , treatment modes , blood flow rates and arterial pressures ) . the applied diagnostic medical device was the non - invasive , bubble counter bcc200 ( gampt gmbh , merseburg , germany ) , a pulsed ultrasonic doppler system with a transmission frequency of 2 mhz , measuring microbubbles in the range of 10500 m . the applied dialysis machines were 5008 haemodialysis systems ( fresenius medical care , germany ) . two types of dialysers were applied : xevonta hi23 ( bbraun , germany ) and fx1000 ( fresenius medical care , germany ) . the standard position of the two sensor heads of the microbubble counter was on the arterial line ( between arterial access and blood pump ) and on the venous line ( between venous bubble catcher and venous access ) . for a consistency check , the arterial and the venous sensor heads of the bcc200 were positioned during a stable treatment at the same blood line in a row . the number of measured microbubbles differed by 8% in the normal and in reversed orders ; thus , the two sensor heads give identical information with a deviation below 10% . some air reservoirs in the blood tubing were obvious , such as in the venous bubble catcher . other potential air reservoirs were magnitudes smaller ; in rare cases , air bubbles with a diameter of a few millimetres were seen , and in most cases , air bubbles were found only after attentive search due to their extremely small size . . the usually high level of blood was not changed due to the purely observational character of our study . bubbles were found at the upper part of the dialyser on the dialysate side, at the dialyser caps on the blood side of the dialyser, in the blood line segment of the roller pump, in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . at the upper part of the dialyser on the dialysate side , at the dialyser caps on the blood side of the dialyser , in the blood line segment of the roller pump , in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . measurements with the bubble counter usually revealed a broad range of sizes , with bubble diameters between 10 and 300 m , mostly between 10 and 200 m . it was repeatedly observed that the start of the blood pump was accompanied by an increase in the bubble count , higher than the level when the blood pump was running continuously . in a single case , we could follow a small visible air volume moving through the heparin line into the main blood line ; this volume vanished downstream of the dialyser , but instead 33 microbubbles were detected by the microbubble counter . obviously , the visible volume had become the source of many microbubbles . in a single case , the two sensor heads were positioned up- and downstream of the arterial luer lock connector . the downstream sensor head measured 46 microbubbles and the upstream sensor head only 2 , indicating that small amounts of air were entering the blood line at this connection . at this part of the tubing system , as planned , a broad range of treatment conditions could be investigated , including the treatment modes online haemodiafiltration and hd , one single - needle treatment , access via cimino fistula , teflon shunt or catheter . arterial pressures were observed in the range between 230 and 80 mmhg . in total , measurements with complete data sets were obtained from 29 treatments , including 21 patients , with an observed treatment time summed up to 7871 min ( 131 h ) . we observed a correlation between the count of microbubbles and the blood flow rate and the arterial pressure , see figures 1 and 2 . the microbubble rate increased with the blood flow rate and the negative arterial pressure . 1.microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . fig . 2.microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . an overview of our observational results concerning microbubble rate is shown in figure 3 . the data were grouped first according to the treatment mode , hd or online haemodiafiltration , and then according to blood access and cannula diameter . additionally , the largest group of 14 treatments with identical blood access , cannula diameter and treatment mode was divided into three groups by arterial pressure . 3.microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm . the number of patients ( n ) as well as the number of treatments ( n ) per group is noted . for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below . microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm . the number of patients ( n ) as well as the number of treatments ( n ) per group is noted . for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below . generally , a large difference between the groups was seen with a spread in microbubble rate up to a factor of 5 . microbubble rates below 40 per minute were observed for hd treatments with catheters at blood flow rates of 200 ml / min ( 1st bar ) , and online haemodiafiltration treatments in patients with goretex shunts ( 3rd bar ) or cimino fistulas with mean blood flow rates < 360 ml / min ( 5th and 6th bars ) . at similar conditions concerning blood access , cannula diameter , blood flow rate and arterial pressure , a much higher microbubble rate was observed during hd than during online haemodiafiltration ( 2nd bar versus 6th bar ) . this observation was possibly due to the substitution fluid applied during online haemodiafiltration , prepared from degassed dialysate . the highest microbubble rates were observed for high blood flow rates and low arterial pressures ( 4th and 7th bars ) . a low arterial pressure ( 210 mmhg ) was observed ( although a large cannula diameter was applied : 2.0 mm ) at the very high blood flow rate ( 550 ml / min ) . at a lower blood flow rate ( 376 ml / min ) with a lower cannula diameter ( 1.8 mm ) , the arterial pressure was also low ( 198 mmhg ) . these observations indicate that a high blood flow rate and a low arterial pressure may lead to a high microbubble rate . a large spread was observed also concerning the number and volume of the injected microbubbles . the lowest microbubble rate was observed in patients with goretex shunts , a cannula diameter of 1.8 mm , treated by online haemodiafiltration at a medium blood flow rate ( 339 ml / min ) and a moderate arterial pressure ( 112 mmhg ) , resulting in a mean value of 3723 microbubbles per treatment with a total volume of 17 l . the highest microbubble rate was observed in patients with cimino fistula , a cannula diameter of 2.0 mm , treated by online haemodiafiltration at a high blood flow rate ( 550 ml / min ) and a low arterial pressure ( 210 mmhg ) , resulting in a mean of 29 889 microbubbles per treatment with a total volume of 232 l . some air reservoirs in the blood tubing were obvious , such as in the venous bubble catcher . other potential air reservoirs were magnitudes smaller ; in rare cases , air bubbles with a diameter of a few millimetres were seen , and in most cases , air bubbles were found only after attentive search due to their extremely small size . . the usually high level of blood was not changed due to the purely observational character of our study . bubbles were found at the upper part of the dialyser on the dialysate side, at the dialyser caps on the blood side of the dialyser, in the blood line segment of the roller pump, in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . at the upper part of the dialyser on the dialysate side , at the dialyser caps on the blood side of the dialyser , in the blood line segment of the roller pump , in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . measurements with the bubble counter usually revealed a broad range of sizes , with bubble diameters between 10 and 300 m , mostly between 10 and 200 m . it was repeatedly observed that the start of the blood pump was accompanied by an increase in the bubble count , higher than the level when the blood pump was running continuously . in a single case , we could follow a small visible air volume moving through the heparin line into the main blood line ; this volume vanished downstream of the dialyser , but instead 33 microbubbles were detected by the microbubble counter . obviously , the visible volume had become the source of many microbubbles . in a single case , the two sensor heads were positioned up- and downstream of the arterial luer lock connector . the downstream sensor head measured 46 microbubbles and the upstream sensor head only 2 , indicating that small amounts of air were entering the blood line at this connection . at this part of the tubing system , as planned , a broad range of treatment conditions could be investigated , including the treatment modes online haemodiafiltration and hd , one single - needle treatment , access via cimino fistula , teflon shunt or catheter . arterial pressures were observed in the range between 230 and 80 mmhg . in total , measurements with complete data sets were obtained from 29 treatments , including 21 patients , with an observed treatment time summed up to 7871 min ( 131 h ) . we observed a correlation between the count of microbubbles and the blood flow rate and the arterial pressure , see figures 1 and 2 . the microbubble rate increased with the blood flow rate and the negative arterial pressure . 1.microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . fig . 2.microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . an overview of our observational results concerning microbubble rate is shown in figure 3 . the data were grouped first according to the treatment mode , hd or online haemodiafiltration , and then according to blood access and cannula diameter . additionally , the largest group of 14 treatments with identical blood access , cannula diameter and treatment mode was divided into three groups by arterial pressure . 3.microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm . the number of patients ( n ) as well as the number of treatments ( n ) per group is noted . for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below . microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm . the number of patients ( n ) as well as the number of treatments ( n ) per group is noted . for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below . generally , a large difference between the groups was seen with a spread in microbubble rate up to a factor of 5 . microbubble rates below 40 per minute were observed for hd treatments with catheters at blood flow rates of 200 ml / min ( 1st bar ) , and online haemodiafiltration treatments in patients with goretex shunts ( 3rd bar ) or cimino fistulas with mean blood flow rates < 360 ml / min ( 5th and 6th bars ) . at similar conditions concerning blood access , cannula diameter , blood flow rate and arterial pressure , a much higher microbubble rate was observed during hd than during online haemodiafiltration ( 2nd bar versus 6th bar ) . this observation was possibly due to the substitution fluid applied during online haemodiafiltration , prepared from degassed dialysate . the highest microbubble rates were observed for high blood flow rates and low arterial pressures ( 4th and 7th bars ) . a low arterial pressure ( 210 mmhg ) was observed ( although a large cannula diameter was applied : 2.0 mm ) at the very high blood flow rate ( 550 ml / min ) . at a lower blood flow rate ( 376 ml / min ) with a lower cannula diameter ( 1.8 mm ) , the arterial pressure was also low ( 198 mmhg ) . these observations indicate that a high blood flow rate and a low arterial pressure may lead to a high microbubble rate . a large spread was observed also concerning the number and volume of the injected microbubbles . the lowest microbubble rate was observed in patients with goretex shunts , a cannula diameter of 1.8 mm , treated by online haemodiafiltration at a medium blood flow rate ( 339 ml / min ) and a moderate arterial pressure ( 112 mmhg ) , resulting in a mean value of 3723 microbubbles per treatment with a total volume of 17 l . the highest microbubble rate was observed in patients with cimino fistula , a cannula diameter of 2.0 mm , treated by online haemodiafiltration at a high blood flow rate ( 550 ml / min ) and a low arterial pressure ( 210 mmhg ) , resulting in a mean of 29 889 microbubbles per treatment with a total volume of 232 l . our observations increased the knowledge on formation kinetic and quantification of microbubbles in hd . in interventional studies , more microbubbles were observed for a low blood level in the venous bubble catcher than for a high level [ 7 , 12 , 13 ] . air might be dragged below the blood level , and at low blood levels , the buoyant force might not be sufficient to separate small bubbles from downwards flowing blood . then , small bubbles may be injected into the patient [ 6 , 7 , 1214 ] . due to the non - interventional character of our study , the blood level in the venous bubble catcher remained without change ; we could not observe that the air above the high blood level contributes to the formation of microbubbles . the persistent air bubbles found on the dialysate side of the dialyser did not vanish possibly due to a slow degradation of large bubbles at the air fluid interface by the degassed dialysate ( the dissolution time for air bubbles in water strongly increases with larger bubble diameter ) . the observed split of a visible bubble into smaller fragments that were no longer visible gave cause for three concerns concerning patient safety . first , as already demonstrated [ 6 , 7 , 1214 ] , microbubbles may pass the safety system against air infusion without triggering an alarm . second , microbubbles may be transported into the patient , and if small enough , pass the lung capillaries , and be distributed throughout the body . larger gas bubbles have a higher dissolution time and a higher chance to get stuck in a capillary . moreover , microbubbles may increase or decrease depending on surrounding pressure ( e.g. sparkling water effect ) . consequently , different scenarios concerning the lifetime of microbubbles inside the human body are possible . the microbubbles measured upstream of the arterial luer lock connector were possibly a remnant of these microbubbles inside the patient 's body . third , the total surface area increases . if a bubble is divided into smaller bubbles , then the surface area increases ( e.g. for a split into 64 bubbles , the surface area increases by the factor of 4 ) . each increase in the surface area may also mean an increase in interaction of air with blood and the endothelium of the capillaries . microbubbles may originate from the remaining air of an incomplete initial filling with insufficient rinsing of the extracorporeal circuit . if the remaining air was the only source of the microbubbles , then the microbubble count should monotonously decrease . in the beginning of the treatments , the mean microbubble count was an order of magnitude larger than during the rest of the treatment . even in long treatments ( e.g. nocturnal dialysis ) , the number of microbubbles was not decreasing towards the end of the treatment , indicating either other sources of microbubble generation or large stores of initially not removed air somewhere in the blood tubing . an increase in bubble count was repeatedly observed at the start of the blood pump , possibly due to a sudden displacement of the remaining bubbles in the blood line , suction of bubbles from a sideline ( e.g. heparin line ) or from the roller pump segment into the main line . our simultaneous measurement up- and downstream of the arterial luer lock connector indicated that , in the negative pressure region , small amounts of air may enter the blood line . this single observation at the arterial connector did not allow generalization concerning prevalence of incomplete tightness . the involved volume was so small that the safety system against air infusion did not react . generally , the tightness of luer lock connectors is under discussion [ 1720 ] . the substitution fluid is made from degassed dialysis fluid , thus should be counteracting the microbubbles ( e.g. by diluting ) . on the other hand , online haemodiafiltration is often performed with a high blood flow rate and a low negative arterial pressure . possibly , a high microbubble rate may not be due to the online haemodiafiltration per se , but due to a low negative arterial pressure ( e.g. by small cannula diameter ) . a large randomized clinical trial demonstrated the survival benefit of online haemodiafiltration ; a major influence of microbubbles as a confounding factor on the primary outcome parameter all - cause mortality was unlikely , because the blood flow rate was similar in both the study groups ( differences only between 2 and 5% ) . we did not observe treatments without microbubbles , although the measurements covered different conditions ; thus , it seems that microbubbles are always present during a standard hd treatment . this observation is in line with other investigations , and microbubbles were observed during treatments with the dialysis machines of all manufacturers [ 4 , 8 , 11 , 12 ] , indicating that microbubbles are a general side effect of hd . but the range of the microbubble counts was large ; obviously , in the standard hd setting , there are treatments with a low generation of microbubbles . there are several limitations of this non - interventional study : the study was performed in a single centre with a small number of patients and treatments . nevertheless , some preventive actions were identified ( partially mentioned in earlier studies ) : the cannula diameter should be adjusted to the blood flow rate. the arterial luer lock connector should be tightened. careful filling and sufficient rinsing in the preparation phase might reduce remaining gas bubbles. syringes filled with fluids for injections ( such as heparin or iron ) and side lines should be given special care. the blood in the venous bubble catcher should be kept at a high level [ 6 , 7 , 12 , 13]. extreme blood flow rates and arterial pressures should be avoided.from the visible bubbles ( at blood pump segment , dialyser cap , venous chamber and heparin line ) , the total volume was estimated to a few microliters , obviously not enough to be the only source of microbubbles . careful filling and sufficient rinsing in the preparation phase might reduce remaining gas bubbles . syringes filled with fluids for injections ( such as heparin or iron ) and side lines should be given special care . the blood in the venous bubble catcher should be kept at a high level [ 6 , 7 , 12 , 13 ] . systematic investigations in this field are scarce . considering the clinical observations , one might hypothesize that a degassing or cavitation phenomenon of blood may contribute in generating microbubbles , even if the threshold pressure gradient is not achieved . the results should be further investigated in sound laboratory experiments , in vitro and clinical studies . generally , during hd , microbubbles are infused in large numbers into the patients , but the involved volume is extremely small . if there is an impact on the patient 's health , it seems scarcely due to the miniscule volume of an individual microbubble , because this volume is many orders of magnitude below any volume inducing air embolism . the large surface areas of the microbubbles and their enormous number appear to be more eligible candidates . during hd , a large number of microbubbles may reach the patients , but the involved volume is extremely small . origin and pathophysiologic consequences of this phenomenon are not well understood but deserve further study and , if necessary , a new blood line design aiming to minimize blood air interfaces .

backgroundthe infusion of microbubbles as a side effect of haemodialysis was repeatedly demonstrated in recent publications , but the knowledge on the source of microbubbles and on microbubble formation is scarce.methodsmicrobubbles in the range of 10500 m were measured by a non - invasive bubble counter based on a pulsed ultrasonic doppler system in a non - interventional study of a single centre . totally , 29 measurements were performed in standard treatments covering a broad range of patient and treatment conditions ( types of blood access , treatment modes , blood flow rates and arterial pressures).resultsseveral possible sources of microbubbles could be identified such as an arterial luer lock connector at negative pressure and remnant bubbles from insufficient priming , but some sources of microbubbles remain unknown . microbubbles were found in all treatments , haemodialysis ( hd ) and online haemodiafiltration . the lowest average microbubble rates ( 17 16 microbubbles per minute ) were observed in patients treated by online haemodiafiltration at medium blood flow rates and moderate arterial pressures and the highest average microbubble rates ( 117 63 microbubbles per minute ) at high blood flow rates ( 550 ml / min ) and low arterial pressures ( 210 mmhg ) . generally , the microbubble rate correlated to both blood flow rate ( correlation coefficient r = 0.45 ) and negative arterial pressure ( r = 0.67).conclusionsmicrobubbles are a general side effect of hd ; origin and pathophysiologic consequences of this phenomenon are not well understood , and deserve further study .

Introduction Materials and methods Results Observation of visible bubbles Detection of microbubbles Discussion Conclusion Conflict of interest statement

gaseous microemboli are a concern during surgery with cardiopulmonary bypass as potential cause of neurocognitive deficit since these microbubbles were verified by ultrasonic microbubble counters operating with blood flow rates up to 6 l / min [ 1 , 2 ] . in haemodialysis ( hd ) with much lower blood flow rates ( up to 0.6 l / min ) , microbubbles have also been a concern , but proper measurement was difficult . it was speculated that microbubbles lodge in the capillary bed of various organs , mainly the lungs , and that they obstruct the blood flow in the capillaries , causing tissue ischaemia and micro - infarct , followed by inflammatory reaction , complement activation and tissue damage . the autopsy revealed a closed foramen ovale ; the microscopic investigation verified microembolies of air that were surrounded by fibrin in the lungs , the heart and the brain . a study by british investigators confirmed microemboli in the arteriovenous fistulae of hd patients during their treatment , but found no evidence of microembolization in the middle cerebral artery . microbubbles were determined at the site of the arteriovenous access for different blood levels in the venous bubble catcher in a randomized , double - blinded study : the lowest number of microbubbles was observed during high blood levels , confirmed in a second study . recently , an australian group published a computational analysis of microbubbles in hd : most bubbles with a diameter of < 50 m , and many bubbles in the range of 50200 m pass through the venous bubble catcher . the aim of our investigation was to detect possible sources of the generated microbubbles and to increase the knowledge on microbubble formation . the study was designed as purely observational , non - interventional investigation in a single centre , without documentation of patient data . twenty - nine treatments of 21 patients were observed , covering a broad range of patient and treatment conditions ( types of blood access , treatment modes , blood flow rates and arterial pressures ) . the applied diagnostic medical device was the non - invasive , bubble counter bcc200 ( gampt gmbh , merseburg , germany ) , a pulsed ultrasonic doppler system with a transmission frequency of 2 mhz , measuring microbubbles in the range of 10500 m . the standard position of the two sensor heads of the microbubble counter was on the arterial line ( between arterial access and blood pump ) and on the venous line ( between venous bubble catcher and venous access ) . for a consistency check , the arterial and the venous sensor heads of the bcc200 were positioned during a stable treatment at the same blood line in a row . the number of measured microbubbles differed by 8% in the normal and in reversed orders ; thus , the two sensor heads give identical information with a deviation below 10% . some air reservoirs in the blood tubing were obvious , such as in the venous bubble catcher . other potential air reservoirs were magnitudes smaller ; in rare cases , air bubbles with a diameter of a few millimetres were seen , and in most cases , air bubbles were found only after attentive search due to their extremely small size . bubbles were found at the upper part of the dialyser on the dialysate side, at the dialyser caps on the blood side of the dialyser, in the blood line segment of the roller pump, in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . at the upper part of the dialyser on the dialysate side , at the dialyser caps on the blood side of the dialyser , in the blood line segment of the roller pump , in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . measurements with the bubble counter usually revealed a broad range of sizes , with bubble diameters between 10 and 300 m , mostly between 10 and 200 m . it was repeatedly observed that the start of the blood pump was accompanied by an increase in the bubble count , higher than the level when the blood pump was running continuously . in a single case , we could follow a small visible air volume moving through the heparin line into the main blood line ; this volume vanished downstream of the dialyser , but instead 33 microbubbles were detected by the microbubble counter . obviously , the visible volume had become the source of many microbubbles . in a single case , the two sensor heads were positioned up- and downstream of the arterial luer lock connector . at this part of the tubing system , as planned , a broad range of treatment conditions could be investigated , including the treatment modes online haemodiafiltration and hd , one single - needle treatment , access via cimino fistula , teflon shunt or catheter . arterial pressures were observed in the range between 230 and 80 mmhg . we observed a correlation between the count of microbubbles and the blood flow rate and the arterial pressure , see figures 1 and 2 . the microbubble rate increased with the blood flow rate and the negative arterial pressure . 1.microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . 2.microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . the data were grouped first according to the treatment mode , hd or online haemodiafiltration , and then according to blood access and cannula diameter . additionally , the largest group of 14 treatments with identical blood access , cannula diameter and treatment mode was divided into three groups by arterial pressure . 3.microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm . for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below . microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm . for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below . generally , a large difference between the groups was seen with a spread in microbubble rate up to a factor of 5 . microbubble rates below 40 per minute were observed for hd treatments with catheters at blood flow rates of 200 ml / min ( 1st bar ) , and online haemodiafiltration treatments in patients with goretex shunts ( 3rd bar ) or cimino fistulas with mean blood flow rates < 360 ml / min ( 5th and 6th bars ) . at similar conditions concerning blood access , cannula diameter , blood flow rate and arterial pressure , a much higher microbubble rate was observed during hd than during online haemodiafiltration ( 2nd bar versus 6th bar ) . the highest microbubble rates were observed for high blood flow rates and low arterial pressures ( 4th and 7th bars ) . a low arterial pressure ( 210 mmhg ) was observed ( although a large cannula diameter was applied : 2.0 mm ) at the very high blood flow rate ( 550 ml / min ) . at a lower blood flow rate ( 376 ml / min ) with a lower cannula diameter ( 1.8 mm ) , the arterial pressure was also low ( 198 mmhg ) . these observations indicate that a high blood flow rate and a low arterial pressure may lead to a high microbubble rate . the lowest microbubble rate was observed in patients with goretex shunts , a cannula diameter of 1.8 mm , treated by online haemodiafiltration at a medium blood flow rate ( 339 ml / min ) and a moderate arterial pressure ( 112 mmhg ) , resulting in a mean value of 3723 microbubbles per treatment with a total volume of 17 l . the highest microbubble rate was observed in patients with cimino fistula , a cannula diameter of 2.0 mm , treated by online haemodiafiltration at a high blood flow rate ( 550 ml / min ) and a low arterial pressure ( 210 mmhg ) , resulting in a mean of 29 889 microbubbles per treatment with a total volume of 232 l . other potential air reservoirs were magnitudes smaller ; in rare cases , air bubbles with a diameter of a few millimetres were seen , and in most cases , air bubbles were found only after attentive search due to their extremely small size . bubbles were found at the upper part of the dialyser on the dialysate side, at the dialyser caps on the blood side of the dialyser, in the blood line segment of the roller pump, in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . at the upper part of the dialyser on the dialysate side , at the dialyser caps on the blood side of the dialyser , in the blood line segment of the roller pump , in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . measurements with the bubble counter usually revealed a broad range of sizes , with bubble diameters between 10 and 300 m , mostly between 10 and 200 m . it was repeatedly observed that the start of the blood pump was accompanied by an increase in the bubble count , higher than the level when the blood pump was running continuously . in a single case , we could follow a small visible air volume moving through the heparin line into the main blood line ; this volume vanished downstream of the dialyser , but instead 33 microbubbles were detected by the microbubble counter . obviously , the visible volume had become the source of many microbubbles . in a single case , the two sensor heads were positioned up- and downstream of the arterial luer lock connector . the downstream sensor head measured 46 microbubbles and the upstream sensor head only 2 , indicating that small amounts of air were entering the blood line at this connection . at this part of the tubing system , as planned , a broad range of treatment conditions could be investigated , including the treatment modes online haemodiafiltration and hd , one single - needle treatment , access via cimino fistula , teflon shunt or catheter . arterial pressures were observed in the range between 230 and 80 mmhg . we observed a correlation between the count of microbubbles and the blood flow rate and the arterial pressure , see figures 1 and 2 . the microbubble rate increased with the blood flow rate and the negative arterial pressure . 1.microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . 2.microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients . the data were grouped first according to the treatment mode , hd or online haemodiafiltration , and then according to blood access and cannula diameter . additionally , the largest group of 14 treatments with identical blood access , cannula diameter and treatment mode was divided into three groups by arterial pressure . 3.microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm . for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below . microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm . for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below . microbubble rates below 40 per minute were observed for hd treatments with catheters at blood flow rates of 200 ml / min ( 1st bar ) , and online haemodiafiltration treatments in patients with goretex shunts ( 3rd bar ) or cimino fistulas with mean blood flow rates < 360 ml / min ( 5th and 6th bars ) . at similar conditions concerning blood access , cannula diameter , blood flow rate and arterial pressure , a much higher microbubble rate was observed during hd than during online haemodiafiltration ( 2nd bar versus 6th bar ) . the highest microbubble rates were observed for high blood flow rates and low arterial pressures ( 4th and 7th bars ) . a low arterial pressure ( 210 mmhg ) was observed ( although a large cannula diameter was applied : 2.0 mm ) at the very high blood flow rate ( 550 ml / min ) . at a lower blood flow rate ( 376 ml / min ) with a lower cannula diameter ( 1.8 mm ) , the arterial pressure was also low ( 198 mmhg ) . these observations indicate that a high blood flow rate and a low arterial pressure may lead to a high microbubble rate . the lowest microbubble rate was observed in patients with goretex shunts , a cannula diameter of 1.8 mm , treated by online haemodiafiltration at a medium blood flow rate ( 339 ml / min ) and a moderate arterial pressure ( 112 mmhg ) , resulting in a mean value of 3723 microbubbles per treatment with a total volume of 17 l . the highest microbubble rate was observed in patients with cimino fistula , a cannula diameter of 2.0 mm , treated by online haemodiafiltration at a high blood flow rate ( 550 ml / min ) and a low arterial pressure ( 210 mmhg ) , resulting in a mean of 29 889 microbubbles per treatment with a total volume of 232 l . our observations increased the knowledge on formation kinetic and quantification of microbubbles in hd . in interventional studies , more microbubbles were observed for a low blood level in the venous bubble catcher than for a high level [ 7 , 12 , 13 ] . air might be dragged below the blood level , and at low blood levels , the buoyant force might not be sufficient to separate small bubbles from downwards flowing blood . due to the non - interventional character of our study , the blood level in the venous bubble catcher remained without change ; we could not observe that the air above the high blood level contributes to the formation of microbubbles . the microbubbles measured upstream of the arterial luer lock connector were possibly a remnant of these microbubbles inside the patient 's body . in the beginning of the treatments , the mean microbubble count was an order of magnitude larger than during the rest of the treatment . nocturnal dialysis ) , the number of microbubbles was not decreasing towards the end of the treatment , indicating either other sources of microbubble generation or large stores of initially not removed air somewhere in the blood tubing . an increase in bubble count was repeatedly observed at the start of the blood pump , possibly due to a sudden displacement of the remaining bubbles in the blood line , suction of bubbles from a sideline ( e.g. our simultaneous measurement up- and downstream of the arterial luer lock connector indicated that , in the negative pressure region , small amounts of air may enter the blood line . generally , the tightness of luer lock connectors is under discussion [ 1720 ] . on the other hand , online haemodiafiltration is often performed with a high blood flow rate and a low negative arterial pressure . possibly , a high microbubble rate may not be due to the online haemodiafiltration per se , but due to a low negative arterial pressure ( e.g. a large randomized clinical trial demonstrated the survival benefit of online haemodiafiltration ; a major influence of microbubbles as a confounding factor on the primary outcome parameter all - cause mortality was unlikely , because the blood flow rate was similar in both the study groups ( differences only between 2 and 5% ) . this observation is in line with other investigations , and microbubbles were observed during treatments with the dialysis machines of all manufacturers [ 4 , 8 , 11 , 12 ] , indicating that microbubbles are a general side effect of hd . but the range of the microbubble counts was large ; obviously , in the standard hd setting , there are treatments with a low generation of microbubbles . there are several limitations of this non - interventional study : the study was performed in a single centre with a small number of patients and treatments . the arterial luer lock connector should be tightened. syringes filled with fluids for injections ( such as heparin or iron ) and side lines should be given special care. extreme blood flow rates and arterial pressures should be avoided.from the visible bubbles ( at blood pump segment , dialyser cap , venous chamber and heparin line ) , the total volume was estimated to a few microliters , obviously not enough to be the only source of microbubbles . generally , during hd , microbubbles are infused in large numbers into the patients , but the involved volume is extremely small . during hd , a large number of microbubbles may reach the patients , but the involved volume is extremely small . origin and pathophysiologic consequences of this phenomenon are not well understood but deserve further study and , if necessary , a new blood line design aiming to minimize blood air interfaces .

breast cancer is the most common cancer among iranian women , comprising nearly 21.4% of all cancers in this group . studies show that the trend of breast cancer incidence among iranian women is increasing , and the age of breast cancer incidence is lower in comparison to women in other countries . although reports vary from 60% to 82% for actual percentages , widespread agreement exists among health - care professionals that the majority of iranian women with breast cancer are diagnosed at advanced stages of the disease . a family history of breast cancer , especially among first - degree relatives , is a strong risk factor for this disease . women who have one first - degree relative with a history of breast cancer have 1.8 times higher risk of developing the disease than women with no such family history ; furthermore , the risk tends to rise as the number of affected family members increase . although studies in other countries demonstrate that women with a family history of breast cancer are more adherent to screening programs , iranian women with a family history of breast cancer tend to participate in this screening at a lower rate than the average rate among their peers . mammography provides the main tool for breast cancer screening ; however , clinical breast exam ( cbe ) as performed by a physician or other trained health professional can also be useful . cbe has been reported to yield a much lower accuracy in detecting breast lesions compared to mammography , but its availability and low cost render it a supplementary approach for breast cancer screening , especially in low- to middle - income countries such as iran . according to the american cancer society recommendations in 2012 , cbe should be started at age 20 . women between ages of 20 and 40 should receive a cbe every 3 years , and those over 40-year - old should undergo cbe each year . behavioral studies have shown that a patient 's education , perceived level of susceptibility to the disease , acceptance or rejection of screening techniques , and perceived level of potential benefit , or harm from the screening techniques all influence their willingness to participate in cancer screening programs , such as screening mammography or cbe . show that an optimistic attitude toward breast cancer screening programs is associated with higher rates of participation in them . other studies have demonstrated that women who have a positive mindset about cbe have sought care which includes a cbe more often nojomi et al . report that iranian women who perceive fewer barriers and more benefits about cbe are more likely to undergo it . many studies have applied health belief model ( hbm ) to evaluate factors associated with breast cancer screening method such as cbe . according to this model , people will follow healthy behavior , if they perceive susceptibility and seriousness of that situation and if they believe that benefits of doing the behavior are more than its barriers . hence , in this study , hbm has been used as the conceptual framework to determine variables associated with cbe . moreover , because decision - making processes related to cbe breast cancer screening are very complex , the stage of change of ttm of behavior was used as a clue for the study . the transtheoretical model ( ttm ) , among the most important behavioral models , was first developed by prochaska and diclemente . as modified by the rakoweski classification system , these stages are : precontemplation : never had a cbe and is not thinking about receiving a cbe in the next 12 months ( for women over 40-year - old ) or the next 36 months ( for women under 40-year - old ) , contemplation : never had a cbe but is thinking about receiving a cbe in the next 12 months ( for women over 40-year - old ) or the next 36 months ( for women under 40-year - old ) , action : had a cbe before and is going to receive another cbe in the next 12 months ( for women over 40-year - old ) or the next 36 months ( for women under 40-year - old ) , maintenance : had two or more cbe and is going to receive another cbe in the next 12 months ( for women over 40-year - old ) or the next 36 months ( for women under 40-year - old ) , relapse : had one or more cbe previously but is not going to receive a cbe in the next 12 months ( for women over 40-year - old ) or the next 36 months ( for women under 40-year - old ) . lee - lin study showed chinese american immigrant women who were in precontemplation stage of mammography screening have significantly higher perceived barrier and lower perceived susceptibility than women in contemplation stage . according to our literature review , few studies evaluated factors associated with iranian women participation in cbe screening program , and these studies were related to women who did not have a family history of breast cancer . because of the higher incidence of breast cancer among women with a family history of breast cancer , it is important to identify factors influencing cbe screening behaviors . these factors are principal to designing suitable interventions to promote cbe screening among this at - risk group . in this study , we evaluated ttm stages as applied to cbe in iranian women with a family history of breast cancer . we have also assessed different demographic features that may be related to women 's ttm stages as applied to cbe . we have also assessed the association between the hbm of iranian women and their ttm stage as applied to cbe . this is a cross - sectional study with a convenience sampling method which was conducted between june and august 2015 in sayed - al - shohada hospital , the main cancer treatment center in isfahan province in iran . inclusion criteria were women aged 20 years and older residing in isfahan , first- or second - degree relatives of breast cancer patients who referred to sayed - al - shohada hospital who was accompanied with patient , with no personal history of breast cancer . women who do not desire to participate in the study were excluded from the study . most of the known cancerous patients with their family members referred to sayed - al - shohada hospital , so the selection bias seems likely to be negligible . sample size was estimated 192 by considering 80% for study power , = 0.05 , p = 48% , effect size = 7.7% , and 20% no response of participants . the face - to - face interview was conducted for eligible women who accompanied a breast cancer patient referring to s. al - shohada hospital sections in isfahan . initially , the interviewer explained the purpose of the study and voluntary participation to each of the women . each interview took 2030 min in a quiet room which was considered for this purpose at certain times of the day . the study continued until the sample size completed ( between june and august 2015 ) . of the 200 women who were first- or second - degree relatives of patients referring to hospital during the study period , only 162 women were desire to participate . for the ethical reason , the information collected was kept confidential . furthermore , informed consent from the participants was obtained . this study approved by ethical committee of isfahan university of medical sciences ( ethical number : 394130 ) . three categories of information were collected for each participant : demographic questions , ttm stages as applied to cbe , and hbm scores . demographic characteristics were obtained by collecting information about participant 's age , level of education , marital status , health insurance status , income , employment status , history of breast disease , and history of breast self - examination ( bse ) . to evaluate ttm stages as applied to cbe , we categorized women into five stages according to rakoweski classification checklist : precontemplation , contemplation , action , maintenance , and relapse . to determine women 's hbm about breast cancer screening , we used the latest persian version of champion 's revised hbm scale which is the standard for iranian women by taymoori . this scale includes 61-items in eight categories from which we used five subscales in this study . we assessed women 's health beliefs about breast cancer and cbe , considering perceived sensitivity ( 3-item , cronbach 's = 0.82 ) , perceived severity ( 7-item , cronbach 's = 0.84 ) , perceived benefits ( 6-item , cronbach 's = 0.72 ) , and perceived barriers ( 10-item cronbach 's = 0.73 ) , alongside health motivation ( 7-item , cronbach 's = 0.77 ) . all of the hbm subscales questions were scored based on a 5-point likert scoring system , ranging from strongly disagree ( 1 ) to strongly agree ( 5 ) . except for barriers toward cbe , for all other categories , the score of each five scales was calculated separately . statistical package for the social sciences version 27 . chi - square test was used to detect an association between demographic variables and ttm stages ( precontemplation / relapse , contemplation , and action / maintenance ) . analysis of covariance ( ancova ) was used to compare hbm components ( perceived sensitivity , perceived severity , perceived barrier , perceived benefit , and health motivation ) among different ttm stages . independent t - test was used to determine which ttm stage is significantly different from the others considering hbm components . the level of significance for p < 0.05 . this is a cross - sectional study with a convenience sampling method which was conducted between june and august 2015 in sayed - al - shohada hospital , the main cancer treatment center in isfahan province in iran . inclusion criteria were women aged 20 years and older residing in isfahan , first- or second - degree relatives of breast cancer patients who referred to sayed - al - shohada hospital who was accompanied with patient , with no personal history of breast cancer . women who do not desire to participate in the study were excluded from the study . most of the known cancerous patients with their family members referred to sayed - al - shohada hospital , so the selection bias seems likely to be negligible . sample size was estimated 192 by considering 80% for study power , = 0.05 , p = 48% , effect size = 7.7% , and 20% no response of participants . the face - to - face interview was conducted for eligible women who accompanied a breast cancer patient referring to s. al - shohada hospital sections in isfahan . initially , the interviewer explained the purpose of the study and voluntary participation to each of the women . each interview took 2030 min in a quiet room which was considered for this purpose at certain times of the day . the study continued until the sample size completed ( between june and august 2015 ) . of the 200 women who were first- or second - degree relatives of patients referring to hospital during the study period , only 162 women were desire to participate . for the ethical reason , the information collected was kept confidential . furthermore , informed consent from the participants was obtained . this study approved by ethical committee of isfahan university of medical sciences ( ethical number : 394130 ) . three categories of information were collected for each participant : demographic questions , ttm stages as applied to cbe , and hbm scores . demographic characteristics were obtained by collecting information about participant 's age , level of education , marital status , health insurance status , income , employment status , history of breast disease , and history of breast self - examination ( bse ) . to evaluate ttm stages as applied to cbe , we categorized women into five stages according to rakoweski classification checklist : precontemplation , contemplation , action , maintenance , and relapse . to determine women 's hbm about breast cancer screening , we used the latest persian version of champion 's revised hbm scale which is the standard for iranian women by taymoori . this scale includes 61-items in eight categories from which we used five subscales in this study . we assessed women 's health beliefs about breast cancer and cbe , considering perceived sensitivity ( 3-item , cronbach 's = 0.82 ) , perceived severity ( 7-item , cronbach 's = 0.84 ) , perceived benefits ( 6-item , cronbach 's = 0.72 ) , and perceived barriers ( 10-item cronbach 's = 0.73 ) , alongside health motivation ( 7-item , cronbach 's = 0.77 ) . all of the hbm subscales questions were scored based on a 5-point likert scoring system , ranging from strongly disagree ( 1 ) to strongly agree ( 5 ) . except for barriers toward cbe , for all other categories , chi - square test was used to detect an association between demographic variables and ttm stages ( precontemplation / relapse , contemplation , and action / maintenance ) . analysis of covariance ( ancova ) was used to compare hbm components ( perceived sensitivity , perceived severity , perceived barrier , perceived benefit , and health motivation ) among different ttm stages . independent t - test was used to determine which ttm stage is significantly different from the others considering hbm components . the level of significance for p < 0.05 . of the 200 potential participants , 38 people were excluded due to their unwillingness to fill out the questionnaire ( response rate of 81% ) . as a result , our final sample size was 162 women . in this study , we did not have missing data because an interviewer completed the questionnaire for each of the participants . moreover , we did not have drop out data because we did not have any intervention or follow - up period . the mean age of the participants was 37.6 years ( standard deviation = 11.16 , range from 20 to 73 ) . thirty - two ( 20% ) of participants had a history of benign breast disease . approximately , 117 ( 72% ) of the participants had a first - degree relative with a history of breast cancer , while 45 ( 28% ) had a second - degree relative with a history of breast cancer . women 's characteristics with a family history of breast cancer , isfahan ( n=162 ) the majority of women were in contemplation stage ( 68 [ 42% ] ) , followed by women in action ( 28 [ 17.3% ] ) , maintenance ( 27 [ 16.7% ] ) , precontemplation ( 25 [ 15.4% ] ) , and relapse stages ( 14 [ 8.6% ] ) , respectively . to report the results , we combined relapse and precontemplation stages together , since women in these stages were not planning to undergo cbe . we also combined action stage with maintenance stage because women in these stages were planning to receive cbe [ figure 1 ] . transtheoretical model stages applied to clinical breast exam for women with a family history of breast cancer ( n = 162 ) our results showed that distribution of age group , health insurance status , bse status , first - degree relative with a history of breast cancer ( in comparison to second - degree relative ) , and personal history of breast disease have a significant difference in women 's ttm stage as applied to cbe ( p < 0.05 ) . association between transtheoretical model stage applied to clinical breast exam and women 's characteristics result showed that older women , women who had performed bse at least once , women had a health insurance , women with a personal history of breast cancer , and women with a family history of breast cancer in their first - degree relatives were significantly more in action / maintenance stages of cbe screening behavior ( p < 0.05 ) . after adjusting for confounder variables ( including age group , personal history of breast disease , bse status , history of breast cancer in first - degree relative versus second - degree relative , and health insurance status ) , ancova showed there is a significant difference between mean score of perceived barriers in different ttm stages as applied to cbe [ table 3 ] . independent t - test showed women in action / maintenance stages had significantly fewer perceived barriers in comparison to women in precontemplation / relapse stages ( p < 0.001 ) and contemplation stages ( p < 0.001 ) . in addition , women in contemplation stage had fewer barriers than women in precontemplation / relapse stages ( p = 0.018 ) . no significant difference was detected in the mean score of other hbm categories between ttm stages after adjusting for mentioned confounders [ table 3 ] . mean of health belief model components applying to clinical breast examination among different stages of transtheoretical model of the 200 potential participants , 38 people were excluded due to their unwillingness to fill out the questionnaire ( response rate of 81% ) . as a result , our final sample size was 162 women . in this study , we did not have missing data because an interviewer completed the questionnaire for each of the participants . moreover , we did not have drop out data because we did not have any intervention or follow - up period . the mean age of the participants was 37.6 years ( standard deviation = 11.16 , range from 20 to 73 ) . thirty - two ( 20% ) of participants had a history of benign breast disease . approximately , 117 ( 72% ) of the participants had a first - degree relative with a history of breast cancer , while 45 ( 28% ) had a second - degree relative with a history of breast cancer . the majority of women were in contemplation stage ( 68 [ 42% ] ) , followed by women in action ( 28 [ 17.3% ] ) , maintenance ( 27 [ 16.7% ] ) , precontemplation ( 25 [ 15.4% ] ) , and relapse stages ( 14 [ 8.6% ] ) , respectively . to report the results , we combined relapse and precontemplation stages together , since women in these stages were not planning to undergo cbe . we also combined action stage with maintenance stage because women in these stages were planning to receive cbe [ figure 1 ] . transtheoretical model stages applied to clinical breast exam for women with a family history of breast cancer ( n = 162 ) our results showed that distribution of age group , health insurance status , bse status , first - degree relative with a history of breast cancer ( in comparison to second - degree relative ) , and personal history of breast disease have a significant difference in women 's ttm stage as applied to cbe ( p < 0.05 ) . association between transtheoretical model stage applied to clinical breast exam and women 's characteristics result showed that older women , women who had performed bse at least once , women had a health insurance , women with a personal history of breast cancer , and women with a family history of breast cancer in their first - degree relatives were significantly more in action / maintenance stages of cbe screening behavior ( p < 0.05 ) . after adjusting for confounder variables ( including age group , personal history of breast disease , bse status , history of breast cancer in first - degree relative versus second - degree relative , and health insurance status ) , ancova showed there is a significant difference between mean score of perceived barriers in different ttm stages as applied to cbe [ table 3 ] . independent t - test showed women in action / maintenance stages had significantly fewer perceived barriers in comparison to women in precontemplation / relapse stages ( p < 0.001 ) and contemplation stages ( p < 0.001 ) . in addition , women in contemplation stage had fewer barriers than women in precontemplation / relapse stages ( p = 0.018 ) . no significant difference was detected in the mean score of other hbm categories between ttm stages after adjusting for mentioned confounders [ table 3 ] . mean of health belief model components applying to clinical breast examination among different stages of transtheoretical model to our knowledge , this is the first study which has evaluated ttm stages as applied to cbe among iranian women with a family history of breast cancer . in this survey , we evaluated ttm stages as applied to cbe in iranian women with a family history of breast cancer and factors associated with cbe screening behavior . our finding showed that most of the participants were in contemplation stage of ttm stages as applied to cbe and only one - third of them were in action / maintenance stage of cbe behavior . however , having an older age , have a health insurance , history of the bse , first - degree relative with breast cancer patient ( in comparison to second - degree relative ) , and a personal history of breast disease seemed to have a significant association with being in action / maintenance stage of ttm stages as applied to cbe . moreover , women in action / maintenance stage of cbe screening behavior had significantly fewer barriers compared with two other groups . this study has shown that only one - third of the women who had a family history of breast cancer are in action / maintenance stages of ttm which is lower than the 48% reported by tu et al . among cambodian american women in 2002 however , tu et al . used a different classification for ttm stages as applied to cbe and did not limit their study to women with a family history of breast cancer . however , a lack of active screening in iran health system and little knowledge about breast cancer risk factors may justify this result . our study reviled older women are more likely to be in action / maintenance stages . this finding is consistent with studies conducted by tu et al . , ahmad and stewart , price et al . , and parsa and kandiah which showed undergoing cbe increases as women become older . this may be due to an increase in women 's perceived risk of breast cancer as they get older . in contrast , siahpush and singh showed older women are less likely to engage in breast cancer screening programs . however , that study was conducted more than a decade ago , and this difference may be due to the gradual increase in participation of older iranian women in breast cancer screening programs during the last decade . similar to most of the earlier studies , we found out that performing bse is associated with participating in other breast cancer screening programs such as cbe . we also found out that women with a history of breast disease are more likely to be in action / maintenance stages . other studies have shown similar results . in women with a personal history of benign breast disease , becoming more educated about breast cancer screening programs , alongside their excessive anxiety about the possibility of breast cancer in the future , may lead to their adherence to screening programs . in our study , having a health insurance was associated with being in action / maintenance stages for undergoing cbe . ahmadian and samah have reported similar results in their review article about factors influencing breast cancer screening . however , our findings are not compatible with the results from the study conducted by parsa and kandiah in malaysian women . it is possible that unique economic and health - care structure of each country plays a role in this variation . in this study , however , since there were few participants who did not have any health insurance at all , we can not be too certain about this finding . furthermore , our results showed women with a family history of breast cancer in their first - degree relatives are more likely to be in action / maintenance stages compared to those with a family history of breast cancer in their second - degree relatives . finney rutten and iannotti have also reported a positive correlation between a family history of breast cancer in closer relatives and compliance with breast cancer screening . it seems the anxiety caused by the history of breast cancer in the first - degree relatives , promotes breast cancer screening participation in these women . in our study , from different hbm elements , only level of perceived barriers was significantly lower among women in action / maintenance stages compared to women in precontemplation / relapse and contemplation stages . this means women in action / maintenance stage have fewer barriers about time of cbe performing , place , pain , cost , and method of cbe performing . moreover , they feel less embarrassed from cbe performing . furthermore , women in contemplation stage had fewer barriers than those in precontemplation / relapse stages . however , other studies have reported that level of perceived susceptibility to breast cancer and perceived benefits from breast cancer screening are the most important determinants of willingness to participate in these programs . however , the different designs of studies , such as target group risk factors can justify observed differences . our survey was performed in a referral hospital in isfahan in which a wide range of patients with various types of sociodemographic situation refers to ; therefore , our findings can nearly represent cbe behavior among women with a family history of breast cancer . however , according to inclusion criteria , our finding can be extended only to women with a family history of breast cancer and not to the general population . this may have led to recall bias , so we suggest using of recorded data for future studies . furthermore , we have used a cross - sectional design for our study , so we could not find any causal association between research variables . our finding clarified that the rate of women with a family history of breast cancer that is in action / maintenance stage of cbe behavior is low . these factors should be used in designing efficient interventions by focusing on changeable cbe barriers . further collaboration among government , health - care sections , and insurance companies is required to provide more education , alongside with social and financial supports to increase participation in breast cancer screening programs . ffd contributed in the conception of the work , analysis , interpretation of data for the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the workzf contributed in the conception of the work , conducting the study , analysis , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the worksh contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the workrs contributed in interpretation of data for the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the worknt contributed in analyze of data , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the workrr contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . ffd contributed in the conception of the work , analysis , interpretation of data for the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work zf contributed in the conception of the work , conducting the study , analysis , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work sh contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work rs contributed in interpretation of data for the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work nt contributed in analyze of data , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work rr contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .

background : participation of iranian women with a family history of breast cancer in breast cancer screening programs is low . this study evaluates the compliance of women having a family history of breast cancer with clinical breast exam ( cbe ) according to the stage of transtheoretical model ( ttm ) and health belief model ( hbm).materials and methods : in this cross - sectional study , we used persian version of champion 's hbm scale to collect factors associated with ttm stages applied to screening from women over 20 years and older . the obtained data were analyzed by spss , using descriptive statistics , chi - square test , independent t - test , and analysis of covariance.results:final sample size was 162 women . thirty - three percent were in action / maintenance stage . older women , family history of breast cancer in first - degree relatives , personal history of breast disease , insurance coverage , and a history of breast self - examination were associated with action / maintenance stage . furthermore , women in action / maintenance stages had significantly fewer perceived barriers in terms of cbe in comparison to women in other stages ( p < 0.05 ) . there was no significant difference in other hbm subscales scores between various stages of cbe screening behavior ( p > 0.05).conclusion : the finding indicates that the rate of women in action / maintenance stage of cbe is low . moreover , results show a strong association between perceived barriers and having a regular cbe . these clarify the necessity of promoting national target programs for breast cancer screening , which should be considered as the first preference for reducing cbe barriers .

INTRODUCTION MATERIALS AND METHODS Study design and sample Ethics Instrument Statistics RESULTS Demographic characteristics Distribution of participants among transtheoretical model stages Factors influencing participants transtheoretical model stages as applied to clinical breast exam Relationship between women's transtheoretical model stages as applied to clinical breast exam and health belief model categories (perceived sensitivity, severity, benefit, barrier, and health motivation) DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest AUTHORS CONTRIBUTION

breast cancer is the most common cancer among iranian women , comprising nearly 21.4% of all cancers in this group . studies show that the trend of breast cancer incidence among iranian women is increasing , and the age of breast cancer incidence is lower in comparison to women in other countries . although reports vary from 60% to 82% for actual percentages , widespread agreement exists among health - care professionals that the majority of iranian women with breast cancer are diagnosed at advanced stages of the disease . a family history of breast cancer , especially among first - degree relatives , is a strong risk factor for this disease . women who have one first - degree relative with a history of breast cancer have 1.8 times higher risk of developing the disease than women with no such family history ; furthermore , the risk tends to rise as the number of affected family members increase . although studies in other countries demonstrate that women with a family history of breast cancer are more adherent to screening programs , iranian women with a family history of breast cancer tend to participate in this screening at a lower rate than the average rate among their peers . mammography provides the main tool for breast cancer screening ; however , clinical breast exam ( cbe ) as performed by a physician or other trained health professional can also be useful . cbe has been reported to yield a much lower accuracy in detecting breast lesions compared to mammography , but its availability and low cost render it a supplementary approach for breast cancer screening , especially in low- to middle - income countries such as iran . according to the american cancer society recommendations in 2012 , cbe should be started at age 20 . behavioral studies have shown that a patient 's education , perceived level of susceptibility to the disease , acceptance or rejection of screening techniques , and perceived level of potential benefit , or harm from the screening techniques all influence their willingness to participate in cancer screening programs , such as screening mammography or cbe . show that an optimistic attitude toward breast cancer screening programs is associated with higher rates of participation in them . many studies have applied health belief model ( hbm ) to evaluate factors associated with breast cancer screening method such as cbe . hence , in this study , hbm has been used as the conceptual framework to determine variables associated with cbe . moreover , because decision - making processes related to cbe breast cancer screening are very complex , the stage of change of ttm of behavior was used as a clue for the study . the transtheoretical model ( ttm ) , among the most important behavioral models , was first developed by prochaska and diclemente . lee - lin study showed chinese american immigrant women who were in precontemplation stage of mammography screening have significantly higher perceived barrier and lower perceived susceptibility than women in contemplation stage . according to our literature review , few studies evaluated factors associated with iranian women participation in cbe screening program , and these studies were related to women who did not have a family history of breast cancer . because of the higher incidence of breast cancer among women with a family history of breast cancer , it is important to identify factors influencing cbe screening behaviors . in this study , we evaluated ttm stages as applied to cbe in iranian women with a family history of breast cancer . we have also assessed different demographic features that may be related to women 's ttm stages as applied to cbe . we have also assessed the association between the hbm of iranian women and their ttm stage as applied to cbe . this is a cross - sectional study with a convenience sampling method which was conducted between june and august 2015 in sayed - al - shohada hospital , the main cancer treatment center in isfahan province in iran . inclusion criteria were women aged 20 years and older residing in isfahan , first- or second - degree relatives of breast cancer patients who referred to sayed - al - shohada hospital who was accompanied with patient , with no personal history of breast cancer . sample size was estimated 192 by considering 80% for study power , = 0.05 , p = 48% , effect size = 7.7% , and 20% no response of participants . of the 200 women who were first- or second - degree relatives of patients referring to hospital during the study period , only 162 women were desire to participate . three categories of information were collected for each participant : demographic questions , ttm stages as applied to cbe , and hbm scores . demographic characteristics were obtained by collecting information about participant 's age , level of education , marital status , health insurance status , income , employment status , history of breast disease , and history of breast self - examination ( bse ) . to evaluate ttm stages as applied to cbe , we categorized women into five stages according to rakoweski classification checklist : precontemplation , contemplation , action , maintenance , and relapse . to determine women 's hbm about breast cancer screening , we used the latest persian version of champion 's revised hbm scale which is the standard for iranian women by taymoori . this scale includes 61-items in eight categories from which we used five subscales in this study . we assessed women 's health beliefs about breast cancer and cbe , considering perceived sensitivity ( 3-item , cronbach 's = 0.82 ) , perceived severity ( 7-item , cronbach 's = 0.84 ) , perceived benefits ( 6-item , cronbach 's = 0.72 ) , and perceived barriers ( 10-item cronbach 's = 0.73 ) , alongside health motivation ( 7-item , cronbach 's = 0.77 ) . chi - square test was used to detect an association between demographic variables and ttm stages ( precontemplation / relapse , contemplation , and action / maintenance ) . analysis of covariance ( ancova ) was used to compare hbm components ( perceived sensitivity , perceived severity , perceived barrier , perceived benefit , and health motivation ) among different ttm stages . independent t - test was used to determine which ttm stage is significantly different from the others considering hbm components . the level of significance for p < 0.05 . this is a cross - sectional study with a convenience sampling method which was conducted between june and august 2015 in sayed - al - shohada hospital , the main cancer treatment center in isfahan province in iran . inclusion criteria were women aged 20 years and older residing in isfahan , first- or second - degree relatives of breast cancer patients who referred to sayed - al - shohada hospital who was accompanied with patient , with no personal history of breast cancer . sample size was estimated 192 by considering 80% for study power , = 0.05 , p = 48% , effect size = 7.7% , and 20% no response of participants . of the 200 women who were first- or second - degree relatives of patients referring to hospital during the study period , only 162 women were desire to participate . three categories of information were collected for each participant : demographic questions , ttm stages as applied to cbe , and hbm scores . demographic characteristics were obtained by collecting information about participant 's age , level of education , marital status , health insurance status , income , employment status , history of breast disease , and history of breast self - examination ( bse ) . to evaluate ttm stages as applied to cbe , we categorized women into five stages according to rakoweski classification checklist : precontemplation , contemplation , action , maintenance , and relapse . to determine women 's hbm about breast cancer screening , we used the latest persian version of champion 's revised hbm scale which is the standard for iranian women by taymoori . we assessed women 's health beliefs about breast cancer and cbe , considering perceived sensitivity ( 3-item , cronbach 's = 0.82 ) , perceived severity ( 7-item , cronbach 's = 0.84 ) , perceived benefits ( 6-item , cronbach 's = 0.72 ) , and perceived barriers ( 10-item cronbach 's = 0.73 ) , alongside health motivation ( 7-item , cronbach 's = 0.77 ) . except for barriers toward cbe , for all other categories , chi - square test was used to detect an association between demographic variables and ttm stages ( precontemplation / relapse , contemplation , and action / maintenance ) . analysis of covariance ( ancova ) was used to compare hbm components ( perceived sensitivity , perceived severity , perceived barrier , perceived benefit , and health motivation ) among different ttm stages . independent t - test was used to determine which ttm stage is significantly different from the others considering hbm components . the level of significance for p < 0.05 . as a result , our final sample size was 162 women . in this study , we did not have missing data because an interviewer completed the questionnaire for each of the participants . thirty - two ( 20% ) of participants had a history of benign breast disease . approximately , 117 ( 72% ) of the participants had a first - degree relative with a history of breast cancer , while 45 ( 28% ) had a second - degree relative with a history of breast cancer . women 's characteristics with a family history of breast cancer , isfahan ( n=162 ) the majority of women were in contemplation stage ( 68 [ 42% ] ) , followed by women in action ( 28 [ 17.3% ] ) , maintenance ( 27 [ 16.7% ] ) , precontemplation ( 25 [ 15.4% ] ) , and relapse stages ( 14 [ 8.6% ] ) , respectively . transtheoretical model stages applied to clinical breast exam for women with a family history of breast cancer ( n = 162 ) our results showed that distribution of age group , health insurance status , bse status , first - degree relative with a history of breast cancer ( in comparison to second - degree relative ) , and personal history of breast disease have a significant difference in women 's ttm stage as applied to cbe ( p < 0.05 ) . association between transtheoretical model stage applied to clinical breast exam and women 's characteristics result showed that older women , women who had performed bse at least once , women had a health insurance , women with a personal history of breast cancer , and women with a family history of breast cancer in their first - degree relatives were significantly more in action / maintenance stages of cbe screening behavior ( p < 0.05 ) . after adjusting for confounder variables ( including age group , personal history of breast disease , bse status , history of breast cancer in first - degree relative versus second - degree relative , and health insurance status ) , ancova showed there is a significant difference between mean score of perceived barriers in different ttm stages as applied to cbe [ table 3 ] . independent t - test showed women in action / maintenance stages had significantly fewer perceived barriers in comparison to women in precontemplation / relapse stages ( p < 0.001 ) and contemplation stages ( p < 0.001 ) . in addition , women in contemplation stage had fewer barriers than women in precontemplation / relapse stages ( p = 0.018 ) . no significant difference was detected in the mean score of other hbm categories between ttm stages after adjusting for mentioned confounders [ table 3 ] . mean of health belief model components applying to clinical breast examination among different stages of transtheoretical model of the 200 potential participants , 38 people were excluded due to their unwillingness to fill out the questionnaire ( response rate of 81% ) . as a result , our final sample size was 162 women . in this study , we did not have missing data because an interviewer completed the questionnaire for each of the participants . thirty - two ( 20% ) of participants had a history of benign breast disease . approximately , 117 ( 72% ) of the participants had a first - degree relative with a history of breast cancer , while 45 ( 28% ) had a second - degree relative with a history of breast cancer . the majority of women were in contemplation stage ( 68 [ 42% ] ) , followed by women in action ( 28 [ 17.3% ] ) , maintenance ( 27 [ 16.7% ] ) , precontemplation ( 25 [ 15.4% ] ) , and relapse stages ( 14 [ 8.6% ] ) , respectively . transtheoretical model stages applied to clinical breast exam for women with a family history of breast cancer ( n = 162 ) our results showed that distribution of age group , health insurance status , bse status , first - degree relative with a history of breast cancer ( in comparison to second - degree relative ) , and personal history of breast disease have a significant difference in women 's ttm stage as applied to cbe ( p < 0.05 ) . association between transtheoretical model stage applied to clinical breast exam and women 's characteristics result showed that older women , women who had performed bse at least once , women had a health insurance , women with a personal history of breast cancer , and women with a family history of breast cancer in their first - degree relatives were significantly more in action / maintenance stages of cbe screening behavior ( p < 0.05 ) . after adjusting for confounder variables ( including age group , personal history of breast disease , bse status , history of breast cancer in first - degree relative versus second - degree relative , and health insurance status ) , ancova showed there is a significant difference between mean score of perceived barriers in different ttm stages as applied to cbe [ table 3 ] . independent t - test showed women in action / maintenance stages had significantly fewer perceived barriers in comparison to women in precontemplation / relapse stages ( p < 0.001 ) and contemplation stages ( p < 0.001 ) . in addition , women in contemplation stage had fewer barriers than women in precontemplation / relapse stages ( p = 0.018 ) . no significant difference was detected in the mean score of other hbm categories between ttm stages after adjusting for mentioned confounders [ table 3 ] . mean of health belief model components applying to clinical breast examination among different stages of transtheoretical model to our knowledge , this is the first study which has evaluated ttm stages as applied to cbe among iranian women with a family history of breast cancer . in this survey , we evaluated ttm stages as applied to cbe in iranian women with a family history of breast cancer and factors associated with cbe screening behavior . our finding showed that most of the participants were in contemplation stage of ttm stages as applied to cbe and only one - third of them were in action / maintenance stage of cbe behavior . however , having an older age , have a health insurance , history of the bse , first - degree relative with breast cancer patient ( in comparison to second - degree relative ) , and a personal history of breast disease seemed to have a significant association with being in action / maintenance stage of ttm stages as applied to cbe . moreover , women in action / maintenance stage of cbe screening behavior had significantly fewer barriers compared with two other groups . this study has shown that only one - third of the women who had a family history of breast cancer are in action / maintenance stages of ttm which is lower than the 48% reported by tu et al . used a different classification for ttm stages as applied to cbe and did not limit their study to women with a family history of breast cancer . our study reviled older women are more likely to be in action / maintenance stages . in contrast , siahpush and singh showed older women are less likely to engage in breast cancer screening programs . however , that study was conducted more than a decade ago , and this difference may be due to the gradual increase in participation of older iranian women in breast cancer screening programs during the last decade . similar to most of the earlier studies , we found out that performing bse is associated with participating in other breast cancer screening programs such as cbe . we also found out that women with a history of breast disease are more likely to be in action / maintenance stages . in women with a personal history of benign breast disease , becoming more educated about breast cancer screening programs , alongside their excessive anxiety about the possibility of breast cancer in the future , may lead to their adherence to screening programs . in our study , having a health insurance was associated with being in action / maintenance stages for undergoing cbe . in this study , however , since there were few participants who did not have any health insurance at all , we can not be too certain about this finding . furthermore , our results showed women with a family history of breast cancer in their first - degree relatives are more likely to be in action / maintenance stages compared to those with a family history of breast cancer in their second - degree relatives . finney rutten and iannotti have also reported a positive correlation between a family history of breast cancer in closer relatives and compliance with breast cancer screening . it seems the anxiety caused by the history of breast cancer in the first - degree relatives , promotes breast cancer screening participation in these women . in our study , from different hbm elements , only level of perceived barriers was significantly lower among women in action / maintenance stages compared to women in precontemplation / relapse and contemplation stages . this means women in action / maintenance stage have fewer barriers about time of cbe performing , place , pain , cost , and method of cbe performing . furthermore , women in contemplation stage had fewer barriers than those in precontemplation / relapse stages . our survey was performed in a referral hospital in isfahan in which a wide range of patients with various types of sociodemographic situation refers to ; therefore , our findings can nearly represent cbe behavior among women with a family history of breast cancer . however , according to inclusion criteria , our finding can be extended only to women with a family history of breast cancer and not to the general population . furthermore , we have used a cross - sectional design for our study , so we could not find any causal association between research variables . our finding clarified that the rate of women with a family history of breast cancer that is in action / maintenance stage of cbe behavior is low . further collaboration among government , health - care sections , and insurance companies is required to provide more education , alongside with social and financial supports to increase participation in breast cancer screening programs . ffd contributed in the conception of the work , analysis , interpretation of data for the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the workzf contributed in the conception of the work , conducting the study , analysis , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the worksh contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the workrs contributed in interpretation of data for the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the worknt contributed in analyze of data , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the workrr contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . ffd contributed in the conception of the work , analysis , interpretation of data for the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work zf contributed in the conception of the work , conducting the study , analysis , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work sh contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work rs contributed in interpretation of data for the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work nt contributed in analyze of data , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work rr contributed in the conception of the work , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work .

the term epigenetics was coined some 70 years ago by sir conrad waddington , who theorized the existence of a necessary layer of molecular complexity beyond the genome that must be responsible for producing distinct and variable cellular phenotypes from a singular genome . instincts and genetic programs as useful conceptualizations of the ontogeny of behavior and , importantly , have initiated an appreciation of the multitude of complex influences on phenotypic expression throughout development . epigenetics in its current formulation is more narrowly defined as the perpetuation of genetic information from a cell to its descendants without any necessary change to the genetic code itself , and has been posited as a molecular bridge between the information contained in the genotype and what emerges as a complex and evermodifiable phenotype . the revolution in molecular biology that began in the 1950s , following shortly after waddington 's theoretical formulation of epigenetics , has provided scientists with tools capable of characterizing and understanding the mechanisms that comprise this layer of complexity beyond the genome ( ie , the epigenome ) . dna exists in a continuum of variably compacted states controlled by the structural state of chromatin , ie , the dna and the histone proteins around which it is wrapped . alterations to the structural state of the chromatin can have profound and persistent effects on gene expression . simply put , by establishing and maintaining the structural state of the chromatin , epigenetic processes regulate the ease in which transcription factors and other proteins can access their dna substrates . for example , the amino acid tails of histone proteins are subjected to various post - translational modifications ( eg , acetylation , methylation , phosphorylation ) that render the chromatin relatively compact and transcriptionally inactive ( ie , heterochromatin ) or less compact and transcriptionally active ( ie , euchromatin ) . methylation at the 5-position of cytosine nucleotides within cpg dinucelotides is the only direct epigenetic modification of dna and is associated with transcriptional silencing . epigenetic processes have long been recognized as indispensable for appropriate embryonic and early postnatal development . more recently it has come to light that these same mechanisms that drive critical processes in development and in mitotic cells throughout the lifespan remain dynamic in neurons that , once differentiated , are incapable of mitosis . describes processes that utilize the same mechanisms classically defined as epigenetic , but are clearly for functionally distinct purposes . moreover , there is now recognition that dna methylation itself , once thought to be the most stable of epigenetic marks , may switch between methylated and unmethylated states that render stretches of the chromatin a dynamic canvas on which epigenetic and other mechanisms can work to promote forms of plasticity necessary for long - term information storage . the hypothesis that the processes underlying stable transmission of chromatin states in dividing cells remain active in neurons for the purpose of long - term information storage is gaining significant traction and interest within neuroscience , and a better understanding of these processes is necessary for a more complete conceptualization of neural plasticity and memory . alterations in the structural state of chromatin appear to be highly conserved mechanisms underlying information storage in invertebrate ( eg , crab , honeybee ) and vertebrate ( eg , rat , mouse , human ) central nervous systems . the primary focus of the current review is to highlight the accumulating data suggesting that dynamic dna methylation and demethylation , and the enzymes responsible for methylating and demethylating dna , are critically involved in memory formation and behavioral plasticity . while the recognition that the structures of chromatin and dna are rapidly modifiable in the brain adds a significant amount of complexity to our understanding of behavioral and neuronal plasticity , it also suggests a heretofore largely untapped therapeutic potential for alleviating a wide range of neurological , and other , disorders . dna methylation plays an essential role in several developmental processes ( eg , genomic imprinting , x - chromosome inactivation ) , in the maintenance of genome stability by silencing repetitive elements , and in maintaining tissue - specific and appropriate patterns of gene expression through cell division . during embryonic and early postnatal development coordinated waves of methylation and demethylation ensure temporally specific patterns of gene expression that act to establish and perpetuate tissue appropriate cellular identities . once established in somatic cells , methylation patterns have traditionally been considered immutable . a seminal study in 2004 by meaney and colleagues showed that variable early postnatal levels of maternal care ( eg , nursing , grooming ) could alter dna methylation patterns in neurons and that these alterations persisted into adulthood and influenced behavioral and neural responses to stress . methylation of a cytosine nucleotide ( 5mc ) is a thermodynamically very stable modification that is endowed with robust power to influence gene expression . for example , methylation of a single site in a brain - derived neurotrophic factor ( bdnf ) exon promoter can silence the gene . transcriptional silencing is thought to occur via one of two nonmutually exclusive mechanisms ; 5mc can physically restrict transcription factor and rna polymerase ii binding , or 5mc can recruit transcriptional repressor protein complexes . until recently , it was believed that 5mc occurred only in the context of cg dinucleotides ; however , new findings have demonstrated the existence of substantial levels of mch methylation where h represents an a , t , or c. like 5mc , mch is depleted in expressed genes and inversely proportional to the level of expressed transcript . the enzymes responsible for catalyzing the transfer of a methyl group to cytosine nucleotides , using dietary sources of s - adenosyl - l - methoinine as the methyl donor , are the dna methyltransferases ( dnmts ) and are broadly subdivided into two categories : the de novo dnmts , dnmt3a and dnmt3b , establish initial methylation patterns on unmethlyated dna ; and the maintenance dnmt , dnmt1 , recreates already established methylation patterns on hemimethylated replicating dna . dnmts are essential to normal development as evidenced by the embryonic or early postnatal lethality of constitutive knockout of dnmt1 or dnmt3a . dnmt1 , dnmt3a , and dnmt3b are all expressed in the postnatal developing rat brain . dnmt1 and dnmt3a are expressed in adult neurons and oligodendrocytes , and dnmt3b expression is detectable as well , although not to the extent of dnmt3a . dnmt mrna generally reaches its highest level at around 1 week postnatal and subsequently decreases in brain . conditional brain - specific dnmt knockouts have yielded insights into the roles of the dnmts in the central nervous system . a conditional dnmt1 knockout induced during embryonic development using the cre - lox system upregulated apoptotic genes and led to degeneration of the cortex and hippocampus , abnormal morphology of dendrites , and alterations in the resting electrophysiological properties of neurons . the conditional forebrain knockout mice survived to adulthood but , not surprisingly , evidenced severe learning and memory impairments , as they failed to show any learning curve in a spatial memory test following 13 days of training . another study that assayed neurological phenotypes in conditional dnmt1 knockout mice in which the knockout occurred at embryonic day 12 ( e12 ) reported that dnmt1 deficiency led to hypomethylation in differentiated neurons and apoptosis of neurons prior to postnatal day 21 in mosaic animals . dnmt3a null mice are essentially normal at birth , but quickly deteriorate and die in early postnatal development . these mice exhibit impaired postnatal neurogenesis accompanied by dramatic alterations in gene expression profiles in neural stem cells with 1253 genes upregulated and 1022 downregulated , effects likely mediated by impaired polycomb repression of neurogenic genes . gene - specific dna methylation as well as neuronal expression of dnmt enzymes , fluctuates as a result of experiences ranging from intake of drugs of abuse , associative and nonassociative learning experiences , cellular insults , and prenatal stressors . furthermore , in several brain pathologies the expression of the dnmts , as well as methylation of specific gene promoters , appears aberrant . endres et al reported that an increase in dna methylation was associated with more robust brain lesions following induction of stroke using the middle cerebral artery occlusion ( mcao ) model . treatment with the nonspecific dnmt inhibitor 5-aza-2'-deoxycytodine ( 5aza ) or mice heterozygous for dnmt evidenced a reduction in neuronal damage following mcao . a more recent study in gerbils determined that 5 minutes of ischemia induced by bilateral common carotid artery occlusion ( 2vo ) significantly upregulated dnmt1 expression specifically in hippocampal ca1 gabaergic neurons as well as in astrocytes 4 days after the occlusion . ninety days of chronic brain hypoperfusion induced by 2vo promoted a decrease in global dna methylation accompanied by a decrease in expression of dnmt3a in parietal lobe cortex with no change in dnmt1 expression . these findings suggest that acute vs chronic ischemic insults may differentially affect dna methylation and expression of de novo and maintenance dnmts in adult brain , and that targeted dnmt inhibition may offer some therapeutic potential in restricting or preventing brain damage invoked by a cerebrovascular accident . temporal lobe epilepsy is associated with aberrant dna methylation of specific genes as well as increased expression patterns of dnmt isoforms in postmortem tissue from human epileptics and in animal models of the disorder . studies using postmortem tissue from patients with intractable epilepsy demonstrate that dnmt1 and dnmt3a protein expression are robustly increased in hippocampal tissue from epileptics , and hypermethylation of the reelin gene promoter , presumably mediated by a dnmt , has been reported . using a rat model , parrish et al recently found that kainic acidinduced epileptic activity in the hippocampus led to increased global dna methylation in the ca1 and ca3 and decreased methylation in the dentate gyrus 6 weeks after kainate treatment . interestingly , distinct patterns of dnmt1 and dnmt3a expression were observed in hippocampal subregions immediately after ( 1 hour ) and 6 weeks after induction of epilepsy . decreased expression of dnmt3a persisted 6 weeks after induced seizure , whereas immediate decreases in dnmt1 expression normalized by 6 weeks . importantly , intrahippocampal treatment with the nonspecific dnmt inhibitor zebularine decreased the latency to seizure onset and prevented the changes in global methylation and promoter methylation of the grin2b / nr2b , a gene known to play a role in epilepsy . collectively , the results from ischemic and epileptic models suggest that wide - ranging insults can influence dna methylation in brain . in the case of epilepsy it is not yet clear if the changes in expression are involved in the etiology of epilepsy or result from the pathology . these findings nevertheless demonstrate that genomic methylation is indeed plastic and likely plays a key role in neurological disorders and neurological responses to insult . prenatal stressors as well as stress paradigms administered in adulthood have repeatedly been shown to influence neuronal dna methylation . indeed , differential methylation of the corticotropin - releasing factor gene promoter follows maternal deprivation stress , prenatal stress , and chronic mild stress . variation in the diet of mice during gestation or later in development can alter the methylation status of dna in a persistent fashion . an increase in dietary l - methionine or treatment with the nonspecific histone deacetylase inhibitor trichostatin a ( tsa ) reverses the effects of poor maternal care behavior on dna methylation and hypothalamic - pituitary - adrenal axis , and behavioral responses to stress , providing further evidence that dna methylation marks in neurons are modifiable . exposure to a cat increases bdnf gene methylation in the dorsal hippocampus in rats , while simultaneously favoring a decrease in methylation of the bdnf gene in the ventral hippocampus with no change in bdnf methylation in the basolateral amygdala or the prefrontal cortex . the functional significance of bidirectional methylation of the same gene in different brain regions , or how this is mediated , is not yet clear . prenatal exposure to the environmental toxin methyl mercury hypermethylates the bdnf promoter region in the hippocampal dentate gyrus , with an accompanying hypoacetylation of histone h3 and a decrease in bdnf mrna expression . these epigenetic alterations are associated with increased depressive - like behavior in adulthood , as assessed by the forced swim test . interestingly , bdnf expression in the hippocampal dentate gyrus is necessary for antidepressant efficacy in the forced swim test , thus suggesting a mechanism whereby various stressors experienced in the intrauterine environment may relate to deleterious behavioral phenotypes in adult animals . tian et al reported that conditioned place preference to cocaine led to global methylation in the prefrontal cortex , and that methionine supplementation prevented the establishment of conditioned place preference for cocaine , but not morphine or food reward . bodetto et al have confirmed a role for dna methylation in mediating the rewarding effects of cocaine . in their study , cocaine increased dna methylation at the protein phosphatase 1 ( pp1 ) gene promoter , a memory suppressor gene , and increased expression of dnmt3a . dnmt3a overexpression specifically in the nucleus accumbens , a brain region often implicated in behavioral responses to drugs of abuse , has been shown to attenuate cocaine reward and increase dendritic spine density of thin spines in nucleus accumbens neurons . by contrast , nucleus accumbens - specific knockout of dnmt3a potentiated conditioned place preference for cocaine . acute vs chronic cocaine use has opposite effects on dnmt3a expression in nucleus accumbens as acute treatment increases , whereas chronic treatment decreases , dnmt3a expression in the accumbens . administration of the dnmt inhibitor rg108 blocks cocaine 's effect on spine density in the nucleus accumbens and enhances conditioned place preference for cocaine . ethanol exposure during all 3 trimesters of embryonic development similarly has been shown to upregulate the expression of dnmt3a as well as dnmt1 and the methyl - binding protein methyl - cpg - binding protein 2 ( mecp2 ) in the hippocampus . importantly , cellular insults , prenatal stressors , or exposure to aversive stimuli are not the only experience - driven changes in dna methylation or dnmt expression . single running wheel exercise sessions or week - long access to a running wheel , known to be a rewarding activity in rodents , demethylate the bdnf exon iv promoter , increase bdnf mrna and protein in the hippocampus of sprague - dawley rats , and can elevate levels of phosphorylated mecp2 , and subsequently silence the associated gene . phosphorylation of mecp2 can lead to its dissociation from chromatin , which may favor transcriptional activation of bdnf . single exercise sessions decreased dnmt3b and dnmt1 in hippocampus in young , but not old , rats . interestingly , long - term exercise is associated with improved learning in rodents and humans , and in enhanced hippocampal plasticity in rodent models , an effect that may be related to the increases in bdnf . in summary , highly variable and biologically relevant environmental experiences appear to alter the methylation state of specific regions of the genome and promote increases or decreases in the associated mrna and protein . long thought to be a paragon of biological stability , gene methylation , at least in the brain , may be a rather dynamic process that is altered as a result of environmental input . broadly speaking , epigenetic processes have been implicated in behavioral adaptations that rely on associative and nonassociative learning processes as well as in the subsequent storage of putative memory traces in the central nervous system . the notion that long - term memories are encoded in the methylation state of the dna was initially proposed by griffith and mahler in a theoretical paper published in nature ( the dna ticketing theory of memory ) , in 1969 . they proposed that the physical basis of memory could lie in the enzymatic modification of the dna of nerve cells . the events in the nervous system that are necessary for the formation of long - term memories are complex and not completely understood . memory formation requires the orchestration of precise and temporally coordinated changes in gene expression , transcription factor activation and inactivation , and bidirectional changes in the expression and activity of chromatin and dna modifying enzymes . these molecular events coalesce to produce de novo changes in the synaptic strength and connectivity within specific circuitry underlying the formation and long - term storage of new information . moreover , the specific neural pattern responsible for the storage of the memory is retrievable in the presence or absence of the stimuli that promoted its formation . importantly , the memory trace must be self - perpetuating , must persist in spite of the continual turnover of molecules involved in its genesis , and can potentially last the lifetime of an organism . the idea that dynamic changes in dna methylation are necessary for long - term memory formation was first given empirical support by sweatt and colleagues . initially the sweatt laboratory reported that treatment with nonspecific dnmt inhibitors impaired the formation of contextual fear associations . a follow - up study found that experience in associative fear learning to context , a paradigm in which an animal is exposed to contiguous presentations of a novel and initially innocuous environmental context paired with an aversive footshock , could rapidly ( ie , within 30 minutes ) increase the methylation of the memory suppressor gene protein phosphatase 1 ( pp1 ) , while concurrently demethylating the promoter region of the plasticity - related gene reelin . experience in fear learning upregulated the expression of dnmt3a and dnmt3b in areas of the brain necessary for learning ( eg , hippocampus ) , with no effect on dnmt1 . moreover , nonspecific inhibition of dnmts impaired memory and prevented the methylation of pp1 , while enhancing the demethylation of reelin . following training , the methylation of reelin and pp1 returned to normal within 24 hours , leading the authors to conclude that dna methylation , while critical for memory formation , is not likely to be a mechanism of longterm storage , at least in the hippocampus . further studies have expanded on these initial findings and implicated methylation of the bdnf gene in associative fear learning . experience in a fear learning paradigm demethylates the bdnf exon iii and exon iv promoters in the hippocampus , and these effects are blocked by application of the nmda receptor antagonist mk801 , indicating that they are activity - driven . in accordance with the aforementioned miller and sweatt study , the effects on methylation of bdnf in the hippocampus were relatively transient and observable 30 minutes and 24 hours after training . although methylation changes in the hippocampus are relatively transient , methylation of the memory suppressor calcineurin is increased in the prefrontal cortex 7 days after fear conditioning , and this hypermethylation migrates to the anterior cingulate cortex as long as 1 month following initial training . infusion of dnmt inhibitors directly into the anterior cingulate cortex blocks memory retrieval 30 days after training . these data are consistent with the known roles of these brain regions in memory formation vs storage , and suggest that transient and long - lasting methylation / demethylation in distinct brain circuits is important in establishing long - term memory of fearful stimuli . day et al have demonstrated that the importance of dna methylation / demethylation in memory formation is not restricted to aversive events ( eg , fear conditioning , morris water maze ) . using a cued - sucrose delivery associative reward learning paradigm , the authors found that experience in the learning task increased the expression of the immediate early genes erg1 and c - fos , which were demethylated following learning . in a neuronal culture preparation , kc1 depolarization did not change dnmt3a or dnmt3b expression ; however , it did increase dnmt3a binding at the genomic sites ( ergl and c - fos ) that underwent de novo methylation in the in vivo reward - learning experiments . dnmt inhibition before kc1 treatment prevented depolarization - induced changes in dna methylation , and pharmacological inhibition of dnmts in the ventral tegmental area in vivo blocked reward learning without influencing motivation in general . interestingly , treatment with dnmt inhibitors can prevent the memory enhancing effects induced by other compounds , as dnmt inhibition has been shown to prevent estrogen - induced improvements in memory . estrogen treatments increase the hippocampal expression of dnmt3a and dnmt3b , but not dnmt1 . using mice with conditional forebrain - specific double knockout of dnmt1 and dnmt3a in neurons , feng et al reported learning and ltp deficits that were not apparent with a single knockout of either dnmt1 or dnmt3a . therefore , although experience in associative learning tasks and other stimuli appear to differentially affect de novo vs maintenance dnmt expression , it seems that each of these distinct isoforms can compensate for the lack of the other . the culmination of the molecular events that promote long - term memory formation leads to structural changes at synapses in brain - region specific circuits that underlie learning and memory . therefore , it is not surprising that epigenetic modifications of chromatin have been implicated in regulating the forms of synapse plasticity believed to establish memory . initial studies implicating epigenetic processes in synaptic plasticity focused on histone modifications such as acetylation and deacetylation . subsequently , manipulation of dnmts in dissociated neuronal cultures and in acute brain slice experiments have implicated the process underlying addition or removal of 5mc in regulating basal neuronal function as well as plasticity within brain circuits . levenson et al found that treatment of a hippocampal slice preparation with the dnmt inhibitors 5aza or zebularine impaired the magnitude of long - term potentiation ( ltp ) at the schaeffer collateral - ca1 pathway . ltp , widely believed to be a cellular correlate of learning , is typically induced via electrical stimulation of brain slices with robust high - frequency stimuli , which causes a demonstrable increase in synaptic responses subsequent to the high - frequency induction protocol . -burst stimulation , a physiologically relevant means of inducing ltp , led to robust and enduring potentiation ( 3 hours ) in vehicle - treated slices ; however , treatment with either dnmt inhibitor impaired ltp magnitude and maintenance . in a follow - up study , it was shown that dnmt - inhibitor induced ltp deficits were rescued by slice application of the histone deacetylase inhibitor sodium butyrate , suggesting a crosstalk between dna methylation and histone acetylation in the regulation of hippocampal synaptic plasticity . in accordance with the observation that nonspecific dnmt inhibition impairs ltp magnitude and maintenance , feng et al have shown that forebrain - specific conditional double knockout of both dnmt1 and dnmt3a led to similar ltp impairments . somewhat surprisingly , no effects on synaptic function were observed in dnmt1 or dnmt3a single knockouts . depolarization of hippocampal neuron cultures with 50 mm kc1 downregulates dnmt1 and dnmt3a expression , an effect that is prevented by the sodium - channel blockers tetrodotoxin and veratridine . kc1-induced increases in neuronal activity demethylate the regulatory region of the bdnf exon iv promoter , and promote the dissociation of a corepressor complex composed of mecp2 , histone deacetylases , and sin3a from the bdnf promoter . bdnf has been broadly implicated in neuronal viability , synaptic plasticity , synaptogenesis , and memory , suggesting important activity - dependent functional consequences of bdnf demethylation . although basal synapse function is normal in acute hippocampal slices treated with dnmt inhibitors , dnmt inhibitors administered to dissociated hippocampal neuron cultures decrease the frequency of spontaneous miniature excitatory postsynaptic currents ( mepscs ) and demethylate the bdnf i promoter . these effects are activity - dependent , as the nmda receptor antagonist ap5 prevented bdnf promoter demethylation . although most studies assessing the role of histone modifications and dna methylation on ltp have examined the hippocampus , sui et al found that high frequency stimulation - induced ltp led to demethylation of the reelin and bdnf gene promoters in the prefrontal cortex and increased acetylation of histone 3 and histone 4 , marks of active transcription . dnmt inhibitor treatment impaired ltp in prefrontal cortex and prevented the alterations in histone acetylation . in future experiments it will be interesting to determine if the electrophysiological correlates of memory formation ( eg , ltp ) migrate from brain regions necessary for memory formation ( eg , hippocampus ) to areas more involved in memory storage ( eg , anterior cingulate cortex , prefrontal cortex ) and if such changes require dna methylation status updates within this geography . any role for rapid dna methylation / demethylation in memory formation necessitates the existence of an active mechanism for removing 5mc from specific genes involved in plasticity and memory . in other words there switch on specific cytosine nucleotides that is responsive to environmental contingencies that promote associative and nonassociative forms of learning . among the strongest candidates as molecular agents of demethylation tet1 , tet2 , and tet.3 are known to be bona fide mediators of 5mc demethylation in plants , and in mammalian tissue exhibit a strong preference for cpg - rich motifs . the pathway responsible for conversion of 5mc to cytosine is thought to involve successive oxidation of 5mc to 5-hydroxymethylcytosine ( 5hmc ) to 5-formylcytosine ( 5fc ) to 5-carboxylcytosine ( 5cac ) . the presence of 5hmc in the brain is significantly diminished when tet proteins are inhibited . all three members of the tet protein family are capable of converting 5mc to 5hmc as well as subsequent oxidation of 5hmc to 5fc and 5cac . the modified bases can then be further subjected to deamination , glycosylation , and base excision repair to result in final conversion back to a cytosine base . reconversion back to cytosine may require the activity of base - excision repair mechanisms . 5hmc can be deaminated to 5hmu by activation - induced deaminase ( aid ) , with subsequent removal of 5hmu by thymine dna glycosylase ( tdg ) , methyl - binding domain protein 4 ( mdb4 ) , and single strand - specific monofunctional uracil dna glycosylase 1 ( smug1 ) . thymidine glycosylase can also directly target 5fc and 5cac ; however , any in vivo role of tdg in demethylation remains unclear . interestingly , it was recently shown that dnmts may also act as demethylases capable of converting 5hmc to c , possibly by a direct interaction with tdg . methyl - binding domain protein 2 ( mdb2 ) can directly demethylate dna containing 5mc by a reaction that releases formaldehyde . guo et al have demonstrated activity - dependent demethylation of two plasticity - related genes , fibroblast growth factor 1 ( fgf1 ) and bdnf following electrical stimuli capable of inducing epileptiform activity . in the hippocampus , mice with reduced levels of tet1 were incapable of demethylating bdnf and fgf genes following seizure - inducing stimuli . tet1 knockout mice exhibit downregulated expression of the neuronal activity - related genes npas4 , c - fos , and arc and a reduction in 5hmc levels in the hippocampus and cortex with no change in 5mc . tet1 knockouts develop normally without any observable brain abnormalities , which may suggest that tet2 or tet3 can compensate for loss of tet1 . tet1 knockouts also have impaired short - term spatial memory formation and abnormal neuro - genesis in neural precursor cells , but do not appear to have robust long - term memory impairments . the knockouts surprisingly have abnormally enhanced long - term depression elicited in the hippocampus and an impaired ability to extinguish previously acquired associative memories ( ie , extinction ) . expression of several plasticity - related genes were shown to be diminished , however , including c - fos , arc , npas4 , and erg2 in hippocampus , likely resulting from hypermethylation of those genes . tet1 knockout elevated promoter methylation of 478 genes and decreased methylation in 38 genes , with an overlap of 39 that were both hypermethylated and downregulated . by contrast , hippocampaltargeted overexpression of tet1 upregulated memory - associated genes including c - fos , arc , erg1 , homer1 , and nr4a2 and impaired contextual fear learning . interestingly , overexpression of a catalytically inactive form of tet1 also increased expression of those genes and impaired fear learning suggesting demethylase - dependent and -independent effects on learning and gene expression . recent studies have also characterized the role of other potential demethylation factors in brain function . the growth arrest and dna - inducible 45 ( gadd45 ) family of enzymes may bind to and focus the enzymatic activity of cytidine deaminases and thymidine glycosylases to specific gene promoters , thereby tagging specific genes for active demethylation . ma et al suggested that neuronal activity may focus base excision repair mechanisms to cpg promoters and this may be mediated by gadd45 enzymes . leach et al reported that gadd45b knockout mice are impaired in contextual fear conditioning , whereas sultan and coworkers found improvements in contextual fear learning at 24 hours and 28 days post - training , and an enhanced late - phase ltp . the reasons behind the contrasting results are not yet clear ; however , in both cases the studies suggest that manipulations of putative demethylases can alter normal learning and memory . any role for rapid dna methylation / demethylation in memory formation necessitates the existence of an active mechanism for removing 5mc from specific genes involved in plasticity and memory . in other words there must exist a switch on specific cytosine nucleotides that is responsive to environmental contingencies that promote associative and nonassociative forms of learning . among the strongest candidates as molecular agents of demethylation tet1 , tet2 , and tet.3 are known to be bona fide mediators of 5mc demethylation in plants , and in mammalian tissue exhibit a strong preference for cpg - rich motifs . the pathway responsible for conversion of 5mc to cytosine is thought to involve successive oxidation of 5mc to 5-hydroxymethylcytosine ( 5hmc ) to 5-formylcytosine ( 5fc ) to 5-carboxylcytosine ( 5cac ) . the presence of 5hmc in the brain is significantly diminished when tet proteins are inhibited . all three members of the tet protein family are capable of converting 5mc to 5hmc as well as subsequent oxidation of 5hmc to 5fc and 5cac . the modified bases can then be further subjected to deamination , glycosylation , and base excision repair to result in final conversion back to a cytosine base . reconversion back to cytosine may require the activity of base - excision repair mechanisms . 5hmc can be deaminated to 5hmu by activation - induced deaminase ( aid ) , with subsequent removal of 5hmu by thymine dna glycosylase ( tdg ) , methyl - binding domain protein 4 ( mdb4 ) , and single strand - specific monofunctional uracil dna glycosylase 1 ( smug1 ) . thymidine glycosylase can also directly target 5fc and 5cac ; however , any in vivo role of tdg in demethylation remains unclear . interestingly , it was recently shown that dnmts may also act as demethylases capable of converting 5hmc to c , possibly by a direct interaction with tdg . methyl - binding domain protein 2 ( mdb2 ) can directly demethylate dna containing 5mc by a reaction that releases formaldehyde . guo et al have demonstrated activity - dependent demethylation of two plasticity - related genes , fibroblast growth factor 1 ( fgf1 ) and bdnf following electrical stimuli capable of inducing epileptiform activity . in the hippocampus , mice with reduced levels of tet1 were incapable of demethylating bdnf and fgf genes following seizure - inducing stimuli . tet1 knockout mice exhibit downregulated expression of the neuronal activity - related genes npas4 , c - fos , and arc and a reduction in 5hmc levels in the hippocampus and cortex with no change in 5mc . tet1 knockouts develop normally without any observable brain abnormalities , which may suggest that tet2 or tet3 can compensate for loss of tet1 . tet1 knockouts also have impaired short - term spatial memory formation and abnormal neuro - genesis in neural precursor cells , but do not appear to have robust long - term memory impairments . the knockouts surprisingly have abnormally enhanced long - term depression elicited in the hippocampus and an impaired ability to extinguish previously acquired associative memories ( ie , extinction ) . expression of several plasticity - related genes were shown to be diminished , however , including c - fos , arc , npas4 , and erg2 in hippocampus , likely resulting from hypermethylation of those genes . tet1 knockout elevated promoter methylation of 478 genes and decreased methylation in 38 genes , with an overlap of 39 that were both hypermethylated and downregulated . by contrast , hippocampaltargeted overexpression of tet1 upregulated memory - associated genes including c - fos , arc , erg1 , homer1 , and nr4a2 and impaired contextual fear learning . interestingly , overexpression of a catalytically inactive form of tet1 also increased expression of those genes and impaired fear learning suggesting demethylase - dependent and -independent effects on learning and gene expression . recent studies have also characterized the role of other potential demethylation factors in brain function . the growth arrest and dna - inducible 45 ( gadd45 ) family of enzymes may bind to and focus the enzymatic activity of cytidine deaminases and thymidine glycosylases to specific gene promoters , thereby tagging specific genes for active demethylation . ma et al suggested that neuronal activity may focus base excision repair mechanisms to cpg promoters and this may be mediated by gadd45 enzymes . leach et al reported that gadd45b knockout mice are impaired in contextual fear conditioning , whereas sultan and coworkers found improvements in contextual fear learning at 24 hours and 28 days post - training , and an enhanced late - phase ltp . the reasons behind the contrasting results are not yet clear ; however , in both cases the studies suggest that manipulations of putative demethylases can alter normal learning and memory . the brain exhibits dynamic patterns of dna methylation and dnmt expression during aging , and the transcription of key memory - related genes declines in aging . consistent with this observation is that in aged animals impairments in ltp magnitude and maintenance are observed when weak ltp induction protocols ( ie , near - threshold ) are used . siegmund et al have shown changing patterns of dna methylation patterns at various gene loci in human brain , with a general trend for increasing methylation over the lifespan in the 50 loci examined . inappropriate methylation of the activity - related cytoskeleton - associated protein ( arc ) , a known actor in synaptic plasticity and memory , may play a role in age - related memory impairments . old rats have higher levels of methylation of the arc promoter than do adult rats , and the transcription of arc is reduced in the aged hippocampus after learning events relative to younger animals . in addition , inhibition of arc interferes with maintenance of ltp , likely due to its role in synaptic -amino-3-hydroxy5-methyl-4 isoxazolepropionic acid ( ampa ) receptor trafficking . oliviera et al have demonstrated an aging - associated decrease in the expression of dnmt3a2 , one of two transcripts from the dnmt3a gene , in the hippocampus of aged mice . dnmt3a2 is structurally identical to dnmt3al except it lacks 219 amino acids in the n- terminus , is associated with euchromatin , and appears to act as an immediate early gene . learninginduced activation of dnmt3a2 was shown to be impaired in aged mice and overexpression of dnmt3a2 , which increased global dna methylation in the hippocampus , improved performance in fear learning and object location memory tasks . specific gene methylation alterations have been shown in postmortem brain tissue from alzheimer 's disease patients , and these patients exhibit an accelerated rate of age - related change in methylation . alzheimer's - related alterations in dna methylation may be complex and vary by brain region , as global hypomethylation has been shown in entorhinal cortex in postmortem alzheimer 's disease tissue , as well as hypermethylation in the dorsolateral prefrontal cortex . debilitating psychiatric disorders including schizophrenia , bipolar disorder , and major depressive disorder have been linked to aberrant dna methylation . mill and coworkers examined genomic dna from 125 postmortem brains of schizophrenics , bipolar , and nonpsychiatric patients and concluded that dna methylation is significantly altered in major psychiatric disorders . the reelin and gad1 promoter regions are hypermethylated in the brains of schizophrenic patients , and dnmt1 expression is upregulated . the gad1 gene may be of particular interest as it codes for glutamic acid decarboxylase , the enzyme responsible for synthesis of -aminobutyric acid ( gaba ) , the major inhibitory neurotransmitter in the brain . noh et al demonstrated that an antisense - driven knock - down of dnmt1 in mouse cortical neuron cultures was accompanied by an increase in reelin expression and suggest that reduced reelin and gad67 protein may be due to dnmt1-mediated hypermethylation of their promoters . treatment with the histone deacetylase inhibitors tsa or valproate can decrease gad1 promoter methylation , decrease dnmt1 expression in mouse cortex , and increase the expression of reelin and gad67 . increased reelin and gad67 expression were associated with the dissociation of mecp2containing corepressor complexes from their promoter regions . deficits in the ability to inhibit a startle response to an auditory stimulus when that stimulus is preceded by a smaller magnitude auditory stimulus are observed in human schizophrenic patients as well as in animal models of schizophrenia . our laboratory has recently found a dissociable impact of conditional forebrain knockout of dnmt1 and dnmt3a on prepulse inhibition in mice ( unpublished data ) . dnmt1 knockout mice showed an enhanced inhibition of their startle response at all prepulse stimulus magnitudes tested , effects in opposition to the impairments in prepulse inhibition observed in schizophrenia models . significant stressors experienced in the prenatal environment may predispose an individual to the development of a psychiatric disorder in adulthood . restraint stress experienced by a pregnant mouse leads to increases in dnmt1 and mecp2 binding at the reelin and gad1 promoter regions , changes that resemble those observed in postmortem samples from schizophrenic brain . polymorphisms in the dnmt3b gene were recently found to be associated with suicide attempts in depressed patients . mcgowan et al reported increased methylation of the glucocorticoid receptor gene in postmortem brain tissue from suicide victims that had experienced child abuse , findings that are consistent with the behavioral abnormalities observed in animal models of insufficient postnatal care . overexpression of dnmt3a in the nucleus accumbens has been demonstrated to induce depressive - like behavior in mice , whereas inhibition of dnmts in the nucleus accumbens has antidepressantlike effects in a chronic social defeat model as well as in the forced swim test . one hurdle in the way of a better understanding of the role of dna methylation , and chromatin modification in general , in memory is the robust and dynamic interplay between the various enzymes and proteins capable of altering the chromatin . 5mc , by poorly understood signaling pathways , is able to recruit methyl - binding proteins and subsequently large chromatin remodeling complexes that are believed to stably mark stretches of the genome . the methyl - binding protein mecp2 recognizes single 5mc sites and is thought to further recruit transcriptional compressor complexes . interestingly , approximately 95% of cases of the neurodevelopmental mental retardation syndrome rett syndrome are caused by mutations of mecp2 , with some similar phenotypes apparent in cases of mecp2 duplication syndrome , which involves a duplication of the xq28 chromosomal region harboring mecp2 . using a mouse model of mecp2 duplication syndrome , our laboratory has shown that a 50% increase in mecp2 in brain promotes motor coordination deficits , anxiety , learning and memory , and ltp impairments . therefore , the molecular events proceeding from dna methylation followed by binding of mecp2 appear to be of critical importance for cognitive function and synaptic plasticity . dnmt inhibition can impair memory formation as well as ltp in hippocampal slices , and these effects are reversed by treatment with nonspecific histone deacetylase inhibitors . drugs that promote the acetylation of histones may facilitate the loosening of chromatin by leading to the release of methyl - binding proteins ( eg , mecp2 ) . our laboratory has shown that pharmacological inhibition of dnmts in cultured hippocampal neurons decreases mepscs , however this effect is occluded in mecp2 knockout neurons . another large gap remaining in the pursuit of a more complete understanding of the role of dna methylation in memory formation is determining the mechanisms both upstream and downstream of the epigenetic alterations as well as how individual epigenetic modifiers are activated in distinct environmental and cellular contexts . for instance how are dnmts or tet proteins directed in a sequence - specific manner ? although the factors that guide a dnmt to a specific methylated site are not known for certain , it is believed that interactions with transcription factors and other chromatin proteins play a critical role . heterochromatin is characterized by distinct epigenetic marks , eg , methylated lysines 9 ( h3k9 ) and 27 ( h3k27 ) , and dna cpg methylation , which are associated with further recruitment of methyl - binding proteins , histone deacetylases , and other proteins . these histone modifications and the enzymes that catalyze their formation are known to interact with dnmts and methyl - binding proteins . for example , the histone protein hid recruits dnmt1 and dnmt3b , and the histone methyltransferases suv39h1 and g9a recruit dnmt3a . it has previously been shown that the mir-29 family of micro rnas is capable of targeting dnmt3a , dnmt3b , and the tet proteins , potentially establishing a balance between methylation and demethylation at specific genomic targets . vire et al have highlighted the importance of polycomb group proteins as links between the methylation of histones and dna methylation . trimethylation of histone 3 at lysine 9 ( h3k9 ) and h4k20 appears to be a prerequisite for subsequent methylation of a gene . following h3k9 binding , heterochromatin protein 1 ( hp1 ) can associate with dnmt3a by direct binding to its plant homeodomain ( phd ) motif . how dynamic are chromatin states and the molecular agents that confer specific states resulting from environmental experience ? are there distinct roles for different dnmt or tet isoforms in methylation and demethylation in specific tissues or in response to distinct signals ? in spite of the inherent complexity of the epigenome , great strides are being made to determine its role in the central nervous system . these advances are , and will continue to be , essential for understanding diverse processes including memory formation , responsiveness to stressors and neurological insults , and the etiology of psychiatric disorders .

dynamic regulation of chromatin structure in postmitotic neurons plays an important role in learning and memory . methylation of cytosine nucleotides has historically been considered the strongest and least modifiable of epigenetic marks . accumulating recent data suggest that rapid and dynamic methylation and demethylation of specific genes in the brain may play a fundamental role in learning , memory formation , and behavioral plasticity . the current review focuses on the emergence of data that support the role of dna methylation and demethylation , and its molecular mediators in memory formation .

Introduction DNA methylation is indispensable for normal organismal development DNA methylation and DNMT expression are responsive to environmental stressors and cellular insults DNA methylation, DNMTs, and memory DNA methylation and synaptic function Determining an active demethylation process DNA methylation in disorders presenting with cognitive impairment Future questions

epigenetics in its current formulation is more narrowly defined as the perpetuation of genetic information from a cell to its descendants without any necessary change to the genetic code itself , and has been posited as a molecular bridge between the information contained in the genotype and what emerges as a complex and evermodifiable phenotype . the revolution in molecular biology that began in the 1950s , following shortly after waddington 's theoretical formulation of epigenetics , has provided scientists with tools capable of characterizing and understanding the mechanisms that comprise this layer of complexity beyond the genome ( ie , the epigenome ) . dna exists in a continuum of variably compacted states controlled by the structural state of chromatin , ie , the dna and the histone proteins around which it is wrapped . methylation at the 5-position of cytosine nucleotides within cpg dinucelotides is the only direct epigenetic modification of dna and is associated with transcriptional silencing . moreover , there is now recognition that dna methylation itself , once thought to be the most stable of epigenetic marks , may switch between methylated and unmethylated states that render stretches of the chromatin a dynamic canvas on which epigenetic and other mechanisms can work to promote forms of plasticity necessary for long - term information storage . the hypothesis that the processes underlying stable transmission of chromatin states in dividing cells remain active in neurons for the purpose of long - term information storage is gaining significant traction and interest within neuroscience , and a better understanding of these processes is necessary for a more complete conceptualization of neural plasticity and memory . alterations in the structural state of chromatin appear to be highly conserved mechanisms underlying information storage in invertebrate ( eg , crab , honeybee ) and vertebrate ( eg , rat , mouse , human ) central nervous systems . the primary focus of the current review is to highlight the accumulating data suggesting that dynamic dna methylation and demethylation , and the enzymes responsible for methylating and demethylating dna , are critically involved in memory formation and behavioral plasticity . while the recognition that the structures of chromatin and dna are rapidly modifiable in the brain adds a significant amount of complexity to our understanding of behavioral and neuronal plasticity , it also suggests a heretofore largely untapped therapeutic potential for alleviating a wide range of neurological , and other , disorders . dna methylation plays an essential role in several developmental processes ( eg , genomic imprinting , x - chromosome inactivation ) , in the maintenance of genome stability by silencing repetitive elements , and in maintaining tissue - specific and appropriate patterns of gene expression through cell division . during embryonic and early postnatal development coordinated waves of methylation and demethylation ensure temporally specific patterns of gene expression that act to establish and perpetuate tissue appropriate cellular identities . a seminal study in 2004 by meaney and colleagues showed that variable early postnatal levels of maternal care ( eg , nursing , grooming ) could alter dna methylation patterns in neurons and that these alterations persisted into adulthood and influenced behavioral and neural responses to stress . for example , methylation of a single site in a brain - derived neurotrophic factor ( bdnf ) exon promoter can silence the gene . dnmt1 , dnmt3a , and dnmt3b are all expressed in the postnatal developing rat brain . a conditional dnmt1 knockout induced during embryonic development using the cre - lox system upregulated apoptotic genes and led to degeneration of the cortex and hippocampus , abnormal morphology of dendrites , and alterations in the resting electrophysiological properties of neurons . the conditional forebrain knockout mice survived to adulthood but , not surprisingly , evidenced severe learning and memory impairments , as they failed to show any learning curve in a spatial memory test following 13 days of training . gene - specific dna methylation as well as neuronal expression of dnmt enzymes , fluctuates as a result of experiences ranging from intake of drugs of abuse , associative and nonassociative learning experiences , cellular insults , and prenatal stressors . furthermore , in several brain pathologies the expression of the dnmts , as well as methylation of specific gene promoters , appears aberrant . these findings suggest that acute vs chronic ischemic insults may differentially affect dna methylation and expression of de novo and maintenance dnmts in adult brain , and that targeted dnmt inhibition may offer some therapeutic potential in restricting or preventing brain damage invoked by a cerebrovascular accident . temporal lobe epilepsy is associated with aberrant dna methylation of specific genes as well as increased expression patterns of dnmt isoforms in postmortem tissue from human epileptics and in animal models of the disorder . using a rat model , parrish et al recently found that kainic acidinduced epileptic activity in the hippocampus led to increased global dna methylation in the ca1 and ca3 and decreased methylation in the dentate gyrus 6 weeks after kainate treatment . importantly , intrahippocampal treatment with the nonspecific dnmt inhibitor zebularine decreased the latency to seizure onset and prevented the changes in global methylation and promoter methylation of the grin2b / nr2b , a gene known to play a role in epilepsy . collectively , the results from ischemic and epileptic models suggest that wide - ranging insults can influence dna methylation in brain . indeed , differential methylation of the corticotropin - releasing factor gene promoter follows maternal deprivation stress , prenatal stress , and chronic mild stress . variation in the diet of mice during gestation or later in development can alter the methylation status of dna in a persistent fashion . an increase in dietary l - methionine or treatment with the nonspecific histone deacetylase inhibitor trichostatin a ( tsa ) reverses the effects of poor maternal care behavior on dna methylation and hypothalamic - pituitary - adrenal axis , and behavioral responses to stress , providing further evidence that dna methylation marks in neurons are modifiable . exposure to a cat increases bdnf gene methylation in the dorsal hippocampus in rats , while simultaneously favoring a decrease in methylation of the bdnf gene in the ventral hippocampus with no change in bdnf methylation in the basolateral amygdala or the prefrontal cortex . tian et al reported that conditioned place preference to cocaine led to global methylation in the prefrontal cortex , and that methionine supplementation prevented the establishment of conditioned place preference for cocaine , but not morphine or food reward . in their study , cocaine increased dna methylation at the protein phosphatase 1 ( pp1 ) gene promoter , a memory suppressor gene , and increased expression of dnmt3a . ethanol exposure during all 3 trimesters of embryonic development similarly has been shown to upregulate the expression of dnmt3a as well as dnmt1 and the methyl - binding protein methyl - cpg - binding protein 2 ( mecp2 ) in the hippocampus . single running wheel exercise sessions or week - long access to a running wheel , known to be a rewarding activity in rodents , demethylate the bdnf exon iv promoter , increase bdnf mrna and protein in the hippocampus of sprague - dawley rats , and can elevate levels of phosphorylated mecp2 , and subsequently silence the associated gene . interestingly , long - term exercise is associated with improved learning in rodents and humans , and in enhanced hippocampal plasticity in rodent models , an effect that may be related to the increases in bdnf . in summary , highly variable and biologically relevant environmental experiences appear to alter the methylation state of specific regions of the genome and promote increases or decreases in the associated mrna and protein . long thought to be a paragon of biological stability , gene methylation , at least in the brain , may be a rather dynamic process that is altered as a result of environmental input . memory formation requires the orchestration of precise and temporally coordinated changes in gene expression , transcription factor activation and inactivation , and bidirectional changes in the expression and activity of chromatin and dna modifying enzymes . these molecular events coalesce to produce de novo changes in the synaptic strength and connectivity within specific circuitry underlying the formation and long - term storage of new information . the idea that dynamic changes in dna methylation are necessary for long - term memory formation was first given empirical support by sweatt and colleagues . a follow - up study found that experience in associative fear learning to context , a paradigm in which an animal is exposed to contiguous presentations of a novel and initially innocuous environmental context paired with an aversive footshock , could rapidly ( ie , within 30 minutes ) increase the methylation of the memory suppressor gene protein phosphatase 1 ( pp1 ) , while concurrently demethylating the promoter region of the plasticity - related gene reelin . moreover , nonspecific inhibition of dnmts impaired memory and prevented the methylation of pp1 , while enhancing the demethylation of reelin . following training , the methylation of reelin and pp1 returned to normal within 24 hours , leading the authors to conclude that dna methylation , while critical for memory formation , is not likely to be a mechanism of longterm storage , at least in the hippocampus . experience in a fear learning paradigm demethylates the bdnf exon iii and exon iv promoters in the hippocampus , and these effects are blocked by application of the nmda receptor antagonist mk801 , indicating that they are activity - driven . in accordance with the aforementioned miller and sweatt study , the effects on methylation of bdnf in the hippocampus were relatively transient and observable 30 minutes and 24 hours after training . although methylation changes in the hippocampus are relatively transient , methylation of the memory suppressor calcineurin is increased in the prefrontal cortex 7 days after fear conditioning , and this hypermethylation migrates to the anterior cingulate cortex as long as 1 month following initial training . these data are consistent with the known roles of these brain regions in memory formation vs storage , and suggest that transient and long - lasting methylation / demethylation in distinct brain circuits is important in establishing long - term memory of fearful stimuli . day et al have demonstrated that the importance of dna methylation / demethylation in memory formation is not restricted to aversive events ( eg , fear conditioning , morris water maze ) . dnmt inhibition before kc1 treatment prevented depolarization - induced changes in dna methylation , and pharmacological inhibition of dnmts in the ventral tegmental area in vivo blocked reward learning without influencing motivation in general . the culmination of the molecular events that promote long - term memory formation leads to structural changes at synapses in brain - region specific circuits that underlie learning and memory . in a follow - up study , it was shown that dnmt - inhibitor induced ltp deficits were rescued by slice application of the histone deacetylase inhibitor sodium butyrate , suggesting a crosstalk between dna methylation and histone acetylation in the regulation of hippocampal synaptic plasticity . kc1-induced increases in neuronal activity demethylate the regulatory region of the bdnf exon iv promoter , and promote the dissociation of a corepressor complex composed of mecp2 , histone deacetylases , and sin3a from the bdnf promoter . bdnf has been broadly implicated in neuronal viability , synaptic plasticity , synaptogenesis , and memory , suggesting important activity - dependent functional consequences of bdnf demethylation . although most studies assessing the role of histone modifications and dna methylation on ltp have examined the hippocampus , sui et al found that high frequency stimulation - induced ltp led to demethylation of the reelin and bdnf gene promoters in the prefrontal cortex and increased acetylation of histone 3 and histone 4 , marks of active transcription . in future experiments it will be interesting to determine if the electrophysiological correlates of memory formation ( eg , ltp ) migrate from brain regions necessary for memory formation ( eg , hippocampus ) to areas more involved in memory storage ( eg , anterior cingulate cortex , prefrontal cortex ) and if such changes require dna methylation status updates within this geography . any role for rapid dna methylation / demethylation in memory formation necessitates the existence of an active mechanism for removing 5mc from specific genes involved in plasticity and memory . among the strongest candidates as molecular agents of demethylation tet1 , tet2 , and tet.3 are known to be bona fide mediators of 5mc demethylation in plants , and in mammalian tissue exhibit a strong preference for cpg - rich motifs . the presence of 5hmc in the brain is significantly diminished when tet proteins are inhibited . thymidine glycosylase can also directly target 5fc and 5cac ; however , any in vivo role of tdg in demethylation remains unclear . tet1 knockout mice exhibit downregulated expression of the neuronal activity - related genes npas4 , c - fos , and arc and a reduction in 5hmc levels in the hippocampus and cortex with no change in 5mc . recent studies have also characterized the role of other potential demethylation factors in brain function . the growth arrest and dna - inducible 45 ( gadd45 ) family of enzymes may bind to and focus the enzymatic activity of cytidine deaminases and thymidine glycosylases to specific gene promoters , thereby tagging specific genes for active demethylation . the reasons behind the contrasting results are not yet clear ; however , in both cases the studies suggest that manipulations of putative demethylases can alter normal learning and memory . any role for rapid dna methylation / demethylation in memory formation necessitates the existence of an active mechanism for removing 5mc from specific genes involved in plasticity and memory . among the strongest candidates as molecular agents of demethylation tet1 , tet2 , and tet.3 are known to be bona fide mediators of 5mc demethylation in plants , and in mammalian tissue exhibit a strong preference for cpg - rich motifs . the presence of 5hmc in the brain is significantly diminished when tet proteins are inhibited . 5hmc can be deaminated to 5hmu by activation - induced deaminase ( aid ) , with subsequent removal of 5hmu by thymine dna glycosylase ( tdg ) , methyl - binding domain protein 4 ( mdb4 ) , and single strand - specific monofunctional uracil dna glycosylase 1 ( smug1 ) . in the hippocampus , mice with reduced levels of tet1 were incapable of demethylating bdnf and fgf genes following seizure - inducing stimuli . tet1 knockout mice exhibit downregulated expression of the neuronal activity - related genes npas4 , c - fos , and arc and a reduction in 5hmc levels in the hippocampus and cortex with no change in 5mc . recent studies have also characterized the role of other potential demethylation factors in brain function . the reasons behind the contrasting results are not yet clear ; however , in both cases the studies suggest that manipulations of putative demethylases can alter normal learning and memory . the brain exhibits dynamic patterns of dna methylation and dnmt expression during aging , and the transcription of key memory - related genes declines in aging . siegmund et al have shown changing patterns of dna methylation patterns at various gene loci in human brain , with a general trend for increasing methylation over the lifespan in the 50 loci examined . inappropriate methylation of the activity - related cytoskeleton - associated protein ( arc ) , a known actor in synaptic plasticity and memory , may play a role in age - related memory impairments . old rats have higher levels of methylation of the arc promoter than do adult rats , and the transcription of arc is reduced in the aged hippocampus after learning events relative to younger animals . dnmt3a2 is structurally identical to dnmt3al except it lacks 219 amino acids in the n- terminus , is associated with euchromatin , and appears to act as an immediate early gene . learninginduced activation of dnmt3a2 was shown to be impaired in aged mice and overexpression of dnmt3a2 , which increased global dna methylation in the hippocampus , improved performance in fear learning and object location memory tasks . alzheimer's - related alterations in dna methylation may be complex and vary by brain region , as global hypomethylation has been shown in entorhinal cortex in postmortem alzheimer 's disease tissue , as well as hypermethylation in the dorsolateral prefrontal cortex . debilitating psychiatric disorders including schizophrenia , bipolar disorder , and major depressive disorder have been linked to aberrant dna methylation . mill and coworkers examined genomic dna from 125 postmortem brains of schizophrenics , bipolar , and nonpsychiatric patients and concluded that dna methylation is significantly altered in major psychiatric disorders . the reelin and gad1 promoter regions are hypermethylated in the brains of schizophrenic patients , and dnmt1 expression is upregulated . the gad1 gene may be of particular interest as it codes for glutamic acid decarboxylase , the enzyme responsible for synthesis of -aminobutyric acid ( gaba ) , the major inhibitory neurotransmitter in the brain . overexpression of dnmt3a in the nucleus accumbens has been demonstrated to induce depressive - like behavior in mice , whereas inhibition of dnmts in the nucleus accumbens has antidepressantlike effects in a chronic social defeat model as well as in the forced swim test . one hurdle in the way of a better understanding of the role of dna methylation , and chromatin modification in general , in memory is the robust and dynamic interplay between the various enzymes and proteins capable of altering the chromatin . using a mouse model of mecp2 duplication syndrome , our laboratory has shown that a 50% increase in mecp2 in brain promotes motor coordination deficits , anxiety , learning and memory , and ltp impairments . dnmt inhibition can impair memory formation as well as ltp in hippocampal slices , and these effects are reversed by treatment with nonspecific histone deacetylase inhibitors . another large gap remaining in the pursuit of a more complete understanding of the role of dna methylation in memory formation is determining the mechanisms both upstream and downstream of the epigenetic alterations as well as how individual epigenetic modifiers are activated in distinct environmental and cellular contexts . heterochromatin is characterized by distinct epigenetic marks , eg , methylated lysines 9 ( h3k9 ) and 27 ( h3k27 ) , and dna cpg methylation , which are associated with further recruitment of methyl - binding proteins , histone deacetylases , and other proteins . it has previously been shown that the mir-29 family of micro rnas is capable of targeting dnmt3a , dnmt3b , and the tet proteins , potentially establishing a balance between methylation and demethylation at specific genomic targets . vire et al have highlighted the importance of polycomb group proteins as links between the methylation of histones and dna methylation . are there distinct roles for different dnmt or tet isoforms in methylation and demethylation in specific tissues or in response to distinct signals ? in spite of the inherent complexity of the epigenome , great strides are being made to determine its role in the central nervous system . these advances are , and will continue to be , essential for understanding diverse processes including memory formation , responsiveness to stressors and neurological insults , and the etiology of psychiatric disorders .

ayurveda advocates therapeutic uses of mineral and metallic preparations in many diseases since century in clinical practice . knowing the possibilities of toxic effects , seers emphasized on following set of exclusive pharmaceutical procedures such as shodhana ( purification and/or detoxification ) , marana ( incineration and/or calcination ) , and amritikarana that converts the metals and minerals into bhasma ( calcined powders ) , . bhasmas are unique preparations which are safely being practiced in ayurveda without any noticeable side effects can be considered as a testimony to their safety , but no objective verifiable data exist to support many such claims . preclinical studies of ayurvedic drug provide scientific basis for their traditional use and to prove that they are safe and efficacious . tamra bhasma ( tb ) ( incinerated copper ) is one among such ayurvedic herbo - metallic preparation used in the treatment of udara ( ascites ) , pandu ( anemia ) , svasa ( asthma ) , and amlapitta ( hyperacidity ) . though wide therapeutic utility of tb has been mentioned in classics , it is reported as poison as or more than that if not processed or purified properly as per classical methods . to indicate its toxic potential , ashtamahadoshas ( eight major ill effects ) though number of studies have been carried out in direction of safety of bhasmas , concerns are always being raised on ayurvedic formulations for the presence of heavy metals , . amritikarana is exclusively mentioned for tb and abhrak bhasma which is said to eliminate all the blemishes from the end product . there is a need to provide scientific basis to establish the impact of this procedure . hence , the present study is aimed to evaluate the acute and subchronic toxicity studies of tb prepared with and without amritikarana in rats . two samples of tb with and without amritikarana were prepared by following standard guidelines as prescribed in ayurvedic classics . copper scraps with 99.89% pure copper was procured from local industrial area , jamnagar , india . it was subjected to general and specific purification procedure followed by incineration after mixed with purified sulfur , kajjali ( black sulfide of mercury ) , and juice of citrus jambhiri lush . in amritikarana , it was mixed with half part purified sulfur and juice of c. jambhiri lush , kept in the corm of amorphophallus campanulatus linn . it was subjected to heat treatment and labeled as tamra bhasma with amritikarana ( tba ) . wistar strain albino rats of either sex , weighing 200 20 g , were used as per the guidelines of the institutional animal ethics committee ( iaec ) . the animals were obtained from the animal house attached to the pharmacology laboratory , ipgt and ra , jamnagar . the animals were maintained under ideal husbandry conditions in terms of standard conditions of temperature ( 23 2 c ) , relative humidity ( 5060% ) and exposed to 12 h light and dark cycles . all animals were exposed to the same environmental conditions and were maintained on standard diet and drinking water ad libitum . the experimental protocol was approved by the iaec/14/2013/16 as per guideline of committee for the purpose of control and supervision of experiments on animals in india . as per classical guideline , the therapeutic clinical dose of tb is 30 mg twice a day ( 60 mg / day ) . the suitable dose for rats was calculated by referring to table of paget and branes and was found to be 5.5 mg / kg body weight of rat ( considered as ted ) . the test drug was administered orally ( licking ) along with honey as adjuvant with the help of oral cannula . young , healthy , nulliparous , and nonpregnant wistar strain albino female rats were selected and acclimatized for 7 days before the experiment . both test drugs along with adjuvant were orally administered at limit dose of 2000 mg / kg to overnight fasted female rats by following organization for economic cooperation and development ( oecd ) 425 guideline . the rats were observed closely for behavioral changes , signs and symptoms of toxicity , and mortality , if any continuously for the first 6 h and thereafter periodically up to 14 days . rats were randomized into six groups , each consisting of six rats comprising three male and three female . group i was kept as control group , received vehicle as honey ( 2 ml / kg , orally ) . group ii to iv were administered with test drug tb without amritikarana along with adjuvant at ted ( 5.5 mg / kg , orally ) , ted 5 ( 27.5 mg / kg , orally ) , and ted 10 ( 55.0 mg / kg , orally ) , respectively . group v to vii were administered with test drug , tba along with adjuvant at ted ( 5.5 mg / kg , orally ) , ted 5 ( 27.5 mg / kg , orally ) , and ted 10 ( 55.0 mg / kg , orally ) , respectively . the suspensions of test drugs were administered orally once a day for 28 consecutive days . general behavioral pattern was observed once a week by exposing each animal to open arena . on the 29th day supraorbital plexus was punctured under light anesthesia and blood was collected by capillary in two different types of tubes , one containing anticoagulant fluid for hematological parameters and another plain tube for serum biochemical investigations . then , the rats were sacrificed and the abdomen was opened through midline incision to record the autopsy changes followed by dissecting out the important organs . hematological analysis was performed by using an automatic hematological analyzer ( swelab , sweden ) . total red blood cell ( trbc ) , hemoglobin ( hb ) , hematocrit , mean corpuscular volume , mean corpuscular hb , mean corpuscular hb concentration , white blood cell ( wbc ) , neutrophils , lymphocyte percentage , eosinophils percentage , monocyte percentage , packed cell volume ( pcv ) , and platelet count were measured from the blood samples . serum biochemical parameters were carried out by using fully automated biochemical random access analyzer ( bs-200 , lilac medicare pvt . ltd . , the studied parameters were blood glucose , urea , creatinine , total cholesterol , high - density lipoprotein ( hdl)-cholesterol , triglyceride , very - low - density lipoprotein ( vldl)-cholesterol , low - density lipoprotein ( ldl)-cholesterol , total protein , albumin , globulin , alkaline phosphatase , serum glutamic oxaloacetic transaminase ( sgot ) , serum glutamic pyruvic transaminase ( sgpt ) , uric acid , direct bilirubin , total bilirubin , and serum calcium . all the important internal organs were carefully dissected namely , liver , kidney , heart , lungs , trachea , intestine spleen , thymus , lymph node , testis , seminal vesicle , prostate , uterus , and ovary . after noting signs of gross lesion and ponderal changes of major organs , all were transferred to 10% phosphate buffered formalin solution for fixation and later on subjected to dehydrating , wax embedding , sectioning , and staining with hematoxylin and eosin for histological evaluation . the slides were viewed under trinocular research carl - zeiss 's microscope at various magnifications to note down the changes in the microscopic features of the tissues . the data are expressed as mean standard error of mean for six rats per experimental group . one - way analysis of variance was used to compare the mean values of quantitative variables among the groups followed by holm sidak multiple t - test for unpaired data by using sigmastat software ( version 3.5 , systat software inc . ) to determine significant difference between groups at p < 0.05 . two samples of tb with and without amritikarana were prepared by following standard guidelines as prescribed in ayurvedic classics . copper scraps with 99.89% pure copper was procured from local industrial area , jamnagar , india . it was subjected to general and specific purification procedure followed by incineration after mixed with purified sulfur , kajjali ( black sulfide of mercury ) , and juice of citrus jambhiri lush . in amritikarana , it was mixed with half part purified sulfur and juice of c. jambhiri lush , kept in the corm of amorphophallus campanulatus linn . it was subjected to heat treatment and labeled as tamra bhasma with amritikarana ( tba ) . wistar strain albino rats of either sex , weighing 200 20 g , were used as per the guidelines of the institutional animal ethics committee ( iaec ) . the animals were obtained from the animal house attached to the pharmacology laboratory , ipgt and ra , jamnagar . the animals were maintained under ideal husbandry conditions in terms of standard conditions of temperature ( 23 2 c ) , relative humidity ( 5060% ) and exposed to 12 h light and dark cycles . all animals were exposed to the same environmental conditions and were maintained on standard diet and drinking water ad libitum . the experimental protocol was approved by the iaec/14/2013/16 as per guideline of committee for the purpose of control and supervision of experiments on animals in india . as per classical guideline , the therapeutic clinical dose of tb is 30 mg twice a day ( 60 mg / day ) . the suitable dose for rats was calculated by referring to table of paget and branes and was found to be 5.5 mg / kg body weight of rat ( considered as ted ) . the test drug was administered orally ( licking ) along with honey as adjuvant with the help of oral cannula . young , healthy , nulliparous , and nonpregnant wistar strain albino female rats were selected and acclimatized for 7 days before the experiment . both test drugs along with adjuvant were orally administered at limit dose of 2000 mg / kg to overnight fasted female rats by following organization for economic cooperation and development ( oecd ) 425 guideline . the rats were observed closely for behavioral changes , signs and symptoms of toxicity , and mortality , if any continuously for the first 6 h and thereafter periodically up to 14 days . rats were randomized into six groups , each consisting of six rats comprising three male and three female . group i was kept as control group , received vehicle as honey ( 2 ml / kg , orally ) . group ii to iv were administered with test drug tb without amritikarana along with adjuvant at ted ( 5.5 mg / kg , orally ) , ted 5 ( 27.5 mg / kg , orally ) , and ted 10 ( 55.0 mg / kg , orally ) , respectively . group v to vii were administered with test drug , tba along with adjuvant at ted ( 5.5 mg / kg , orally ) , ted 5 ( 27.5 mg / kg , orally ) , and ted 10 ( 55.0 mg / kg , orally ) , respectively . the suspensions of test drugs were administered orally once a day for 28 consecutive days . general behavioral pattern was observed once a week by exposing each animal to open arena . supraorbital plexus was punctured under light anesthesia and blood was collected by capillary in two different types of tubes , one containing anticoagulant fluid for hematological parameters and another plain tube for serum biochemical investigations . then , the rats were sacrificed and the abdomen was opened through midline incision to record the autopsy changes followed by dissecting out the important organs . hematological analysis was performed by using an automatic hematological analyzer ( swelab , sweden ) . total red blood cell ( trbc ) , hemoglobin ( hb ) , hematocrit , mean corpuscular volume , mean corpuscular hb , mean corpuscular hb concentration , white blood cell ( wbc ) , neutrophils , lymphocyte percentage , eosinophils percentage , monocyte percentage , packed cell volume ( pcv ) , and platelet count were measured from the blood samples . serum biochemical parameters were carried out by using fully automated biochemical random access analyzer ( bs-200 , lilac medicare pvt . ltd . , the studied parameters were blood glucose , urea , creatinine , total cholesterol , high - density lipoprotein ( hdl)-cholesterol , triglyceride , very - low - density lipoprotein ( vldl)-cholesterol , low - density lipoprotein ( ldl)-cholesterol , total protein , albumin , globulin , alkaline phosphatase , serum glutamic oxaloacetic transaminase ( sgot ) , serum glutamic pyruvic transaminase ( sgpt ) , uric acid , direct bilirubin , total bilirubin , and serum calcium . all the important internal organs were carefully dissected namely , liver , kidney , heart , lungs , trachea , intestine spleen , thymus , lymph node , testis , seminal vesicle , prostate , uterus , and ovary . after noting signs of gross lesion and ponderal changes of major organs , all were transferred to 10% phosphate buffered formalin solution for fixation and later on subjected to dehydrating , wax embedding , sectioning , and staining with hematoxylin and eosin for histological evaluation . the slides were viewed under trinocular research carl - zeiss 's microscope at various magnifications to note down the changes in the microscopic features of the tissues . the data are expressed as mean standard error of mean for six rats per experimental group . one - way analysis of variance was used to compare the mean values of quantitative variables among the groups followed by holm sidak multiple t - test for unpaired data by using sigmastat software ( version 3.5 , systat software inc . ) to determine significant difference between groups at p < 0.05 . the results of acute toxicity showed that both the samples of tb along with adjuvant did not affect any behavioral changes and other parameters during entire experimental period of 14 days . both tb and tba did not show any signs and symptoms of toxicity and mortality when given orally at a dose of 2000 mg / kg . behavioral changes were not observed in treated groups during the course of subchronic toxicity study in comparison to control group . no mortality was observed in both samples of tb at ted 10 , ted 5 , and ted dose levels during the experimental period . effects of tb and tba on change in body weight showed that weight gain was observed in all treated groups , but percentage change in body weight pattern in treated groups did not differ significantly from the changes observed in control groups [ table 1 ] . subchronic administration of tb at different dose levels lead to nonsignificant effect on change in relative weight of nine organs except significant increase was observed in relative weight of heart in tba at ted level [ table 2 ] . tba at all dose levels produced nonsignificant increase in relative weight of thymus , spleen , and testis in a uniform manner while other changes were not distinct in comparison to control group . effect of samples of tb on hematological parameters [ table 2 ] revealed that out of the twelve parameters studied , nonsignificant increase in wbc count and neutrophil in tb at ted 10 dose level while significant increase in monocyte count in tb at ted 5 dose level in comparison to control group . significant increase in hb , pcv , and trbc were observed in tb and tba treated groups at ted and ted 5 dose levels [ table 3 ] . both samples of tb at all dose levels produced nonsignificant increase in platelet count in comparison to control group . among the 18 serum biochemical parameters , significant decrease in blood sugar was found in tb at ted dose level and tba treated drug at ted 5 and ted 10 levels , while significant increase in cholesterol was found in tb and tba treated groups at ted and ted 5 dose levels , while higher dose of both drugs produced nonsignificant increase in serum cholesterol level . significant increase in serum triglyceride , ldl , and vldl level was found in tb at ted 10 treated groups while nonsignificant increase in triglyceride and vldl level in all tba treated groups in comparison to control group . hdl cholesterol was significantly increased in tb treated group at ted dose level and tba treated group at ted and ted 5 treated groups while both at higher dose levels produced nonsignificant increase in comparison to control group . significant increase in albumin the changes observed in remaining biochemical parameters were statistically nonsignificant in comparison to control group [ table 4 ] tb with and without amritikarana at ted 10 dose level did not affect the cytoarchitecture of the spleen , lung , stomach , lymph node , trachea , adrenal , prostate , seminal vesicle , testis , ovary , and uterus . administration of tb at ted 10 causes mild fatty changes in the heart but tba at ted 10 showed normal cytoarchitecture [ fig . 1 ] , while both drugs at higher dose level showed micro fatty changes in liver [ fig . 2 ] . mild cell infiltration and edema in the kidney in tb at ted 10 , while mild fatty changes in tba at ted 10 dose level [ fig . 3 ] were observed and both drugs at higher dose produced mild decrease in cellularity in cytoarchitecture of thymus [ fig . 4 ] however , all these changes were not observed in both the drugs treated groups at ted 5 and ted dose levels in rats that suggest that drug is devoid of drastic toxicity . the results of acute toxicity showed that both the samples of tb along with adjuvant did not affect any behavioral changes and other parameters during entire experimental period of 14 days . both tb and tba did not show any signs and symptoms of toxicity and mortality when given orally at a dose of 2000 mg / kg . behavioral changes were not observed in treated groups during the course of subchronic toxicity study in comparison to control group . no mortality was observed in both samples of tb at ted 10 , ted 5 , and ted dose levels during the experimental period . effects of tb and tba on change in body weight showed that weight gain was observed in all treated groups , but percentage change in body weight pattern in treated groups did not differ significantly from the changes observed in control groups [ table 1 ] . subchronic administration of tb at different dose levels lead to nonsignificant effect on change in relative weight of nine organs except significant increase was observed in relative weight of heart in tba at ted level [ table 2 ] . tba at all dose levels produced nonsignificant increase in relative weight of thymus , spleen , and testis in a uniform manner while other changes were not distinct in comparison to control group . effect of samples of tb on hematological parameters [ table 2 ] revealed that out of the twelve parameters studied , nonsignificant increase in wbc count and neutrophil in tb at ted 10 dose level while significant increase in monocyte count in tb at ted 5 dose level in comparison to control group . significant increase in hb , pcv , and trbc were observed in tb and tba treated groups at ted and ted 5 dose levels [ table 3 ] . both samples of tb at all dose levels produced nonsignificant increase in platelet count in comparison to control group . among the 18 serum biochemical parameters , significant decrease in blood sugar was found in tb at ted dose level and tba treated drug at ted 5 and ted 10 levels , while significant increase in cholesterol was found in tb and tba treated groups at ted and ted 5 dose levels , while higher dose of both drugs produced nonsignificant increase in serum cholesterol level . significant increase in serum triglyceride , ldl , and vldl level was found in tb at ted 10 treated groups while nonsignificant increase in triglyceride and vldl level in all tba treated groups in comparison to control group . hdl cholesterol was significantly increased in tb treated group at ted dose level and tba treated group at ted and ted 5 treated groups while both at higher dose levels produced nonsignificant increase in comparison to control group . significant increase in albumin the changes observed in remaining biochemical parameters were statistically nonsignificant in comparison to control group [ table 4 ] tb with and without amritikarana at ted 10 dose level did not affect the cytoarchitecture of the spleen , lung , stomach , lymph node , trachea , adrenal , prostate , seminal vesicle , testis , ovary , and uterus . administration of tb at ted 10 causes mild fatty changes in the heart but tba at ted 10 showed normal cytoarchitecture [ fig . 1 ] , while both drugs at higher dose level showed micro fatty changes in liver [ fig . 2 ] . mild cell infiltration and edema in the kidney in tb at ted 10 , while mild fatty changes in tba at ted 10 dose level [ fig . 3 ] were observed and both drugs at higher dose produced mild decrease in cellularity in cytoarchitecture of thymus [ fig . 4 ] however , all these changes were not observed in both the drugs treated groups at ted 5 and ted dose levels in rats that suggest that drug is devoid of drastic toxicity . metals and minerals are extensively used in ayurvedic formulations since vedic period , which became an important part of ayurvedic therapeutics . to make them for therapeutic use , they have to pass through a set of classical pharmaceutical processes known as shodhana , marana , etc . the possible impurities that remain in bhasma after marana process are advocated to be removed by another specialized procedure known as amritikarana . in classics the word , amritikarana itself explains the importance of this procedure , i.e. , the process by which amrititva is imparted to the matter . this procedure is recommended for all the bhasmas but claimed to be essential mainly in abhraka and tb . acute toxicity was carried out to record immediate adverse signs and symptoms after the administration of single dose of drug at dose levels that are several folds higher than the therapeutic equivalent dose ( 2000 mg / kg ) . oecd 425 guideline for oral acute toxicity study were employed using a female sex to reduce variability and as a means of minimizing the number of animals used . this is because there is little difference in sensitivity in ld50 test between the sexes ; however , in those cases where differences were observed , females were generally slightly more sensitive . in this study , tb s at the dose level of 2000 mg / kg , orally did not produce any observable toxic effects during the entire duration of acute toxicity study and all female rats survived 14 days of observation suggest that ld50 value may be higher than 2000 mg / kg by oral route . as per un classification , any substance which has oral ld50 of more than 2000 mg / kg is considered as low hazard potential and categorized as un 6.1 pg iii . thus , as per the above criterion , both tb with or without amritikarana can be categorized as substances with low health hazard potential ( class 4 of ghs and un 6.1 pg iii ) . on subchronic administration of tb with or without amritikarana showed significant increase in hb , red blood cell ( rbc ) and pcv but not found to be in dose - dependent manner . in the present study , increased hb content is due to the increase observed in the production of erythrocytes and pcv in these treated groups . the fact that the observed effect was not dose - dependent and is not remarkably deviant from the normal range of values indicates that they do not indicate any serious toxicity potential . previous study also revealed significant increase in total rbc count , pcv , and platelet count at different dose level of tb on administration for 45 days in albino rats . copper is essential for the synthesis of hb and also supports absorption of iron from intestine , which may responsible for increase in rbc related parameters in rats . significant decrease in fasting blood sugar at ted level of tb and higher dose level of tba suggest role of copper in glucose metabolism . copper plays important role in carbohydrate metabolism by stimulating insulin binding , hexose transport , and lipogenesis in vivo and in vitro significant increase in cholesterol level was found in tb and tba treated groups at ted and ted 5 dose levels while higher dose of both drugs produced nonsignificant increase in serum cholesterol level . significant increase in serum triglyceride , ldl , and vldl level was found in tb at ted 10 treated groups while nonsignificant increase in triglyceride and vldl level in all tba treated groups in comparison to control group . this suggests that the test drugs may have effect on cholesterol turnover in the body ; however , parameters were not affected to significant extent at higher dose level in tba treated group . though tendency toward increasing of lipid profile is seen in tba treated group , it may not be indicative of any drastic influence on lipid metabolism , but tb treated rats prone to produce hyperlipidemia and related disorders . hdl - cholesterol was significantly increased in tb treated group at ted dose level and tba treated group at ted and ted 5 treated groups while both at higher dose levels produced nonsignificant increase in comparison to control group . this activity seen at therapeutic dose levels in tb and tba can have good therapeutic application because elevation of hdl - cholesterol level will be quite useful in patients with hypercholesterolemia conditions . however , same was not observed at higher dose levels in both the treated group . parameters related to normal liver functioning such as sgpt , sgot , alkaline phosphatase , and bilirubin were not altered to significant extent at all dose levels in both treated groups in comparison to control group . significant increase in albumin was observed at all dose levels of tb and higher dose level of tba . albumin is a most abundant protein in human blood plasma and synthesized in liver as preproalbumin . however , the values of total protein , globulin , uric acid , calcium , urea , and creatinine in treated groups and control group are still within normal range that suggests that both drugs are devoid of any drastic toxic effect in rats at therapeutic dose level . the results of histopathological studies revealed that mild changes were observed in four visceral organs , i.e. , thymus , heart , liver , and kidney at higher dose level . however , these changes were not observed in both the drug treated groups at lower dose levels in rats , which suggest that the drug is devoid of drastic toxicity . earlier studies with tb also reported safety of the sample at ted level . considering this , it can be said that the bhasma is safe at ted levels , and mild changes can manifest at higher dose levels . overall , effect of both drugs , tb and tba at different dose levels compared with control group . further , there is no significant difference between tb and tba treated groups in any of the observed parameters . tb and tba produced almost same magnitude of effect except tb produced changes on alkaline phosphatase , triglyceride , ldl - cholesterol , and total bilirubin . tba produced more effect on hdl - cholesterol which may have good therapeutic application and will be quite useful in patients with hypercholesterolemia conditions . in histopathological observations , tba showed less magnitude of adverse changes compared to tb . considering all these , it suggests that , though tb is not much toxic at therapeutic dose level has potential to produced adverse effects when administered for longer duration at higher dose levels . from the present study , it is concluded that tba and tb at the dose level of 2000 mg / kg , orally did not produce any observable toxic effects and mortality in acute toxicity study which suggest that ld50 value may be higher than 2000 mg / kg by oral route and can be categorized as substances with low health hazard potential . the results of subchronic toxicity concluded that tb can be relatively safe at therapeutic dose levels in rats . however , tb at ted 10 dose level , equivalent of which are not likely to be ever employed in clinical conditions , has prone to produce toxic changes in liver , kidney , and heart and also impact on immune - related organs such as thymus as revealed by ponderal , hematological , biochemical , and histopathological parameters in rats . at higher dose level , a sample without amritikarana has more magnitude of toxicity than the sample with amritikarana . hence , it can be inferred that amritikarana is must for internal administration of tb in clinical practices . institute for post graduate teaching and research in ayurveda , gujarat ayurved university , jamnagar 361 008 .

backgroundtamra bhasma ( tb ) is one among herbo - metallic preparations extensively used in routine ayurvedic practice . in the present era , bhasma preparations used in ayurvedic system of medicines are always under stern observations for containing heavy metals which may raise the question of safety aspect.objectivein the present study , tb prepared with and without amritikarana was subjected to toxicity study to ascertain the role of amritikarana on safety profile of tb in rats.materials and methodsboth the samples of tb were administered to rats for 28 consecutive days at the doses of 5.5 , 27.5 , and 55 mg / kg . the effects of both drugs were assessed on ponderal changes , hematological , serum biochemical , and histopathology of various organs.resultsresults showed that both the samples of tb did not produce any sign and symptoms of toxicity at therapeutic dose level ( 5.5 mg / kg ) and therapeutic equivalent dose ( ted ) 5 ( 27.5 mg / kg ) while at higher dose of ted 10 ( 55 mg / kg ) tb has mild toxicity in liver , kidney , heart , and thymus on repeated administration for 28 days in rats . the sample without amritikarana has more magnitude of toxicity than the sample with amritikarana.conclusionfrom the present study , it is concluded that tb with amritikarana was found to be relatively safer than tb without amritikarana at different dose levels in rats and hence suggest for safely use in humans at therapeutic dose level . it proves the role of amritikarana in the preparation of tb .

Introduction Materials and methods Drugs and chemicals Animals Dose fixation Acute toxicity study Subchronic toxicity Statistical analysis Results Acute toxicity study Subchronic toxicity study Discussion Conclusion Source of support Conflicts of interest

knowing the possibilities of toxic effects , seers emphasized on following set of exclusive pharmaceutical procedures such as shodhana ( purification and/or detoxification ) , marana ( incineration and/or calcination ) , and amritikarana that converts the metals and minerals into bhasma ( calcined powders ) , . tamra bhasma ( tb ) ( incinerated copper ) is one among such ayurvedic herbo - metallic preparation used in the treatment of udara ( ascites ) , pandu ( anemia ) , svasa ( asthma ) , and amlapitta ( hyperacidity ) . though wide therapeutic utility of tb has been mentioned in classics , it is reported as poison as or more than that if not processed or purified properly as per classical methods . to indicate its toxic potential , ashtamahadoshas ( eight major ill effects ) though number of studies have been carried out in direction of safety of bhasmas , concerns are always being raised on ayurvedic formulations for the presence of heavy metals , . hence , the present study is aimed to evaluate the acute and subchronic toxicity studies of tb prepared with and without amritikarana in rats . two samples of tb with and without amritikarana were prepared by following standard guidelines as prescribed in ayurvedic classics . it was subjected to general and specific purification procedure followed by incineration after mixed with purified sulfur , kajjali ( black sulfide of mercury ) , and juice of citrus jambhiri lush . it was subjected to heat treatment and labeled as tamra bhasma with amritikarana ( tba ) . as per classical guideline , the therapeutic clinical dose of tb is 30 mg twice a day ( 60 mg / day ) . the suitable dose for rats was calculated by referring to table of paget and branes and was found to be 5.5 mg / kg body weight of rat ( considered as ted ) . both test drugs along with adjuvant were orally administered at limit dose of 2000 mg / kg to overnight fasted female rats by following organization for economic cooperation and development ( oecd ) 425 guideline . the rats were observed closely for behavioral changes , signs and symptoms of toxicity , and mortality , if any continuously for the first 6 h and thereafter periodically up to 14 days . group ii to iv were administered with test drug tb without amritikarana along with adjuvant at ted ( 5.5 mg / kg , orally ) , ted 5 ( 27.5 mg / kg , orally ) , and ted 10 ( 55.0 mg / kg , orally ) , respectively . group v to vii were administered with test drug , tba along with adjuvant at ted ( 5.5 mg / kg , orally ) , ted 5 ( 27.5 mg / kg , orally ) , and ted 10 ( 55.0 mg / kg , orally ) , respectively . the suspensions of test drugs were administered orally once a day for 28 consecutive days . , the studied parameters were blood glucose , urea , creatinine , total cholesterol , high - density lipoprotein ( hdl)-cholesterol , triglyceride , very - low - density lipoprotein ( vldl)-cholesterol , low - density lipoprotein ( ldl)-cholesterol , total protein , albumin , globulin , alkaline phosphatase , serum glutamic oxaloacetic transaminase ( sgot ) , serum glutamic pyruvic transaminase ( sgpt ) , uric acid , direct bilirubin , total bilirubin , and serum calcium . all the important internal organs were carefully dissected namely , liver , kidney , heart , lungs , trachea , intestine spleen , thymus , lymph node , testis , seminal vesicle , prostate , uterus , and ovary . after noting signs of gross lesion and ponderal changes of major organs , all were transferred to 10% phosphate buffered formalin solution for fixation and later on subjected to dehydrating , wax embedding , sectioning , and staining with hematoxylin and eosin for histological evaluation . two samples of tb with and without amritikarana were prepared by following standard guidelines as prescribed in ayurvedic classics . it was subjected to general and specific purification procedure followed by incineration after mixed with purified sulfur , kajjali ( black sulfide of mercury ) , and juice of citrus jambhiri lush . in amritikarana , it was mixed with half part purified sulfur and juice of c. jambhiri lush , kept in the corm of amorphophallus campanulatus linn . it was subjected to heat treatment and labeled as tamra bhasma with amritikarana ( tba ) . as per classical guideline , the therapeutic clinical dose of tb is 30 mg twice a day ( 60 mg / day ) . the suitable dose for rats was calculated by referring to table of paget and branes and was found to be 5.5 mg / kg body weight of rat ( considered as ted ) . both test drugs along with adjuvant were orally administered at limit dose of 2000 mg / kg to overnight fasted female rats by following organization for economic cooperation and development ( oecd ) 425 guideline . the rats were observed closely for behavioral changes , signs and symptoms of toxicity , and mortality , if any continuously for the first 6 h and thereafter periodically up to 14 days . group ii to iv were administered with test drug tb without amritikarana along with adjuvant at ted ( 5.5 mg / kg , orally ) , ted 5 ( 27.5 mg / kg , orally ) , and ted 10 ( 55.0 mg / kg , orally ) , respectively . group v to vii were administered with test drug , tba along with adjuvant at ted ( 5.5 mg / kg , orally ) , ted 5 ( 27.5 mg / kg , orally ) , and ted 10 ( 55.0 mg / kg , orally ) , respectively . the suspensions of test drugs were administered orally once a day for 28 consecutive days . all the important internal organs were carefully dissected namely , liver , kidney , heart , lungs , trachea , intestine spleen , thymus , lymph node , testis , seminal vesicle , prostate , uterus , and ovary . after noting signs of gross lesion and ponderal changes of major organs , all were transferred to 10% phosphate buffered formalin solution for fixation and later on subjected to dehydrating , wax embedding , sectioning , and staining with hematoxylin and eosin for histological evaluation . the results of acute toxicity showed that both the samples of tb along with adjuvant did not affect any behavioral changes and other parameters during entire experimental period of 14 days . both tb and tba did not show any signs and symptoms of toxicity and mortality when given orally at a dose of 2000 mg / kg . no mortality was observed in both samples of tb at ted 10 , ted 5 , and ted dose levels during the experimental period . effects of tb and tba on change in body weight showed that weight gain was observed in all treated groups , but percentage change in body weight pattern in treated groups did not differ significantly from the changes observed in control groups [ table 1 ] . subchronic administration of tb at different dose levels lead to nonsignificant effect on change in relative weight of nine organs except significant increase was observed in relative weight of heart in tba at ted level [ table 2 ] . tba at all dose levels produced nonsignificant increase in relative weight of thymus , spleen , and testis in a uniform manner while other changes were not distinct in comparison to control group . effect of samples of tb on hematological parameters [ table 2 ] revealed that out of the twelve parameters studied , nonsignificant increase in wbc count and neutrophil in tb at ted 10 dose level while significant increase in monocyte count in tb at ted 5 dose level in comparison to control group . significant increase in hb , pcv , and trbc were observed in tb and tba treated groups at ted and ted 5 dose levels [ table 3 ] . both samples of tb at all dose levels produced nonsignificant increase in platelet count in comparison to control group . among the 18 serum biochemical parameters , significant decrease in blood sugar was found in tb at ted dose level and tba treated drug at ted 5 and ted 10 levels , while significant increase in cholesterol was found in tb and tba treated groups at ted and ted 5 dose levels , while higher dose of both drugs produced nonsignificant increase in serum cholesterol level . significant increase in serum triglyceride , ldl , and vldl level was found in tb at ted 10 treated groups while nonsignificant increase in triglyceride and vldl level in all tba treated groups in comparison to control group . hdl cholesterol was significantly increased in tb treated group at ted dose level and tba treated group at ted and ted 5 treated groups while both at higher dose levels produced nonsignificant increase in comparison to control group . significant increase in albumin the changes observed in remaining biochemical parameters were statistically nonsignificant in comparison to control group [ table 4 ] tb with and without amritikarana at ted 10 dose level did not affect the cytoarchitecture of the spleen , lung , stomach , lymph node , trachea , adrenal , prostate , seminal vesicle , testis , ovary , and uterus . administration of tb at ted 10 causes mild fatty changes in the heart but tba at ted 10 showed normal cytoarchitecture [ fig . 1 ] , while both drugs at higher dose level showed micro fatty changes in liver [ fig . mild cell infiltration and edema in the kidney in tb at ted 10 , while mild fatty changes in tba at ted 10 dose level [ fig . 3 ] were observed and both drugs at higher dose produced mild decrease in cellularity in cytoarchitecture of thymus [ fig . 4 ] however , all these changes were not observed in both the drugs treated groups at ted 5 and ted dose levels in rats that suggest that drug is devoid of drastic toxicity . the results of acute toxicity showed that both the samples of tb along with adjuvant did not affect any behavioral changes and other parameters during entire experimental period of 14 days . both tb and tba did not show any signs and symptoms of toxicity and mortality when given orally at a dose of 2000 mg / kg . no mortality was observed in both samples of tb at ted 10 , ted 5 , and ted dose levels during the experimental period . effects of tb and tba on change in body weight showed that weight gain was observed in all treated groups , but percentage change in body weight pattern in treated groups did not differ significantly from the changes observed in control groups [ table 1 ] . subchronic administration of tb at different dose levels lead to nonsignificant effect on change in relative weight of nine organs except significant increase was observed in relative weight of heart in tba at ted level [ table 2 ] . effect of samples of tb on hematological parameters [ table 2 ] revealed that out of the twelve parameters studied , nonsignificant increase in wbc count and neutrophil in tb at ted 10 dose level while significant increase in monocyte count in tb at ted 5 dose level in comparison to control group . significant increase in hb , pcv , and trbc were observed in tb and tba treated groups at ted and ted 5 dose levels [ table 3 ] . both samples of tb at all dose levels produced nonsignificant increase in platelet count in comparison to control group . among the 18 serum biochemical parameters , significant decrease in blood sugar was found in tb at ted dose level and tba treated drug at ted 5 and ted 10 levels , while significant increase in cholesterol was found in tb and tba treated groups at ted and ted 5 dose levels , while higher dose of both drugs produced nonsignificant increase in serum cholesterol level . significant increase in serum triglyceride , ldl , and vldl level was found in tb at ted 10 treated groups while nonsignificant increase in triglyceride and vldl level in all tba treated groups in comparison to control group . hdl cholesterol was significantly increased in tb treated group at ted dose level and tba treated group at ted and ted 5 treated groups while both at higher dose levels produced nonsignificant increase in comparison to control group . significant increase in albumin the changes observed in remaining biochemical parameters were statistically nonsignificant in comparison to control group [ table 4 ] tb with and without amritikarana at ted 10 dose level did not affect the cytoarchitecture of the spleen , lung , stomach , lymph node , trachea , adrenal , prostate , seminal vesicle , testis , ovary , and uterus . administration of tb at ted 10 causes mild fatty changes in the heart but tba at ted 10 showed normal cytoarchitecture [ fig . 1 ] , while both drugs at higher dose level showed micro fatty changes in liver [ fig . mild cell infiltration and edema in the kidney in tb at ted 10 , while mild fatty changes in tba at ted 10 dose level [ fig . 3 ] were observed and both drugs at higher dose produced mild decrease in cellularity in cytoarchitecture of thymus [ fig . 4 ] however , all these changes were not observed in both the drugs treated groups at ted 5 and ted dose levels in rats that suggest that drug is devoid of drastic toxicity . metals and minerals are extensively used in ayurvedic formulations since vedic period , which became an important part of ayurvedic therapeutics . acute toxicity was carried out to record immediate adverse signs and symptoms after the administration of single dose of drug at dose levels that are several folds higher than the therapeutic equivalent dose ( 2000 mg / kg ) . in this study , tb s at the dose level of 2000 mg / kg , orally did not produce any observable toxic effects during the entire duration of acute toxicity study and all female rats survived 14 days of observation suggest that ld50 value may be higher than 2000 mg / kg by oral route . as per un classification , any substance which has oral ld50 of more than 2000 mg / kg is considered as low hazard potential and categorized as un 6.1 pg iii . on subchronic administration of tb with or without amritikarana showed significant increase in hb , red blood cell ( rbc ) and pcv but not found to be in dose - dependent manner . in the present study , increased hb content is due to the increase observed in the production of erythrocytes and pcv in these treated groups . previous study also revealed significant increase in total rbc count , pcv , and platelet count at different dose level of tb on administration for 45 days in albino rats . copper is essential for the synthesis of hb and also supports absorption of iron from intestine , which may responsible for increase in rbc related parameters in rats . significant decrease in fasting blood sugar at ted level of tb and higher dose level of tba suggest role of copper in glucose metabolism . copper plays important role in carbohydrate metabolism by stimulating insulin binding , hexose transport , and lipogenesis in vivo and in vitro significant increase in cholesterol level was found in tb and tba treated groups at ted and ted 5 dose levels while higher dose of both drugs produced nonsignificant increase in serum cholesterol level . significant increase in serum triglyceride , ldl , and vldl level was found in tb at ted 10 treated groups while nonsignificant increase in triglyceride and vldl level in all tba treated groups in comparison to control group . this suggests that the test drugs may have effect on cholesterol turnover in the body ; however , parameters were not affected to significant extent at higher dose level in tba treated group . hdl - cholesterol was significantly increased in tb treated group at ted dose level and tba treated group at ted and ted 5 treated groups while both at higher dose levels produced nonsignificant increase in comparison to control group . this activity seen at therapeutic dose levels in tb and tba can have good therapeutic application because elevation of hdl - cholesterol level will be quite useful in patients with hypercholesterolemia conditions . however , same was not observed at higher dose levels in both the treated group . parameters related to normal liver functioning such as sgpt , sgot , alkaline phosphatase , and bilirubin were not altered to significant extent at all dose levels in both treated groups in comparison to control group . significant increase in albumin was observed at all dose levels of tb and higher dose level of tba . however , the values of total protein , globulin , uric acid , calcium , urea , and creatinine in treated groups and control group are still within normal range that suggests that both drugs are devoid of any drastic toxic effect in rats at therapeutic dose level . , thymus , heart , liver , and kidney at higher dose level . however , these changes were not observed in both the drug treated groups at lower dose levels in rats , which suggest that the drug is devoid of drastic toxicity . considering this , it can be said that the bhasma is safe at ted levels , and mild changes can manifest at higher dose levels . overall , effect of both drugs , tb and tba at different dose levels compared with control group . considering all these , it suggests that , though tb is not much toxic at therapeutic dose level has potential to produced adverse effects when administered for longer duration at higher dose levels . from the present study , it is concluded that tba and tb at the dose level of 2000 mg / kg , orally did not produce any observable toxic effects and mortality in acute toxicity study which suggest that ld50 value may be higher than 2000 mg / kg by oral route and can be categorized as substances with low health hazard potential . the results of subchronic toxicity concluded that tb can be relatively safe at therapeutic dose levels in rats . however , tb at ted 10 dose level , equivalent of which are not likely to be ever employed in clinical conditions , has prone to produce toxic changes in liver , kidney , and heart and also impact on immune - related organs such as thymus as revealed by ponderal , hematological , biochemical , and histopathological parameters in rats . at higher dose level , a sample without amritikarana has more magnitude of toxicity than the sample with amritikarana . hence , it can be inferred that amritikarana is must for internal administration of tb in clinical practices .

the ability to detect light flicker and contrast sensitivity function is impacted early in diseases affecting visual system neural tissues such as in alzheimer 's disease ( ad ) , diabetic retinopathy , macular degeneration , and glaucoma . light flicker and contrast sensitivity function reductions often occur before other visual processes are impacted in ad such as snellen acuity . screening tests that use either light flicker or contrast sensitivity or a combination of the two can be utilized to detect disease before it has resulted in substantial vision loss and before the vision loss impacts quality of life . in the case of ad , the use of such screenings of neurodegenerative processes affecting the visual system can also result in the timely and appropriate referrals in order to make diagnosis of a cognitive impairing disease . the frequency doubling technology visual field test ( fdt ) the stripes that are variable contrast are vertical sine wave gratings of low spatial frequency ( 0.25 c / deg ) that undergo counter phase flickering at a high temporal frequency of 25 hz . the fdt is already used in eye screenings and is considered an important tool for the identification of eye disease and prevention of vision loss [ 2 , 3 ] . the fdt has several programs internal to the system to perform either simple screens or more complex threshold visual field screening and assessment . for screening of substantial numbers of patients , such as public health fairs , there is a simple detection program that takes one minute per eye . for our purposes a large scale project used a different type of instrument , a more complex version of fdt , matrix fdt with smaller five degree targets and with more targets , to screen over 2,000 participants forty years of age or older in the national health and nutrition examination survey . the matrix fdt used was found to be reliable in eighty percent of those tested ; with large reductions in acuity , glaucoma , and age reported to be the major contributions to unreliable tests . the fdt tests have a built - in age - specific normative database and the readout results are calculated based on this . the test utilizes a low spatial frequency sinusoidal grating that undergoes a high temporal frequency counter phase flicker . the participant views targets that are striped square areas ten degrees in size in either central vision or the same targets extending up to twenty degrees peripheral to the center viewing fixation . the test has the additional benefit of monitoring patient reliability by fixation monitoring tests with computer - generated readouts of fixation losses , false positive errors , and false negative errors . the test is not complex for the patient and is easy to perform even with cognitive impairment or with patients as young as four years [ 57 ] . while the matrix fdt used for screening in the national health and nutrition examination survey had a reliability rate of 80% , the target in that test is smaller being one / half the size ; the test has 55 targets , and the spatial frequency of the stripes is smaller . the test is more difficult for participants and it takes longer for participants to perform than the fdt used in this study . studies have shown that patients with mild to moderate ad could perform the fdt test despite their reduced cognitive status [ 810 ] . a test is considered a successful reliable screening test if there are fewer than a combination of ; three fixation losses , false positive errors , or false negative errors , in any combination . a successful reliable screening test is considered a failed screening test if it has readout results of one of the seventeen targets being a p < .5% or a p < 1% or any two or more of seventeen targets being a p < 2% or a p < 5% as compared to the age normative database . in an actual screening setting ( not research ) a failed health screening such as the fdt requires triage and potential referral . if the fdt were to be used in screening to identify biomarkers related to ad and a failure was identified , the triage and needed referrals might involve vision care and/or neurologic consultations . figure 1 is the portion of the readout that shows the grey scale and the corresponding p values . the participant with the failed fdt screening shown in figure 2 had a comprehensive dilated eye examination , and there was no ocular pathology within the eye and no pathology identified within the visual system ( other than ad ) to account for the deficit identified with fdt . neurodegenerative diseases that are not part of the normal aging process result in tissue damage and loss of brain cells and neuronal connections . ad is the most common age - related degenerative process affecting neural tissue , primarily brain . one of the early sites of ad damage is the brain 's visual association area , brodmann area 19 , with loss of cells and neuronal connections . the center for disease reported that ad was the sixth leading cause of death in 2007 and diabetes was the seventh leading cause of death . macular degeneration is the leading cause of blindness for those over the age of 60 and it is estimated that 11 million people in the united states have macular degeneration . diabetic retinopathy , macular degeneration , and glaucoma are common diseases associated with aging , and our research population reflected this . the fdt visual field has been used in a screening capacity to detect eye disease , and when a deficit is identified with fdt , a differential diagnosis between eye disease and neurodegenerative processes such as ad is necessary . it is not out of the ordinary for a patient to fail a visual screening due to deficits identified with the fdt and have the eye exam turn out to be considered within normal limits . the fdt as a tool to detect disease originating in the eye early in the visual pathway can also detect disease further along the visual pathway outside the eye such as ad [ 10 , 1719 ] , parkinson 's disease [ 20 , 21 ] , lesions poster to the chiasm , and multiple sclerosis [ 23 , 24 ] . fdt was found to be better than conventional visual fields in detecting axonal injury due to ischemia along the optic nerve . the fdt has long been considered to be underutilized for applications in the screening and diagnosis of neurologic diseases impacting the visual system . this project demonstrated that the fdt when used as a screening tool most likely can contribute to identifying probable ad . loss of contrast sensitivity function is subtle and not readily detected during standard basic visual assessments and eye exams , but it still impacts quality of life . the fdt visual field has variable contrast and can detect such deficits before other traditional vision tests such as acuity or ophthalmoscopy would identify disease . deficits in visual contrast sensitivity occur frequently in ad [ 2830 ] . detection of light flicker is diminished in neurodegenerative diseases affecting the visual system . in a study of ten participants with ad compared to participants with no dementia , mentis and colleagues found significant reductions in the response to light flicker when measured with imaging using positron emission tomography ( pet ) . the stimulus was a grid pattern with increasing temporal frequency . with lower temporal frequencies , there were minimal differences but as the frequencies approached 15 hz , the reduction in neural response in ad became greater compared to age - matched controls . the fdt proposed for use in this project uses a stimulus of 25 hz . another study using the same pet protocol , but investigating the flicker response to light in those with mild dementia in ad , identified a reduced response in participants with mild ad using a high temporal frequency visual stimulation . using a target of flicker , and motion kurylo and colleagues found that the deficits in 14 participants with ad compared to 20 age - matched controls approached significance . in their study of critical flicker tests , curran and colleagues found that those participants with ad had reduced ability to determine flicker in descending mode ( when the flicker rate is gradually decreased until one can detect flicker ) compared to normal elderly . the authors concluded that a test of light licker , as psychophysical threshold , was free from educational and cultural bias and there are no floor or ceiling effects . they also found that light flicker testing had high test retest reliability and no difficulty administering to dementia patients . a flicker test that included contrast found significant reductions in those with parkinson 's disease compared to age - matched controls . the target used in those studies flickered at 25 hz and was ten degrees in size and was identical to that being used in the fdt for the studies being reported in this paper . studies using the fdt instrument have also found deficits in those with parkinson 's disease compared to control participants [ 17 , 21 ] . light flicker in the form of critical flicker frequency has been shown to be useful in the detection of neuropathology of the visual system including lesions of the optic nerve . a five - degree flickering target at a rate of 3040 hz detected functional deficits in those with glaucoma prior to detection with conventional visual fields in 90% of participants . fdt has been reported to be useful in ad as its target utilizes low spatial frequencies . low spatial frequencies are deficit in ad [ 42 , 43 ] and can be considered a biomarker of disease when identified with fdt . in a study using a matrix fdt ; with smaller targets at a slower flicker rate , risacher and colleagues found that among the nine participants with mild ad , twenty - seven with mild cognitive impairment and twenty - five age - matched healthy controls , that the ad and mildly cognitive impaired participants had deficits compared to the age matched controls . while this group did control for ocular pathology , they did not exclude unreliable tests . unreliable tests can include both false positive and false negative tests results , and the work undertaken by risacher discusses this . pepin and colleagues also reported the matrix fdt model to be promising in identifying mild ad , mild cognitive impairment compared to age - matched healthy controls when evaluating the overall visual field deficits . in a study using fdt , there were significant deficits in the inferior portion of the left eye visual field as tested by fdt in six participants with ad compared to age - matched controls . this correlates with reports of superior axonal nerve defects identified by optical coherence tomography imaging which is a structural measure of the neural retinal tissue and optic nerve portion of the visual pathway [ 4648 ] . the representative fdt readout for ad in figure 2 depicts inferior visual field deficits in both eyes not attributed to any identified eye pathology , and the participant had superior never fiber defects as measured by optical coherence tomography , not attributed to any pathology . fdt is an established test to screen for glaucoma but is also an excellent visual field screening test for optic neuropathies unrelated to glaucoma as indicated by studies , and the fdt is considered to be underutilized for screening of neurologic disease in general . fdt was found to have a specificity of 100% and sensitivity of 84% when used to evaluate 138 eyes of 103 patients in identifying neuroophthalmological pathology when using pass fail criteria of two or more points being depressed to a p less than 1% . in another study using fdt to identify neuropathology that had been confirmed with either mri , computed tomography , or neuroophthalmological exam , the specificity and sensitivity were comparable to other standard visual testing . the screening data was drawn from two groups of participants tested with the fdt , 23 with ad and 14 control participants . one group was a convenience sample study of adult patients attending clinics at the boston university neurology associates . anyone attending the clinics at the boston university neurology associates was invited to participate in the visual screening project . the sites for the screening included the doctor 's office building neurology located in boston , ma , usa and the boston university neurology associates in weymouth , ma , usa . family members or other adults accompanying patients were also invited to participate in the screening as control participants . the screening included a brief ocular history , monocular distance visual acuities using a lighthouse feinbloom low vision chart ( a flip chart with testing from 20/10 to 20/1000 that can be administered at variable distances ) , and fdt visual field utilizing the 201 threshold strategy . all ad participants had ophthalmoscopy of the anterior segment to rule out cataract and to confirm pathology as self - reported by participants or the caregivers . trace cataracts not impinging on visual acuity do not impact the fdt testing ; however , more significant cataracts or media opacity that reduce acuities will impact the testing . an aspect of ophthalmoscopy is the assessment of the anterior structures including the cornea and pupil to screen for abnormalities or opacities , and no participant had anterior segment abnormalities that would impact visual functions such as acuity and contrast or peripheral vision . the participants reporting glaucoma in the screening group ( not having dilated eye exams ) had miotic pupils making a clear view of the optic nerve difficult , and without a dilated exam the glaucoma diagnosis could not be definitively confirmed in those reporting glaucoma . the second group of participants was a group of ad and controls that had , as part of a separate protocol , cognitive assessment using minimental status exam and a comprehensive eye examination that included refraction for glasses , ocular motility evaluation , applanation tonometry , slit lamp anterior segment examination , dilated fundus examination , peripheral visual field assessment , and in some cases imaging with optical coherence tomography . a successfully performed screening was considered a fail if any of the seventeen high flicker frequency contrast targets was recorded ( using the instrument normative screening printout ) as having a probability of p < .5% or p < 1% or any two or more targets having a p < 2% or p < 5% as identified in the fdt printout ( results based on the technology internal normative database ) . an fdt test with three or more reliability parameters , fixation loss , false positive or false negative , was considered unreliable and not used in data analysis . a visual acuity of worse than 20/60 was also considered a failure for that eye . the 20/60 acuity was for this project purpose only as it is a screening protocol , and that is what is considered a fail in large scale public health vision screenings . for the population within the group not having a full eye exam , we did not rule out that the reductions in acuity were due to changes in refraction requiring an update in the glasses . in general to determine if an ad - related pathology is contributing to functional deficits in the visual system ( rather than eye disease ) requires a careful refraction and a more rigid cutoff of 20/25 in each eye separately and 20/25 binocularly in order to assure that undetected eye pathology or uncorrected refractive error is not contributing to the reductions in acuity . while binocular acuities , with both eyes tested open only at distance , are frequently used for vision research related to ad , they can not be considered acceptable . if a binocular acuity is the single measure of acuity , acuity measurement can fail to identify an eye that is substantially impaired due to eye pathology because the eye without pathology has not been occluded and is functioning . undetected eye pathology or uncorrected vision can result in research findings that are inappropriately attributed to ad in studies involving neuropsychological testing . further in an assessment involving any near testing ( this study did not include near testing ) , refractions for optimum eyeglass use would be necessary to insure that the most appropriate optical corrections both near and far are being used . when the glasses used for near testing are out of date , not adequate in strength or otherwise inaccurate , this results in inaccurate near performance during neuropsychological testing , and this was demonstrated by bertone and colleagues . the fdt is in general not impacted by uncorrected vision , and the results do not vary depending on refractive errors less than six diopters . the participants used their habitual glasses during the fdt testing . because the screening protocol does not involve any near tasks and the participants did not require optimum correction for accuracy , an assessment for glasses was not done for the convenience group , and refraction was not part of this screening protocol . the age range of the twenty three ad participants was 68 to 88 years with the average being 80 , and the average for successful tests was 81 . there were fourteen females and five males and the gender of four was not recorded . the age range of the fourteen control participants was 60 to 81 with the average age being 71 . there were nine females , and four males , and the gender of one was not recorded . the groups receiving the comprehensive eye examinations were part of a separate protocol involving evaluation with fdt and were recruited from a clinical neurology population , alzheimer 's disease registries , support groups , and enrollees of adult day care . the controls and all but the ad participants enrolled in day care received a comprehensive eye examination on campus by a single provider on the same day as the fdt testing . the day care participants received a comprehensive exam through contract with nursing home eye care providers but did not have peripheral visual field testing as part of the comprehensive evaluation . during a separate session and prior to testing with fdt and prior to having a peripheral visual field evaluation these participants had repeat acuities , ophthalmoscopy , and slit lamp anterior segment evaluations ( these and the visual field were done using customized portable nursing home examination equipment ) the study followed the tenets of the declaration of helsinki , and the protocol was approved by the boston university institutional review board . all participants and , when appropriate , caregivers were provided with written informed consent before participation in the screening study . all participants signed hippa approved record request forms and record release forms in order to triage identified ocular pathology as needed . fdt presents back - lit flashed images viewed on a fixed , flat , shielded screen in front of a stationary participant . the instrument has readouts that are based on normative data , and the internal data is also age normed taking the reported age into account when the internal calculations are made . the participant views targets that are small striped square - shaped areas in either central vision or targets extending up to twenty degrees peripheral to fixation . as with traditional visual field testing , it is not necessary to patch or otherwise cover the untested eye as the instrument is designed for passive occlusion . this is of benefit to an aged cognitively impaired population who may present with agitation . the participants fixate a target directly in front of them and respond by pushing a button each time they see an image flashed anywhere in their visual field . care was taken to assure that there was adequate hand coordination to perform the task . the technology tests for false positive and false negative rates as well as fixation losses . the test has the additional benefit of monitoring patient reliability by fixation monitoring with computer - generated readouts of the fixation losses , false positive errors , and false negative errors . the test is portable , and the testing in the neurology clinic was done in an ancillary conference area . the fdt instrument was set up on a counter height fold stool , and participants were seated in a clinicians stool with a back . the fdt was placed on a desk in the eye examination area during testing of those participants receiving comprehensive evaluations . the clinician 's examination chair with a back was used for testing . in a clinical setting , the standard would be to repeat the unreliable test as there can be attention and learning aspect to the visual field testing , and the second test may yield a more reliable test . when using the fdt for screening in large groups , the test is often not repeated . the fdt was repeated a second time during the same session in the ad participant diagnosed with probable glaucoma and the right eye test of a different ad participant was repeated due to technical problems with the response button . there was a screening failure rate of 87% with the fdt visual field in the participants with ad while only 8% of the control group failed the screening . the ad group had a rate of reported ocular pathology of 37% , and the control group had ocular related pathology at 21% . the breakdown of useable tests appears in table 2 . utilizing a chi - square test of independence , the overall failure rate when screened with fdt and excluding the unreliable tests for those with ad was significantly greater in comparison to the control group , x(1 ) = 25.63 , p < .0000004 . the failure rates , when controlling for pathology by exclusion in addition to excluding unreliable tests , were also significant for those with ad compared to the control group , x(1 ) = 22.56 , p < .000002 . the overall rate of pathology , although higher for the ad group , was not significantly higher x(1 ) = 0.72 , p = .40 . while the fdt has internal age normative database from which results are generated , to assure this was not a factor the data were analyzed using only those ad and control participants 65 and older and 84 years old or younger . the ad group controlled for reliability , pathology , and age still had significantly higher rates of fdt screening failures compared to the control group , x(1 ) = 14.85 , p = .0001 . the screening results from each eye of participants with a diagnosis of ad were obtained with fdt . while 46 eyes were available , five eyes were not tested . both eyes of one participant were untestable due to severe glaucoma and an inability to fixate the fdt target both eyes of another participant were untestable due to age related macular degeneration and one participant had significant vision loss in only one eye secondary to stroke . the inability to test using fdt had more to do with an eye pathology and inability to perceive the targets than an age - related dementia . this can occur with glaucoma due to large deficits in visual field , even when reasonable acuity is identified . visual field loss in eye disease is a factor contributing to unreliable tests with fdt and poor performance . the control participants were drawn from adults not scheduled as a patient in the neurology clinic , but accompanying a patient or from the control participants that were recruited for participation in other studies at the university . there were 14 control participants , and 28 eyes were tested , and only two of the 28 eyes tested were unreliable . of 41 eyes of those with ad tested with the fdt , eleven were unreliable . in this study , some of the unreliable tests may have been secondary to dementia , and unreliable tests were defined as a combination of three or more of fixation loss , false positive , and false negative responses . of the 11 unreliable fdt tests that were successfully done , only one had association with reported ocular pathology , and this was stroke . after the exclusion of unreliable tests , there were final 30 successful fdt screening tests in the ad participant group . criteria for a deficit in visual function with fdt were deficits in two targets and any test showing reductions based on these criteria was considered a successful fdt but a failed screening test . a reduced acuity was also considered a screening failure , but failed acuities were to be expected in a population known to have ocular pathology . the population evaluated with comprehensive eye examination was prescreened for eye disease over the telephone . there were two cases of undiagnosed glaucoma in the prescreened group , one in the ad and one in the control group . the convenience population was not prescreened for pathology . failed acuity is not a biomarker of ad . traditionally , a failed visual screening with fdt indicates pathology in the visual system , but a failure can be in the eye or due to degenerative disease that affects the visual system beyond the eye in the neuroprocessing visual pathways such as ad . twenty - six of the 30 successful reliable fdt tests obtained from ad participants were considered to be failed fdt screening tests . seven failed fdt were attributed to a reported glaucoma ( some confirmed by ophthalmoscopy ) , and two failures could be accounted for by dense cataracts ( confirmed by ophthalmoscopy ) . seventeen fdt failures were not associated with any reported ocular pathology of the eye ( majority confirmed by ophthalmoscopy ) . of failures due to visual acuity , one was related to stroke , two secondary to glaucoma , and two from age - related macular degeneration . two of the failed visual acuity had no association with reported pathology or any pathology detected with screening ophthalmoscopy . in an actual setting screening for eye pathology and neurodegenerative disease , a failed acuity would be considered a sign of eye disease , not ad , and the primary referral would be for a comprehensive eye exam . both of the failed acuity in the ad group eyes had also failed the fdt screening . the results of these screening failures were provided to the participants with the advice to report the results immediately to their eye care provider or primary care physician . of 28 eyes screened with fdt in the control participant group , there were only two unreliable tests and 4 failed fdt . the characteristics of the control participants appear in table 1 , and the characteristics of the control participants are in table 2 . the lower average age of the control group may be reflective of the caregiver role , younger and healthier . however , the age difference is not likely to fully account for the differences in failure rate with fdt , and using data controlled for age , the differences between ad failures compared to controls remain significant . there was a failure rate of 87% in the participants with ad while only 8% of the control group failed the screening . the ad group had a rate of reported ocular pathology of 37% , and the control group had ocular related pathology at 21% . the group participating in this screening study had access to health care , and the majority reported to have comprehensive eye exams in the previous year . after adjusting for unreliable tests and pathology , the failure rate remains high with the ad participant group having a failure rate of 85% compared to the control group failure rate of 1% . when this is corrected for age including only participants between ages 65 and 85 , ad participant failure rate remains similarly high at 85% for the ad group and 1% for the age - matched control group . health screenings are important in groups that have limited resources or difficulty accessing health care . this is true for a population with cognitive impairments . in a different study investigating the use of fdt as a screening tool for eye disease ( not ad ) , it was demonstrated there were higher rates of previously undetected visual dysfunction or disease in testing venues specializing in care of the elderly such as senior citizen centers . such failures may be an indication of ad in the absence of eye pathology after eye examination . the significance of failures with fdt in those participants with ad compared to control participants not having ad demonstrated in this project that fdt identifies a biomarker of ad with the technology . given the ease and efficiency that screening with fdt presents , this instrument may prove to be an effective means of identifying age - related neurodegenerative processes affecting the visual system , specifically ad . ad impacts the visual system , and the biomarker identified with fdt can be part of the differential diagnosis when there are failures during visual screenings utilizing fdt . screening with fdt not only can be an integral part of the early identification of age - related eye disease , but also neurodegenerative disease impacting the visual system such as ad .

this study was to investigate the feasibility of frequency doubling technology ( fdt ) visual field testing in alzheimer 's disease ( ad ) in order to identify early biomarkers of ad in patients already diagnosed with ad and compare the findings to participants not having alzheimer 's disease . this biomarker would be useful in a battery of tests for the early identification of those with ad . it was not the intent to correlate the visual system biomarker with severity of disease , but to determine if the biomarker was present in pass or fail screening criteria . the study showed with very strong significance that the fdt can identify biomarkers of those with ad compared to an age - matched population that does not have ad . fdt is a simple test to take and administer and has been used to screen for eye and retinal diseases such as glaucoma , retinal macular degeneration , and diabetic retinopathy . the results obtained in the fdt readout are analyzed and compared to the age normative database within the system . the fdt ability to screen for ad biomarker in the visual system was significant in those with ad compared to the controls , and the deficits were not related to any ocular pathology .

1. Background 2. Methodology 3. Results 4. Discussion

the ability to detect light flicker and contrast sensitivity function is impacted early in diseases affecting visual system neural tissues such as in alzheimer 's disease ( ad ) , diabetic retinopathy , macular degeneration , and glaucoma . in the case of ad , the use of such screenings of neurodegenerative processes affecting the visual system can also result in the timely and appropriate referrals in order to make diagnosis of a cognitive impairing disease . the frequency doubling technology visual field test ( fdt ) the stripes that are variable contrast are vertical sine wave gratings of low spatial frequency ( 0.25 c / deg ) that undergo counter phase flickering at a high temporal frequency of 25 hz . the fdt is already used in eye screenings and is considered an important tool for the identification of eye disease and prevention of vision loss [ 2 , 3 ] . the fdt has several programs internal to the system to perform either simple screens or more complex threshold visual field screening and assessment . for screening of substantial numbers of patients , such as public health fairs , there is a simple detection program that takes one minute per eye . for our purposes a large scale project used a different type of instrument , a more complex version of fdt , matrix fdt with smaller five degree targets and with more targets , to screen over 2,000 participants forty years of age or older in the national health and nutrition examination survey . the matrix fdt used was found to be reliable in eighty percent of those tested ; with large reductions in acuity , glaucoma , and age reported to be the major contributions to unreliable tests . the fdt tests have a built - in age - specific normative database and the readout results are calculated based on this . while the matrix fdt used for screening in the national health and nutrition examination survey had a reliability rate of 80% , the target in that test is smaller being one / half the size ; the test has 55 targets , and the spatial frequency of the stripes is smaller . the test is more difficult for participants and it takes longer for participants to perform than the fdt used in this study . a successful reliable screening test is considered a failed screening test if it has readout results of one of the seventeen targets being a p < .5% or a p < 1% or any two or more of seventeen targets being a p < 2% or a p < 5% as compared to the age normative database . in an actual screening setting ( not research ) a failed health screening such as the fdt requires triage and potential referral . if the fdt were to be used in screening to identify biomarkers related to ad and a failure was identified , the triage and needed referrals might involve vision care and/or neurologic consultations . the participant with the failed fdt screening shown in figure 2 had a comprehensive dilated eye examination , and there was no ocular pathology within the eye and no pathology identified within the visual system ( other than ad ) to account for the deficit identified with fdt . one of the early sites of ad damage is the brain 's visual association area , brodmann area 19 , with loss of cells and neuronal connections . macular degeneration is the leading cause of blindness for those over the age of 60 and it is estimated that 11 million people in the united states have macular degeneration . diabetic retinopathy , macular degeneration , and glaucoma are common diseases associated with aging , and our research population reflected this . the fdt visual field has been used in a screening capacity to detect eye disease , and when a deficit is identified with fdt , a differential diagnosis between eye disease and neurodegenerative processes such as ad is necessary . the fdt as a tool to detect disease originating in the eye early in the visual pathway can also detect disease further along the visual pathway outside the eye such as ad [ 10 , 1719 ] , parkinson 's disease [ 20 , 21 ] , lesions poster to the chiasm , and multiple sclerosis [ 23 , 24 ] . the fdt has long been considered to be underutilized for applications in the screening and diagnosis of neurologic diseases impacting the visual system . this project demonstrated that the fdt when used as a screening tool most likely can contribute to identifying probable ad . the fdt visual field has variable contrast and can detect such deficits before other traditional vision tests such as acuity or ophthalmoscopy would identify disease . detection of light flicker is diminished in neurodegenerative diseases affecting the visual system . in a study of ten participants with ad compared to participants with no dementia , mentis and colleagues found significant reductions in the response to light flicker when measured with imaging using positron emission tomography ( pet ) . with lower temporal frequencies , there were minimal differences but as the frequencies approached 15 hz , the reduction in neural response in ad became greater compared to age - matched controls . another study using the same pet protocol , but investigating the flicker response to light in those with mild dementia in ad , identified a reduced response in participants with mild ad using a high temporal frequency visual stimulation . using a target of flicker , and motion kurylo and colleagues found that the deficits in 14 participants with ad compared to 20 age - matched controls approached significance . in their study of critical flicker tests , curran and colleagues found that those participants with ad had reduced ability to determine flicker in descending mode ( when the flicker rate is gradually decreased until one can detect flicker ) compared to normal elderly . a flicker test that included contrast found significant reductions in those with parkinson 's disease compared to age - matched controls . the target used in those studies flickered at 25 hz and was ten degrees in size and was identical to that being used in the fdt for the studies being reported in this paper . studies using the fdt instrument have also found deficits in those with parkinson 's disease compared to control participants [ 17 , 21 ] . light flicker in the form of critical flicker frequency has been shown to be useful in the detection of neuropathology of the visual system including lesions of the optic nerve . a five - degree flickering target at a rate of 3040 hz detected functional deficits in those with glaucoma prior to detection with conventional visual fields in 90% of participants . fdt has been reported to be useful in ad as its target utilizes low spatial frequencies . low spatial frequencies are deficit in ad [ 42 , 43 ] and can be considered a biomarker of disease when identified with fdt . in a study using a matrix fdt ; with smaller targets at a slower flicker rate , risacher and colleagues found that among the nine participants with mild ad , twenty - seven with mild cognitive impairment and twenty - five age - matched healthy controls , that the ad and mildly cognitive impaired participants had deficits compared to the age matched controls . while this group did control for ocular pathology , they did not exclude unreliable tests . unreliable tests can include both false positive and false negative tests results , and the work undertaken by risacher discusses this . pepin and colleagues also reported the matrix fdt model to be promising in identifying mild ad , mild cognitive impairment compared to age - matched healthy controls when evaluating the overall visual field deficits . in a study using fdt , there were significant deficits in the inferior portion of the left eye visual field as tested by fdt in six participants with ad compared to age - matched controls . this correlates with reports of superior axonal nerve defects identified by optical coherence tomography imaging which is a structural measure of the neural retinal tissue and optic nerve portion of the visual pathway [ 4648 ] . the representative fdt readout for ad in figure 2 depicts inferior visual field deficits in both eyes not attributed to any identified eye pathology , and the participant had superior never fiber defects as measured by optical coherence tomography , not attributed to any pathology . fdt is an established test to screen for glaucoma but is also an excellent visual field screening test for optic neuropathies unrelated to glaucoma as indicated by studies , and the fdt is considered to be underutilized for screening of neurologic disease in general . the screening data was drawn from two groups of participants tested with the fdt , 23 with ad and 14 control participants . anyone attending the clinics at the boston university neurology associates was invited to participate in the visual screening project . the sites for the screening included the doctor 's office building neurology located in boston , ma , usa and the boston university neurology associates in weymouth , ma , usa . the screening included a brief ocular history , monocular distance visual acuities using a lighthouse feinbloom low vision chart ( a flip chart with testing from 20/10 to 20/1000 that can be administered at variable distances ) , and fdt visual field utilizing the 201 threshold strategy . an aspect of ophthalmoscopy is the assessment of the anterior structures including the cornea and pupil to screen for abnormalities or opacities , and no participant had anterior segment abnormalities that would impact visual functions such as acuity and contrast or peripheral vision . the participants reporting glaucoma in the screening group ( not having dilated eye exams ) had miotic pupils making a clear view of the optic nerve difficult , and without a dilated exam the glaucoma diagnosis could not be definitively confirmed in those reporting glaucoma . the second group of participants was a group of ad and controls that had , as part of a separate protocol , cognitive assessment using minimental status exam and a comprehensive eye examination that included refraction for glasses , ocular motility evaluation , applanation tonometry , slit lamp anterior segment examination , dilated fundus examination , peripheral visual field assessment , and in some cases imaging with optical coherence tomography . a successfully performed screening was considered a fail if any of the seventeen high flicker frequency contrast targets was recorded ( using the instrument normative screening printout ) as having a probability of p < .5% or p < 1% or any two or more targets having a p < 2% or p < 5% as identified in the fdt printout ( results based on the technology internal normative database ) . the 20/60 acuity was for this project purpose only as it is a screening protocol , and that is what is considered a fail in large scale public health vision screenings . for the population within the group not having a full eye exam , we did not rule out that the reductions in acuity were due to changes in refraction requiring an update in the glasses . in general to determine if an ad - related pathology is contributing to functional deficits in the visual system ( rather than eye disease ) requires a careful refraction and a more rigid cutoff of 20/25 in each eye separately and 20/25 binocularly in order to assure that undetected eye pathology or uncorrected refractive error is not contributing to the reductions in acuity . further in an assessment involving any near testing ( this study did not include near testing ) , refractions for optimum eyeglass use would be necessary to insure that the most appropriate optical corrections both near and far are being used . the fdt is in general not impacted by uncorrected vision , and the results do not vary depending on refractive errors less than six diopters . because the screening protocol does not involve any near tasks and the participants did not require optimum correction for accuracy , an assessment for glasses was not done for the convenience group , and refraction was not part of this screening protocol . the age range of the twenty three ad participants was 68 to 88 years with the average being 80 , and the average for successful tests was 81 . there were fourteen females and five males and the gender of four was not recorded . there were nine females , and four males , and the gender of one was not recorded . the groups receiving the comprehensive eye examinations were part of a separate protocol involving evaluation with fdt and were recruited from a clinical neurology population , alzheimer 's disease registries , support groups , and enrollees of adult day care . the controls and all but the ad participants enrolled in day care received a comprehensive eye examination on campus by a single provider on the same day as the fdt testing . the day care participants received a comprehensive exam through contract with nursing home eye care providers but did not have peripheral visual field testing as part of the comprehensive evaluation . during a separate session and prior to testing with fdt and prior to having a peripheral visual field evaluation these participants had repeat acuities , ophthalmoscopy , and slit lamp anterior segment evaluations ( these and the visual field were done using customized portable nursing home examination equipment ) the study followed the tenets of the declaration of helsinki , and the protocol was approved by the boston university institutional review board . all participants signed hippa approved record request forms and record release forms in order to triage identified ocular pathology as needed . the instrument has readouts that are based on normative data , and the internal data is also age normed taking the reported age into account when the internal calculations are made . as with traditional visual field testing , it is not necessary to patch or otherwise cover the untested eye as the instrument is designed for passive occlusion . the test is portable , and the testing in the neurology clinic was done in an ancillary conference area . the fdt instrument was set up on a counter height fold stool , and participants were seated in a clinicians stool with a back . the fdt was placed on a desk in the eye examination area during testing of those participants receiving comprehensive evaluations . in a clinical setting , the standard would be to repeat the unreliable test as there can be attention and learning aspect to the visual field testing , and the second test may yield a more reliable test . the fdt was repeated a second time during the same session in the ad participant diagnosed with probable glaucoma and the right eye test of a different ad participant was repeated due to technical problems with the response button . there was a screening failure rate of 87% with the fdt visual field in the participants with ad while only 8% of the control group failed the screening . the ad group had a rate of reported ocular pathology of 37% , and the control group had ocular related pathology at 21% . utilizing a chi - square test of independence , the overall failure rate when screened with fdt and excluding the unreliable tests for those with ad was significantly greater in comparison to the control group , x(1 ) = 25.63 , p < .0000004 . the failure rates , when controlling for pathology by exclusion in addition to excluding unreliable tests , were also significant for those with ad compared to the control group , x(1 ) = 22.56 , p < .000002 . the overall rate of pathology , although higher for the ad group , was not significantly higher x(1 ) = 0.72 , p = .40 . while the fdt has internal age normative database from which results are generated , to assure this was not a factor the data were analyzed using only those ad and control participants 65 and older and 84 years old or younger . the ad group controlled for reliability , pathology , and age still had significantly higher rates of fdt screening failures compared to the control group , x(1 ) = 14.85 , p = .0001 . while 46 eyes were available , five eyes were not tested . both eyes of one participant were untestable due to severe glaucoma and an inability to fixate the fdt target both eyes of another participant were untestable due to age related macular degeneration and one participant had significant vision loss in only one eye secondary to stroke . the inability to test using fdt had more to do with an eye pathology and inability to perceive the targets than an age - related dementia . visual field loss in eye disease is a factor contributing to unreliable tests with fdt and poor performance . the control participants were drawn from adults not scheduled as a patient in the neurology clinic , but accompanying a patient or from the control participants that were recruited for participation in other studies at the university . of 41 eyes of those with ad tested with the fdt , eleven were unreliable . in this study , some of the unreliable tests may have been secondary to dementia , and unreliable tests were defined as a combination of three or more of fixation loss , false positive , and false negative responses . of the 11 unreliable fdt tests that were successfully done , only one had association with reported ocular pathology , and this was stroke . a reduced acuity was also considered a screening failure , but failed acuities were to be expected in a population known to have ocular pathology . there were two cases of undiagnosed glaucoma in the prescreened group , one in the ad and one in the control group . traditionally , a failed visual screening with fdt indicates pathology in the visual system , but a failure can be in the eye or due to degenerative disease that affects the visual system beyond the eye in the neuroprocessing visual pathways such as ad . seventeen fdt failures were not associated with any reported ocular pathology of the eye ( majority confirmed by ophthalmoscopy ) . of failures due to visual acuity , one was related to stroke , two secondary to glaucoma , and two from age - related macular degeneration . in an actual setting screening for eye pathology and neurodegenerative disease , a failed acuity would be considered a sign of eye disease , not ad , and the primary referral would be for a comprehensive eye exam . both of the failed acuity in the ad group eyes had also failed the fdt screening . the results of these screening failures were provided to the participants with the advice to report the results immediately to their eye care provider or primary care physician . the characteristics of the control participants appear in table 1 , and the characteristics of the control participants are in table 2 . however , the age difference is not likely to fully account for the differences in failure rate with fdt , and using data controlled for age , the differences between ad failures compared to controls remain significant . there was a failure rate of 87% in the participants with ad while only 8% of the control group failed the screening . the ad group had a rate of reported ocular pathology of 37% , and the control group had ocular related pathology at 21% . the group participating in this screening study had access to health care , and the majority reported to have comprehensive eye exams in the previous year . after adjusting for unreliable tests and pathology , the failure rate remains high with the ad participant group having a failure rate of 85% compared to the control group failure rate of 1% . when this is corrected for age including only participants between ages 65 and 85 , ad participant failure rate remains similarly high at 85% for the ad group and 1% for the age - matched control group . in a different study investigating the use of fdt as a screening tool for eye disease ( not ad ) , it was demonstrated there were higher rates of previously undetected visual dysfunction or disease in testing venues specializing in care of the elderly such as senior citizen centers . such failures may be an indication of ad in the absence of eye pathology after eye examination . the significance of failures with fdt in those participants with ad compared to control participants not having ad demonstrated in this project that fdt identifies a biomarker of ad with the technology . given the ease and efficiency that screening with fdt presents , this instrument may prove to be an effective means of identifying age - related neurodegenerative processes affecting the visual system , specifically ad . ad impacts the visual system , and the biomarker identified with fdt can be part of the differential diagnosis when there are failures during visual screenings utilizing fdt . screening with fdt not only can be an integral part of the early identification of age - related eye disease , but also neurodegenerative disease impacting the visual system such as ad .

ammonium uranyl nitrate ( aun ) is an important intermediate product during conversion of uranyl nitrate [ uo2(no3)2 ] solution to uo2 powder for the fabrication of nuclear fuels , the so - called modified direct denitration ( mdd ) process . many conversion processes have been developed , such as , ammonium uranyl carbonate ( auc ) , ammonium diuranate ( adu ) , and an integrated dry route ( idr ) . , the mdd process offers the greatest potential for cost reduction and good product quality for the production of uo2 powder . the modified direct denitration process involves the thermal decomposition of aun double salts , which are prepared from a mixture consisting of a uo2(no3)2 solution and nh4no3 . also , the sintering behavior of uo2 powder can be related to its powder characteristics and processing parameters . it has been observed that the presence of nh4no3 in a uo2(no3)2 feed solution prior to a thermal denitration greatly improved the sintering properties of uo2 powder . three double salts are known for the uo2(no3)2nh4no3h2o system , but there have been only a few studies done on thermal decomposition of these salts . laboratory scale denitration tests showed that nh4uo2(no3)3 decomposes without melting , and thus does not form a dough stage similar to that encountered during a denitration . also , uo2 produced from nh4uo2(no3)3 in these tests appeared to be more active than a corresponding oxide produced from uo2(no3)2 solution . it was also reported that nh4uo2(no3)3 decomposes without melting at 270300 c to give -uo3 powder of an average size of approximately 3 m , with good ceramic properties for its fabrication into uo2 nuclear fuel pellets . in the conversion of aun to uranium oxides , the characteristic of the resulting powder particles depends upon the aun preparation process and also upon the thermal decomposition procedures . amorphous uranium trioxide ( a - uo3 ) is mainly formed in the absence of ammonium and nitrate ions , whereas deamination of the retained ammonia leads to -uo3 . meanwhile , auc decomposes at around 190 c , giving off co2 , nh3 , and h2o with the formation of an amorphous phase . in a n2 , ar , or co2 atmosphere , the amorphous phase crystallizes to -uo3 before decomposing to u3o8 . various reports have been published on thermal analysis studies of the reactions occurring during a decomposition of auc and adu . however , few studies on the thermal decomposition of aun can be found in the literature . therefore , the objective of this study is to investigate the reaction pathways during a thermal decomposition and reduction of aun to achieve a better knowledge of the influence of an aun preparation process and thermal decomposition procedures on uranium oxides under a nitrogen , air , or hydrogen atmosphere . ( nh4)2uo2(no3)42h2o was resulted when the ph of the reaction solution was 2.58 and the mole ratio of nh4/u was 2.14 . and nh4uo2no3 was resulted when the ph of the reaction solution was 2.01 and the mole ratio of nh4/u was 1.07 . the mixed reaction of these two solutions was performed in a heating mantle where the temperature could be automatically controlled . after the reaction had progressed to a point which a precipitate was generated , the heating was stopped and the precipitated solid was filtered . the precipitate remaining on the filter paper was then left at room temperature to dry . as a result , a primary sample was prepared . the primary sample contained unreacted uo2(no3)26h2o and nh4no3 as impurities . to eliminate these impurities , the recrystallization was performed by dissolving the primary sample in distilled water at 40 c , and then cooling it down to room temperature . the ph of the mixed solution was controlled by adding uo3 or concentrated nitric acid to the mixed solution . when the uo3 was added to that , it was dissolved at a temperature of 30 c . to eliminate any undissolved uo3 from the reaction solution , it was filtered , and the filtrate was used as the reaction solution for aun synthesis . the characterization of synthesized aun was reported in previous studies . to analyze the thermal decomposition and reduction pathways of each aun , the respective thermal decomposition and reduction temperature must be determined beforehand to identify any intermediate phase produced from each reaction stage . for this purpose , a thermogravimetric ( tg)/differential thermal analysis ( dta ) experiment was carried out in various atmospherics , which were 100% nitrogen , air , and hydrogen gas . the flow rate for each gas was 50 ml / min , the heating rate was 5 c / min , and the temperature was varied from room temperature to 800 c . about 8 mg of sample was used in each run . each reaction stage was identified on the basis of dta results , and the temperature for the intermediate phase was determined . samples used to acquire the intermediate phase were obtained by heating each aun sample in the thermal analyzer up to the temperature as determined from the dta results . about 40 mg of aun was thermally decomposed and reduced in the thermal analyzer to produce the samples for use in the analyzer in order to identify the intermediate phases . the intermediate reaction phases were determined and identified by tg analysis and x - ray diffraction . the characteristic analyzes of the intermediate phases and sample preparations were performed by a tg - dta unit ( ta instrument , simultaneous sdt 2960 ) . the x - ray diffraction analysis was performed at room temperature at a scan speed of 0.4/min and by varying the value of 2 from 10 to 70. the target was cu , and one slit was used for the divergence and scattering ( rigaku max/3d ) . the dta results from the thermal decomposition of ( nh4)2uo2(no3)42h2o as manufactured under a nitrogen , air , and hydrogen atmosphere are shown in fig . 1 . the reaction under the nitrogen atmosphere started at around 50 c with a dehydration of the hydrate , which is an endothermic reaction . endothermic peaks , which seemed to be a phase change of nh4no3 , were due to the presence of extremely small amount of impurities . an endothermic peak as a result of the first thermal decomposition of ( nh4)2uo2(no3)42h2o into nh4no3 appeared within a temperature range of 168 to 240 c . a second endothermic that was due to the second thermal decomposition of nh4no3 appeared in the temperature range of 268 to 307 c . after the second endothermic peak continued , a weak exothermic reaction took place around 400 c . this reaction continued for a while , and then ended as an endothermic reaction that took place at 579 c.fig . 1dta curves for ( nh4)2uo2(no3)42h2o in a n2 , b air , and c h2 atmosphere dta curves for ( nh4)2uo2(no3)42h2o in a n2 , b air , and c h2 atmosphere the thermal decomposition under the air atmosphere showed a different trend . nh4uo2(no3)3 was thermally decomposed to uo3 at around 275 c , which was about 7 c higher than the corresponding temperature under the nitrogen atmosphere . it is assumed that such a difference in the reaction temperature between an air and a nitrogen atmosphere was due to the different activation energy that was dependent on the atmospheric under which the decomposition was carried out . also , it has been reported that if the composition of a compound is different or an impurity is present , the peak of the dta curve moves toward a higher temperature . under a hydrogen atmosphere , first , the dehydration of ( nh4)2uo2(no3)42h2o occurred , and the thermal decomposition of nh4no3 then took place , which had the same trend as that under nitrogen and air atmospheres . however , both the temperature at which the thermal decomposition took place and the maximum peak temperature shifted toward a lower temperature . like the exothermic peak under the nitrogen and air atmospheres , the exothermic peak appeared at around 400 c . however , the subsequent reactions were different from those under the nitrogen and air atmospheres due to the reduction by hydrogen , which caused the appearance of two different exothermic peaks . an exothermic reaction took place at 440 c , then shortly after another exothermic reaction started at around 590 c . halldahl and nygren also observed a similar phenomenon , the dta curves for decomposition of ( nh4)2uo2(no3)42h2o were dependent upon the atmospheric gas used during the decomposition . halldahl also reported that the maximum peak temperature when auc was thermally decomposed into uo3 under a nitrogen , air , and hydrogen atmosphere was 195 , 198 , and 185 c , respectively . the thermal decomposition temperature became lower in the following order : hydrogen < nitrogen < air . this result is similar to the result observed in the current study , where the maximum peak temperature appeared when ( nh4)2uo2(no3)42h2o was decomposed into uo3 . the dta results for the thermal decomposition and reduction of nh4uo2(no3)3 under different atmospheric gases are shown in fig . 2 . the results were found to be very similar to the dta results of the thermal decomposition and reduction of ( nh4)2uo2(no3)42h2o . plus , in the case of ( nh4)2uo2(no3)42h2o , the endothermic peaks appeared around 84 , 130 , and 155 c due to a phase change of nh4no3 . it may be noted that in the case of nh4uo2(no3)3 , such peaks were very weak.fig . 2dta curves for nh4uo2(no3)3 in a n2 , b air , and c h2 atmosphere dta curves for nh4uo2(no3)3 in a n2 , b air , and c h2 atmosphere the endothermic peak resulting from the thermal decomposition of nh4no3 , which was the first reaction under a nitrogen atmosphere , appeared between 266 and 305 c , and a weak exothermic reaction then took place at around 400 c followed by an endothermic reaction at 578 c . also , in air , an endothermic reaction was observed at 277 c , an exothermic reaction around 400 c , and two endothermic reactions at around 598 c . under a hydrogen atmosphere , one endothermic reaction was observed at 270 c , and three exothermic reactions were then observed at around 400 , 441 , and 590 c . the temperatures at which the endothermic and exothermic reactions started and ended , as shown in the dta curves , were used to determine intermediate phases during thermal decomposition of ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 and during their reduction . the samples for acquiring the intermediate phases were prepared under the respective atmospheres by decomposing ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 thermally at 390 , 480 , and 800 c , under the nitrogen and air atmospheres , and at 390 , 430 , 480 , and 600 c under a hydrogen atmosphere . the samples were further reduced in a thermal analyzer and cooled down to room temperature . the x - ray diffraction analysis of each intermediate phase was performed to gain a better understanding of the intermediate phases.table 1thermal treatment conditions for the preparation of intermediates decomposed from ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 atmospherefinal temperature of thermal treatment for intermediates of ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 ( c)n2 390480800air390480800h2 390430480600 thermal treatment conditions for the preparation of intermediates decomposed from ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 under a nitrogen atmosphere ( nh4)2uo2(no3)4 and nh4uo2(no3)3 were produced from ( nh4)2uo2(no3)42h2o , while nh4no3 was thermally decomposed . 3b , the structure of nh4uo2(no3)3 disappeared completely at a thermal decomposition temperature of 390 c , and amorphous uo3 , a new uranium oxide was formed . in addition , it was also found that in the intermediate phase , amorphous uo3 was subsequently transformed into -uo3 with a crystal structure at 480 c ( see fig . the -uo3 subsequently transformed into u3o8 by a phase change through a weak endothermic reaction at a temperature of 800 c ( see fig . 3x - ray diffraction patterns of intermediates produced from ( nh4)2uo2(no3)42h2o in n2 atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)2uo2(no3)42h2o in n2 atmosphere the intermediate phase obtained after thermal treatment of ( nh4)2uo2(no3)42h2o under the air atmosphere appeared to be the same as that obtained under a nitrogen atmosphere . 4 . however , as identified in the dta results , under an air atmosphere , the intermediate phase was produced at a higher temperature than that at a nitrogen atmosphere.fig . 4x - ray diffraction patterns of intermediates from ( nh4)2uo2(no3)42h2o in air atmosphere x - ray diffraction patterns of intermediates from ( nh4)2uo2(no3)42h2o in air atmosphere under a hydrogen atmosphere ( nh4)2uo2(no3)42h2o produced a different intermediate phase through a thermal decomposition and reduction compared to the phase observed when decomposed under nitrogen and air atmospheres . the uranium oxide produced at a temperature of 390 c under a hydrogen atmosphere was amorphous uo3 as was the case under nitrogen and air atmospheres ( see fig . 5b ) , however , amorphous uo3 was crystallized at 430 c along with the phase change from uo3 to -uo3 ( see fig . furthermore , -uo3 , which went through an exothermic reaction at 430 c , was transformed into -u3o8 by a phase change at 490 c ( see fig . 5d ) , and -u3o8 was finally reduced to uo2 at 600 c ( see fig . 5e).fig . 5x - ray diffraction patterns of intermediates produced from ( nh4)2uo2(no3)42h2o in h2 atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)2uo2(no3)42h2o in h2 atmosphere the dta results , as shown in fig . 1 indicated that the temperature at which uo3 was reduced to -u3o8 under a hydrogen atmosphere was lower than the temperature at which uo3 was reduced to u3o8 under an air atmosphere . when ( nh4)2uo2(no3)42h2o was thermally decomposed to uo3 , nh4 , as a residual substance , was included in the uo3 matrix therefore , as the temperature increased , the ammonia contained in the residual substance was released and further oxidized under an air atmosphere so that the auto reduction from uo3 to u3o8 was suppressed . as such , it would appear that the production of u3o8 from uo3 took place at a lower temperature under a hydrogen atmosphere . generally , the reduction from uo3 to u3o8 through an auto reduction is as follows;\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ 3 { \text{uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } + 1/ 2 { \text{o } } _ { 2}. $ $ \end{document}it is hypothesized that in hydrogen atmosphere , hydrogen was adsorbed on to the matrix and reacted with oxygen ions and then diffused out of the uo3 matrix to produce h2o . the produced h2o desorbed from the uo3 matrix surface , and at the same time , oxygen present in the uo3 rapidly diffused onto the uo3 matrix . meanwhile , in air atmosphere , the thermal decomposition rate of uo3 to u3o8 was slow , because the oxygen present in the uo3 was prevented from being diffused onto the uo3 matrix . as shown in fig . 6 , in the case of nh4uo2(no3)3 , under a nitrogen atmosphere , amorphous uo3 was produced at 390 c , however , decomposition ( nh4)2uo2(no3)42h2o ( see fig . when comparing this phenomenon with the one in which ( nh4)2uo2(no3)42h2o produced -uo3 at a temperature of 480 c , it suggests that nh4uo2(no3)3 was thermally decomposed through a different phase change route compared to ( nh4)2uo2(no3)42h2o decomposition route . subsequently , the -uo3 phase , which went through an endothermic reaction at 540 c , changed into -u3o8 with a crystal structure at a temperature of 800 c ( see fig . the thermal decomposition process under an air atmosphere was found to be the same as that under a nitrogen atmosphere.fig . 6x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in n2 atmospherefig . 7x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in air atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in n2 atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in air atmosphere as can be seen from fig . 8 , the thermal decomposition and reduction of nh4uo2(no3)3 under a hydrogen atmosphere resulted in a final product of uo2 ( see fig . 8e ) with a crystal structure through a series of phase changes to amorphous uo3 ( see fig . 8d ) . in the case of nh4uo2(no3)3 , when amorphous uo3 crystallized , -uo3 was produced in the intermediate phase , whereas in the case of ( nh4)2uo2(no3)42h2o , -uo3 was produced in the intermediate phase.fig . 8x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in h2 atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in h2 atmosphere table 2 shows a comparison of the above x - ray diffraction analysis results and thermal analysis results between adu and auc . specifically , it is worth noting the phase change of uo3 among the intermediate phases of the uranium oxide that are produced during thermal decomposition process of two auns . it may be noted that the sinterability of uo2 powder is significantly related to the physical and chemical characteristics of the precursor and the reaction mechanism of the intermediate phases . in the current study , it was found that -uo3 was produced as an intermediate product when ( nh4)2uo2(no3)42h2o was thermally decomposed irrespective of the atmospheric gas used . however , when nh4uo2(no3)3 was decomposed , different forms of uo3 were produced depending on the atmosphere under which the decomposition was carried out . under nitrogen and air atmospheres , it appears that starting substance , atmospheric gas , and reaction gas discharge affected the decomposition reaction . the thermal decomposition and reduction processes of aun have not been studied adequately , and an understanding of various reaction processes of aun is unavailable . however , various studies with different opinions have been reported in the literature on the reduction adu and auc . woolfrey explained that under a hydrogen atmosphere , adu can be directly reduced to uo2 , under oxygen or nitrogen atmosphere , adu is transformed into uo3 and u3o8 through a calcination process , which can then be reduced to uo2 under a hydrogen atmosphere . cordfunke reported that amorphous uo3 and -uo3 exist in a crystal form during the process of thermal decomposition of adu . landspersky and rodriguez et al . noted that the existence of two forms of uo3 is due to the heating rate during thermal decomposition . landspersky reported that when the heating rate during thermal decomposition is slow ( 1 c / min ) , amorphous uo3 was produced , whereas when the heating rate increases ( 10 c / min ) , -uo3 is produced . so far , it would seem that the forms of uo3 produced during the process of a thermal decomposition of adu are mostly amorphous uo3 and -uo3 , irrespective of the atmospheric gas used . however , kim reported that in the case of thermal decomposition of auc , under an air atmosphere , only amorphous uo3 was produced , while under nitrogen and hydrogen atmospheres , amorphous uo3 and -uo3 are produced . as shown in table 2 , the thermal decomposition and reduction process of auc depend on the atmospheric gas or starting substance used . in particular , the reaction process differed depending on the two forms of aun used in this study . in the case of aun , the starting substance is produced by varying the mole ratio of nh4/u , and adding nh4no3 as a reactant at a specified time . in the case of auc , both nh3 and co2 were used as reactants . in the case of adu , these additives affected the thermal decomposition characteristics of final products.table 2comparison of the decomposition mechanisms of au , auc and aun with previous resultsresearchersdecomposition mechanism of auatmospherewoolfrey \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } { \text{au iii}}/{\text{iv } } \to { \text{au ii } } \to { \text{au i } } \to \beta { \text{-uo } } _ { 3 } \to \\ { \text{a - uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } /{\text{uo } } _ { 2 . 9 } \to { \text{u } } _ { 4 } { \text{o } } _ { 9 } \to { \text{uo } } _ { 2 + x } \\ \end{aligned } $ $ \end{document } h2 kim \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } { \text{au ii}}/{\text{iii } } \to { \text{au ii}}/{\text{i } } \to { \text{au i } } \to { \text{a - uo } } _ { 3 } \to \beta{\text{-uo } } _ { 3 } \to \\ \alpha{\text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{u } } _ { 4 } { \text{o } } _ { 9 } \to { \text{uo } } _ { 2 } \\ \end{aligned } $ $ \end{document } h2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{auc } } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } air \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{auc } } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } n2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{auc } } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } $ $ \end{document } h2 sato \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{au } } \to { \text{uo } } _ { 3 } { \text{nh } } _ { 3 } \to { \text{a - uo } } _ { 3 } \to \beta { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } airprice \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{au } } \to { \text{uo } } _ { 3 } x{\text{nh } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } $ $ \end{document } h2 current study \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } & \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o}}\\ & \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3}\\ & \to \gamma { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8}\\ & { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document } n2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } & \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o}}\\ & \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \\ & \to \gamma { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8}\\ & { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document } air \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } & \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \\ & \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3}\\ & \to \gamma { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } \\ & { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \beta { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } \\ \end{aligned } $ $ \end{document } h2 comparison of the decomposition mechanisms of au , auc and aun with previous results an analysis of the intermediate phases and reaction characteristics has been discussed for each stage by using the data from dta and x - ray diffraction analysis . by comparing these results with the tg analysis results , the respective thermal decomposition and reduction characteristics of ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 were also reconfirmed . as already identified , nh4uo2(no3)3 is decomposed thermally according to the following reaction formula.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{uo } } _ { 3 } + { \text{n } } _ { 2 } { \text{o } } _ { 5 } \uparrow + { \text { n } } _ { 2 } { \text{o } } \uparrow + { \text { 2h } } _ { 2 } { \text{o } } \uparrow $ $ \end{document}the tg curves corresponding to these reactions under different atmospheres are illustrated in figs . thermal decomposition of nh4uo2(no3)3 into uo3 started at 268 , 275 and 260 c under a nitrogen , air , and hydrogen atmosphere , respectively , and ended at about 450 c under nitrogen and air atmospheres , and at about 350 c under a hydrogen atmosphere . the weight loss was 39.26 , 39.16 and 39.05 wt% under a nitrogen , air , and hydrogen atmosphere , respectively , which was different from the theoretical value of 39.67 wt% . a difference was found for nh4uo2(no3)3 between the experimental value and the theoretical value . from the x - ray diffraction analysis the quantitative difference may be due to the existence of a residual substance in the amorphous uo3 , mainly due to nh4 or h2o . sato et al . reported that amorphous uo3 was produced due to such residual substances . halldal and sorensen observed that the decomposition of auc thermally under a hydrogen atmosphere up to 400 c , resulted in the production of uo3(h2o)0.15 , which is amorphous hydrated urania . govindan reported that an amorphous uo3(h2o)x was produced due to the loss of ammonia and thermal disassembly of carbonate oxy - anion during the thermal decomposition of auc . it was found in this study , through an x - ray diffraction analysis , that the amorphous uo3 was crystallized into -uo3 , -uo3 and -uo3 at 480 c under the nitrogen and air atmospheres , and at 430 c under a hydrogen atmosphere , respectively , yet no weight loss took place as shown by the tg curve . therefore , it appears that the crystallizing stage took place simply due to a phase change.fig . 9tg curves of ( nh4)2uo2(no3)42h2o in a n2 , b air , and c h2 atmospherefig . 10tg curves of nh4uo2(no3)3 in a n2 , b air , and c h2 atmosphere tg curves of ( nh4)2uo2(no3)42h2o in a n2 , b air , and c h2 atmosphere tg curves of nh4uo2(no3)3 in a n2 , b air , and c h2 atmosphere the dta results from the thermal decomposition of ( nh4)2uo2(no3)42h2o as manufactured under a nitrogen , air , and hydrogen atmosphere are shown in fig . 1 . the reaction under the nitrogen atmosphere started at around 50 c with a dehydration of the hydrate , which is an endothermic reaction . endothermic peaks , which seemed to be a phase change of nh4no3 , were due to the presence of extremely small amount of impurities . an endothermic peak as a result of the first thermal decomposition of ( nh4)2uo2(no3)42h2o into nh4no3 appeared within a temperature range of 168 to 240 c . a second endothermic that was due to the second thermal decomposition of nh4no3 appeared in the temperature range of 268 to 307 c . after the second endothermic peak continued , a weak exothermic reaction took place around 400 c . this reaction continued for a while , and then ended as an endothermic reaction that took place at 579 c.fig . 1dta curves for ( nh4)2uo2(no3)42h2o in a n2 , b air , and c h2 atmosphere dta curves for ( nh4)2uo2(no3)42h2o in a n2 , b air , and c h2 atmosphere the thermal decomposition under the air atmosphere showed a different trend . nh4uo2(no3)3 was thermally decomposed to uo3 at around 275 c , which was about 7 c higher than the corresponding temperature under the nitrogen atmosphere . it is assumed that such a difference in the reaction temperature between an air and a nitrogen atmosphere was due to the different activation energy that was dependent on the atmospheric under which the decomposition was carried out . also , it has been reported that if the composition of a compound is different or an impurity is present , the peak of the dta curve moves toward a higher temperature . under a hydrogen atmosphere , first , the dehydration of ( nh4)2uo2(no3)42h2o occurred , and the thermal decomposition of nh4no3 then took place , which had the same trend as that under nitrogen and air atmospheres . however , both the temperature at which the thermal decomposition took place and the maximum peak temperature shifted toward a lower temperature . like the exothermic peak under the nitrogen and air atmospheres , the exothermic peak appeared at around 400 c . however , the subsequent reactions were different from those under the nitrogen and air atmospheres due to the reduction by hydrogen , which caused the appearance of two different exothermic peaks . an exothermic reaction took place at 440 c , then shortly after another exothermic reaction started at around 590 c . halldahl and nygren also observed a similar phenomenon , the dta curves for decomposition of ( nh4)2uo2(no3)42h2o were dependent upon the atmospheric gas used during the decomposition . halldahl also reported that the maximum peak temperature when auc was thermally decomposed into uo3 under a nitrogen , air , and hydrogen atmosphere was 195 , 198 , and 185 c , respectively . the thermal decomposition temperature became lower in the following order : hydrogen < nitrogen < air . this result is similar to the result observed in the current study , where the maximum peak temperature appeared when ( nh4)2uo2(no3)42h2o was decomposed into uo3 . the dta results for the thermal decomposition and reduction of nh4uo2(no3)3 under different atmospheric gases are shown in fig . 2 . the results were found to be very similar to the dta results of the thermal decomposition and reduction of ( nh4)2uo2(no3)42h2o . plus , in the case of ( nh4)2uo2(no3)42h2o , the endothermic peaks appeared around 84 , 130 , and 155 c due to a phase change of nh4no3 . it may be noted that in the case of nh4uo2(no3)3 , such peaks were very weak.fig . 2dta curves for nh4uo2(no3)3 in a n2 , b air , and c h2 atmosphere dta curves for nh4uo2(no3)3 in a n2 , b air , and c h2 atmosphere the endothermic peak resulting from the thermal decomposition of nh4no3 , which was the first reaction under a nitrogen atmosphere , appeared between 266 and 305 c , and a weak exothermic reaction then took place at around 400 c followed by an endothermic reaction at 578 c . also , in air , an endothermic reaction was observed at 277 c , an exothermic reaction around 400 c , and two endothermic reactions at around 598 c . under a hydrogen atmosphere , one endothermic reaction was observed at 270 c , and three exothermic reactions were then observed at around 400 , 441 , and 590 c . the temperatures at which the endothermic and exothermic reactions started and ended , as shown in the dta curves , were used to determine intermediate phases during thermal decomposition of ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 and during their reduction . the samples for acquiring the intermediate phases were prepared under the respective atmospheres by decomposing ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 thermally at 390 , 480 , and 800 c , under the nitrogen and air atmospheres , and at 390 , 430 , 480 , and 600 c under a hydrogen atmosphere . the samples were further reduced in a thermal analyzer and cooled down to room temperature . the x - ray diffraction analysis of each intermediate phase was performed to gain a better understanding of the intermediate phases.table 1thermal treatment conditions for the preparation of intermediates decomposed from ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 atmospherefinal temperature of thermal treatment for intermediates of ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 ( c)n2 390480800air390480800h2 390430480600 thermal treatment conditions for the preparation of intermediates decomposed from ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 under a nitrogen atmosphere ( nh4)2uo2(no3)4 and nh4uo2(no3)3 were produced from ( nh4)2uo2(no3)42h2o , while nh4no3 was thermally decomposed . 3b , the structure of nh4uo2(no3)3 disappeared completely at a thermal decomposition temperature of 390 c , and amorphous uo3 , a new uranium oxide was formed . in addition , it was also found that in the intermediate phase , amorphous uo3 was subsequently transformed into -uo3 with a crystal structure at 480 c ( see fig . the -uo3 subsequently transformed into u3o8 by a phase change through a weak endothermic reaction at a temperature of 800 c ( see fig . 3x - ray diffraction patterns of intermediates produced from ( nh4)2uo2(no3)42h2o in n2 atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)2uo2(no3)42h2o in n2 atmosphere the intermediate phase obtained after thermal treatment of ( nh4)2uo2(no3)42h2o under the air atmosphere appeared to be the same as that obtained under a nitrogen atmosphere . however , as identified in the dta results , under an air atmosphere , the intermediate phase was produced at a higher temperature than that at a nitrogen atmosphere.fig . 4x - ray diffraction patterns of intermediates from ( nh4)2uo2(no3)42h2o in air atmosphere x - ray diffraction patterns of intermediates from ( nh4)2uo2(no3)42h2o in air atmosphere under a hydrogen atmosphere ( nh4)2uo2(no3)42h2o produced a different intermediate phase through a thermal decomposition and reduction compared to the phase observed when decomposed under nitrogen and air atmospheres . the uranium oxide produced at a temperature of 390 c under a hydrogen atmosphere was amorphous uo3 as was the case under nitrogen and air atmospheres ( see fig . 5b ) , however , amorphous uo3 was crystallized at 430 c along with the phase change from uo3 to -uo3 ( see fig . furthermore , -uo3 , which went through an exothermic reaction at 430 c , was transformed into -u3o8 by a phase change at 490 c ( see fig . 5d ) , and -u3o8 was finally reduced to uo2 at 600 c ( see fig . 5e).fig . 5x - ray diffraction patterns of intermediates produced from ( nh4)2uo2(no3)42h2o in h2 atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)2uo2(no3)42h2o in h2 atmosphere the dta results , as shown in fig . 1 indicated that the temperature at which uo3 was reduced to -u3o8 under a hydrogen atmosphere was lower than the temperature at which uo3 was reduced to u3o8 under an air atmosphere . when ( nh4)2uo2(no3)42h2o was thermally decomposed to uo3 , nh4 , as a residual substance , was included in the uo3 matrix . therefore , as the temperature increased , the ammonia contained in the residual substance was released and further oxidized under an air atmosphere so that the auto reduction from uo3 to u3o8 was suppressed . as such , it would appear that the production of u3o8 from uo3 took place at a lower temperature under a hydrogen atmosphere . generally , the reduction from uo3 to u3o8 through an auto reduction is as follows;\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ 3 { \text{uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } + 1/ 2 { \text{o } } _ { 2}. $ $ \end{document}it is hypothesized that in hydrogen atmosphere , hydrogen was adsorbed on to the matrix and reacted with oxygen ions and then diffused out of the uo3 matrix to produce h2o . the produced h2o desorbed from the uo3 matrix surface , and at the same time , oxygen present in the uo3 rapidly diffused onto the uo3 matrix . meanwhile , in air atmosphere , the thermal decomposition rate of uo3 to u3o8 was slow , because the oxygen present in the uo3 was prevented from being diffused onto the uo3 matrix . as shown in fig . 6 , in the case of nh4uo2(no3)3 , under a nitrogen atmosphere , amorphous uo3 was produced at 390 c , however , decomposition ( nh4)2uo2(no3)42h2o ( see fig . when comparing this phenomenon with the one in which ( nh4)2uo2(no3)42h2o produced -uo3 at a temperature of 480 c , it suggests that nh4uo2(no3)3 was thermally decomposed through a different phase change route compared to ( nh4)2uo2(no3)42h2o decomposition route . subsequently , the -uo3 phase , which went through an endothermic reaction at 540 c , changed into -u3o8 with a crystal structure at a temperature of 800 c ( see fig . the thermal decomposition process under an air atmosphere was found to be the same as that under a nitrogen atmosphere.fig . 6x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in n2 atmospherefig . 7x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in air atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in n2 atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in air atmosphere as can be seen from fig . 8 , the thermal decomposition and reduction of nh4uo2(no3)3 under a hydrogen atmosphere resulted in a final product of uo2 ( see fig . 8e ) with a crystal structure through a series of phase changes to amorphous uo3 ( see fig . 8d ) . in the case of nh4uo2(no3)3 , when amorphous uo3 crystallized , -uo3 was produced in the intermediate phase , whereas in the case of ( nh4)2uo2(no3)42h2o , -uo3 was produced in the intermediate phase.fig . 8x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in h2 atmosphere x - ray diffraction patterns of intermediates produced from ( nh4)uo2(no3)3 in h2 atmosphere table 2 shows a comparison of the above x - ray diffraction analysis results and thermal analysis results between adu and auc . specifically , it is worth noting the phase change of uo3 among the intermediate phases of the uranium oxide that are produced during thermal decomposition process of two auns . it may be noted that the sinterability of uo2 powder is significantly related to the physical and chemical characteristics of the precursor and the reaction mechanism of the intermediate phases . in the current study , it was found that -uo3 was produced as an intermediate product when ( nh4)2uo2(no3)42h2o was thermally decomposed irrespective of the atmospheric gas used . however , when nh4uo2(no3)3 was decomposed , different forms of uo3 were produced depending on the atmosphere under which the decomposition was carried out . under nitrogen and air atmospheres , it appears that starting substance , atmospheric gas , and reaction gas discharge affected the decomposition reaction . the thermal decomposition and reduction processes of aun have not been studied adequately , and an understanding of various reaction processes of aun is unavailable . however , various studies with different opinions have been reported in the literature on the reduction adu and auc . woolfrey explained that under a hydrogen atmosphere , adu can be directly reduced to uo2 , under oxygen or nitrogen atmosphere , adu is transformed into uo3 and u3o8 through a calcination process , which can then be reduced to uo2 under a hydrogen atmosphere . cordfunke reported that amorphous uo3 and -uo3 exist in a crystal form during the process of thermal decomposition of adu . landspersky and rodriguez et al . noted that the existence of two forms of uo3 is due to the heating rate during thermal decomposition . landspersky reported that when the heating rate during thermal decomposition is slow ( 1 c / min ) , amorphous uo3 was produced , whereas when the heating rate increases ( 10 c / min ) , -uo3 is produced . so far , it would seem that the forms of uo3 produced during the process of a thermal decomposition of adu are mostly amorphous uo3 and -uo3 , irrespective of the atmospheric gas used . however , kim reported that in the case of thermal decomposition of auc , under an air atmosphere , only amorphous uo3 was produced , while under nitrogen and hydrogen atmospheres , amorphous uo3 and -uo3 are produced . as shown in table 2 , the thermal decomposition and reduction process of auc depend on the atmospheric gas or starting substance used . in particular , the reaction process differed depending on the two forms of aun used in this study . in the case of aun , the starting substance is produced by varying the mole ratio of nh4/u , and adding nh4no3 as a reactant at a specified time . in the case of auc , both nh3 and co2 were used as reactants . in the case of adu , these additives affected the thermal decomposition characteristics of final products.table 2comparison of the decomposition mechanisms of au , auc and aun with previous resultsresearchersdecomposition mechanism of auatmospherewoolfrey \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } { \text{au iii}}/{\text{iv } } \to { \text{au ii } } \to { \text{au i } } \to \beta { \text{-uo } } _ { 3 } \to \\ { \text{a - uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } /{\text{uo } } _ { 2 . 9 } \to { \text{u } } _ { 4 } { \text{o } } _ { 9 } \to { \text{uo } } _ { 2 + x } \\ \end{aligned } $ $ \end{document } h2 kim \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } { \text{au ii}}/{\text{iii } } \to { \text{au ii}}/{\text{i } } \to { \text{au i } } \to { \text{a - uo } } _ { 3 } \to \beta{\text{-uo } } _ { 3 } \to \\ \alpha{\text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{u } } _ { 4 } { \text{o } } _ { 9 } \to { \text{uo } } _ { 2 } \\ \end{aligned } $ $ \end{document } h2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{auc } } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } air \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{auc } } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } n2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{auc } } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } $ $ \end{document } h2 sato \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{au } } \to { \text{uo } } _ { 3 } { \text{nh } } _ { 3 } \to { \text{a - uo } } _ { 3 } \to \beta { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } airprice \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{au } } \to { \text{uo } } _ { 3 } x{\text{nh } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } $ $ \end{document } h2 current study \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } & \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o}}\\ & \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3}\\ & \to \gamma { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8}\\ & { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document } n2 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } & \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o}}\\ & \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \\ & \to \gamma { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8}\\ & { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document } air \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } & \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \\ & \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3}\\ & \to \gamma { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } \\ & { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \beta { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } \\ \end{aligned } $ $ \end{document } h2 comparison of the decomposition mechanisms of au , auc and aun with previous results an analysis of the intermediate phases and reaction characteristics has been discussed for each stage by using the data from dta and x - ray diffraction analysis . by comparing these results with the tg analysis results , the respective thermal decomposition and reduction characteristics of ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 were also reconfirmed . as already identified , nh4uo2(no3)3 is decomposed thermally according to the following reaction formula.\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{uo } } _ { 3 } + { \text{n } } _ { 2 } { \text{o } } _ { 5 } \uparrow + { \text { n } } _ { 2 } { \text{o } } \uparrow + { \text { 2h } } _ { 2 } { \text{o } } \uparrow $ $ \end{document}the tg curves corresponding to these reactions under different atmospheres are illustrated in figs . thermal decomposition of nh4uo2(no3)3 into uo3 started at 268 , 275 and 260 c under a nitrogen , air , and hydrogen atmosphere , respectively , and ended at about 450 c under nitrogen and air atmospheres , and at about 350 c under a hydrogen atmosphere . the weight loss was 39.26 , 39.16 and 39.05 wt% under a nitrogen , air , and hydrogen atmosphere , respectively , which was different from the theoretical value of 39.67 wt% . a difference was found for nh4uo2(no3)3 between the experimental value and the theoretical value . from the x - ray diffraction analysis the quantitative difference may be due to the existence of a residual substance in the amorphous uo3 , mainly due to nh4 or h2o . sato et al . reported that amorphous uo3 was produced due to such residual substances . halldal and sorensen observed that the decomposition of auc thermally under a hydrogen atmosphere up to 400 c , resulted in the production of uo3(h2o)0.15 , which is amorphous hydrated urania . govindan reported that an amorphous uo3(h2o)x was produced due to the loss of ammonia and thermal disassembly of carbonate oxy - anion during the thermal decomposition of auc . it was found in this study , through an x - ray diffraction analysis , that the amorphous uo3 was crystallized into -uo3 , -uo3 and -uo3 at 480 c under the nitrogen and air atmospheres , and at 430 c under a hydrogen atmosphere , respectively , yet no weight loss took place as shown by the tg curve . therefore , it appears that the crystallizing stage took place simply due to a phase change.fig . 9tg curves of ( nh4)2uo2(no3)42h2o in a n2 , b air , and c h2 atmospherefig . 10tg curves of nh4uo2(no3)3 in a n2 , b air , and c h2 atmosphere tg curves of ( nh4)2uo2(no3)42h2o in a n2 , b air , and c h2 atmosphere tg curves of nh4uo2(no3)3 in a n2 , b air , and c h2 atmosphere an intermediate phase , amorphous uo3 was produced when ( nh4)2uo2(no3)4 and nh4uo2(no3)3 were thermally decomposed under air , nitrogen , and hydrogen atmosphere , regardless of the atmosphere used . -uo3 was produced as the intermediate product irrespective of the atmospheric gas used during the decomposition of ( nh4)2uo2(no3)42h2o . however , in the case of nh4uo2(no3)3 , when decomposed under the nitrogen and air atmospheres , -uo3 was produced , whereas -uo3 was produced under a hydrogen atmosphere . the reaction paths of ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 under each atmosphere were as follows : under the nitrogen atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma{\text{-uo } } _ { 3 } \to{\text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document}under the air atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document}under the hydrogen atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \beta { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } $ $ \end{document } under the nitrogen atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma{\text{-uo } } _ { 3 } \to{\text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma{\text{-uo } } _ { 3 } \to{\text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } under the air atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } under the hydrogen atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \beta { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } $ $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } \\ \end{aligned } $ $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \beta { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } $ $ \end{document }

two types of ammonium uranyl nitrate ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 , were thermally decomposed and reduced in a tg - dta unit in nitrogen , air , and hydrogen atmospheres . various intermediate phases produced by the thermal decomposition and reduction process were investigated by an x - ray diffraction analysis and a tg / dta analysis . both ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 decomposed to amorphous uo3 regardless of the atmosphere used . the amorphous uo3 from ( nh4)2uo2(no3)42h2o was crystallized to -uo3 regardless of the atmosphere used without a change in weight . the amorphous uo3 obtained from decomposition of nh4uo2(no3)3 was crystallized to -uo3 under a nitrogen and air atmosphere , and to -uo3 under a hydrogen atmosphere without a change in weight . under each atmosphere , the reaction paths of ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 were as follows : under a nitrogen atmosphere : ( nh4)2uo2(no3)42h2o ( nh4)2uo2(no3)4h2o ( nh4)2uo2(no3)4 nh4uo2(no3)3 a - uo3 -uo3 u3o8 , nh4uo2(no3)3 a - uo3 -uo3 u3o8 ; under an air atmosphere : ( nh4)2uo2(no3)42h2o ( nh4)2uo2(no3)4h2o ( nh4)2uo2(no3)4 nh4uo2(no3)3 a - uo3 -uo3 u3o8 , nh4uo2(no3)3 a - uo3 -uo3 u3o8 ; and under a hydrogen atmosphere : ( nh4)2uo2(no3)42h2o ( nh4)2uo2(no3)4h2o ( nh4)2uo2(no3)4 nh4uo2(no3)3 a - uo3 -uo3 -u3o8 uo2 , nh4 uo2(no3)3 a - uo3 -uo3 -u3o8 uo2 .

Introduction Experimental Results and discussion DTA analysis Preparation of samples for analysis of intermediate phases X-ray diffraction analysis of intermediate phases obtained from AUN Conclusions

the reaction paths of ( nh4)2uo2(no3)42h2o and nh4uo2(no3)3 under each atmosphere were as follows : under the nitrogen atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma{\text{-uo } } _ { 3 } \to{\text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document}under the air atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document}under the hydrogen atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \beta { \text{-uo } } _ { 3 } \to \alpha { \text{-u } } _ { 3 } { \text{o } } _ { 8 } \to { \text{uo } } _ { 2 } $ $ \end{document } under the nitrogen atmosphere\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \gamma{\text{-uo } } _ { 3 } \to{\text{u } } _ { 3 } { \text{o } } _ { 8 } \\ \end{aligned } $ $ \end{document}\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - uo } } _ { 3 } \to \alpha { \text{-uo } } _ { 3 } \to { \text{u } } _ { 3 } { \text{o } } _ { 8 } $ $ \end{document } \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \begin{aligned } \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot 2 { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \cdot { \text{h } } _ { 2 } { \text{o } } \to \left ( { { \text{nh } } _ { 4 } } \right ) _ { 2 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 4 } \to \\ { \text{nh } } _ { 4 } { \text{uo } } _ { 2 } \left ( { { \text{no } } _ { 3 } } \right ) _ { 3 } \to { \text{a - 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intracranial aneurysms ( ica ) are localized , pathological dilation or ballooning of a cerebral artery , with an incidence of 25% in the general population . this vascular malformation is very likely to rupture in up to 50% of patients , and only a few risk factors for rupture has been identified so far . ruptured ica results in a number of cerebrovascular complications associated with high morbidity [ 46 ] , leading to a high mortality rate [ 79 ] . ruptured ica with bleb formation ( ricabf ) is a special type of ruptured ica in which a hematoma arises from a ruptured ica and becomes liquefied at the center . the liquified blood is driven by the constant pressure from the aneurysm , and communicates with the true aneurysm through the vascular rupture opening to form a calabash - like , dumbbell - like , or irregularly - shaped lesions . these false aneurysms are prone to rupture and bleeding due to a weak confinement . thus , ricabf is associated with a mortality rate of up to 80% due to rebleeding if inappropriately treated . a number of surgical or interventional modalities have been attempted for the treatment of ricabf . surgical intervention with craniotomy is very challenging in the treatment of ricabf due to the fragile confinement of the lesion . in addition , the feasibility of interventional radiology techniques in the treatment of these false aneurysms is controversial . nevertheless , it has been proposed that endovascular intervention is an effective and minimally invasive treatment modality with less frequent complications . in conventional endovascular coil embolization , . however , endovascular coil embolization is associated with some issues , particularly in wide - neck aneurysms , due to high shear stress . therefore , the primary treatment effect of endovascular intervention should be to decelerate or eliminate the vortex in the aneurysm neck prior to embolization . proposed that it was therapeutically essential to completely compact the true aneurysm cavity and to improve the hemodynamics in the aneurysm neck . there is a lack of studies regarding the mid - term effectiveness and safety of endovascular coil embolization of aneurysm neck for the treatment of ricabf . therefore , we performed a retrospective , single - arm study in 86 patients to investigate the effectiveness and safety of this interventional radiology approach . the study protocol was approved by the institutional review board of the shanghai municipal jingan district central hospital / huashan hospital jingan branch , affiliated to fudan university . one hundred and eighty - three patients with 201 icas were consecutively hospitalized at our interventional radiology center between october 2004 and may 2012 . the angiographic diagnostic criteria of ricabf were : 1 ) lesion showing as calabash - like , dumbbell - like , or irregular on computed tomography angiography ( cta ) , magnetic resonance angiography ( mra ) , or digital subtraction angiography ( dsa ) ; and 2 ) dsa hemodynamic study revealed the coexistence of turbulence and vortex in the ricabf on dsa , the former being located in the proximal part of the aneurysm sac with a relatively higher flow rate , and the latter being located in the distal part of the aneurysm sac with a relatively lower flow rate . from those ica patients , a cohort of 86 patients with ricabf who underwent endovascular coil embolization of aneurysm neck inclusion criteria were : 1 ) age < 80 years ; 2 ) angiography - confirmed ricabf ; 3 ) aneurysm of 515 mm in diameter ; 4 ) subarachnoid hemorrhage ( sah ) ( figure 1 ) identified on computed tomography to arise from the ricabf in the case of multiple aneurysms ; and 5 ) glasgow coma scale ( gcs ) score > 7 points . in each patient , standard preoperative assessment included angiography , chest radiography , electrocardiography , liver and kidney biochemistry , routine hematology , coagulation function , hepatitis b serology , blood typing , and plasma glucose . beijing , china ) was continuously perfused at a dose of 0.81.0 mg / h to prevent cerebral vasospasm . all included patients underwent endovascular coil embolization under general anaesthesia with intubation , and target - controlled perfusion of nimodipine ( 0.81.0 mg / h ) was maintained throughout the interventional treatment . out of these 86 patients , 25 patients ( 28.7% ) received systematic heparinization , whereas 61 patients received no heparinization due to the presence of a medium - size narrow - neck aneurysm , a favorable vascular condition , and an expected operative time of less than 1 hour . the seldinger technique was used to access the femoral artery , and the aneurysm was accessed using a prowler 10 ( cordis neurovascular , inc . , florida , usa ) or an echelon 10 or 14 ( micro therapeutics inc . dba ev3 neurovascular , ca , usa ) microcatheter equipped with a silverspeed 10 or 14 microguidewire ( micro therapeutics inc . the specific technique used for aneurysm neck embolization depended upon the width of the aneurysm neck . narrow - neck aneurysms ( body / neck ratio > 2 or diameter < 4 mm ) were coil - embolized in 59 patients , using a 3-dimensional compass / complex / cosmos or a 2-dimensional helical microplex coiling system ( microvention , inc . , tustin , ca ) , a hydrocoil embolic system ( microvention , inc . , 75a columbia , usa ) , or a hydrosoft / hypersoft / supersoft / cosmos embolic system ( microvention , inc . , . only the sac ( the true aneurysm cavity ) connecting the parent artery , especially the aneurysm neck , was compactly embolized , while the distal sac ( the ricabf ) was not embolized or was loosely embolized . in the case of a large - orifice opening ( > 3 cm ) between the true aneurysm and the ricabf , the first coil might have been deployed into the ricabf , which required more cautious loose embolization . the delivery of the microcatheter tip and microcoils was more cautious if a high delivery friction was present or if the microcatheter tip projected into the aneurysm . if the ricabf ruptured incidentally upon endovascular coil embolization , the rupture opening was immediately coiled without withdrawing the microcatheter until the true aneurysm was completely compacted . upon achieving adequate support for the microcoil , the tip of the microcatheter was withdrawn into the proximal true aneurysm cavity for further compact embolization of the true aneurysm and aneurysm neck ( figure 2 ) . wide - neck aneurysms ( body / neck ratio < 2 or diameter > 4 mm ) , involving the ocular artery ( n=4 ) , posterior communicating artery ( n=17 ) , anterior communicating ( n=1 ) , superior cerebellar artery ( n=1 ) , middle cerebral artery ( n=1 ) , and basilar artery ( n=3 ) , were embolized using microcoils assisted with stenting ( neuroform , boston scientific corporation , natick , ma ; enterprise , cordis corporation , bridgewater township , new jersey ; solitaire ab , ev3 neurovascular , irvine , ca ) , double - microcatheter , semi - deploying stenting , or endovascular ballooning technique . the stent was delivered into the parent artery but not deployed , and the microcatheter was advanced into the first one - third of the aneurysm with the assistance of the guidewire . the stent was cautiously deployed or semi - deployed , or the endovascular balloon was dilated , to fix the microcatheter between the stent and the artery wall . using a double - microcatheter technique , a microcatheter was advanced into the aneurysm , and the coil was delivered but not released ; a second microcatheter was advanced into the aneurysm , and more microcoils were successively delivered until the aneurysm neck was compacted . briefly , at least 7 ml of contrast media ( yangtze river pharmaceutical group co. ltd . , taizhou , china ) , at a concentration of more than 250 angiographies were continuously captured for more than 8 sec , at a frame rate of 6 frames per second . compact aneurysm embolization was successful if the aneurysm was not visualized on at least 2 projections during dynamic angiography . intravenous perfusion of nimodipine was continued , and blood pressure was maintained at 110120/7080 mmhg . prophylactic antimicrobial agents , sedatives , and neurotrophic or cerebroprotective medication were given , as indicated . aspirin ( bayer healthcare , morristown , nj ) or clopidogrel ( sanofi winthrop industrie s.a . , france ) was prescribed for patients undergoing stent - assisted embolization to inhibit platelet aggregation and consequent endovascular thrombosis . following the intervention , the patients received a loading dose of 150 mg of clopidogrel and 200 mg aspirin on day 1 , followed by a dose of 75 mg clopidogrel or 100 mg aspirin per day . all patients were followed - up using the glasgow outcome scale ( extended ) ( gos - e ) and by cerebral angiography within 12 months after the operation . a recurrence was defined as any increase in the size of the aneurysm or its neck , or as a new hemorrhagic event of the embolized aneurysm . when a recurrence was found in a patient who was considered protected from rebleeding , or when retreatment was judged to be inappropriate , the patient did not undergo additional surgery , and another follow - up angiography was done within 12 months . in each patient , baseline characteristics were assessed , including demography , symptoms , and angiographic characteristics at presentation . treatment outcomes included secondary rupture / bleeding rate , rate of aneurysm neck embolization , aneurysm recurrence / residual , intraoperative incidents , and post - embolization complications , as well as gos - e improvement . continuous variables are presented as means standard deviation ( sd ) if the data were normally distributed . the study protocol was approved by the institutional review board of the shanghai municipal jingan district central hospital / huashan hospital jingan branch , affiliated to fudan university . one hundred and eighty - three patients with 201 icas were consecutively hospitalized at our interventional radiology center between october 2004 and may 2012 . the angiographic diagnostic criteria of ricabf were : 1 ) lesion showing as calabash - like , dumbbell - like , or irregular on computed tomography angiography ( cta ) , magnetic resonance angiography ( mra ) , or digital subtraction angiography ( dsa ) ; and 2 ) dsa hemodynamic study revealed the coexistence of turbulence and vortex in the ricabf on dsa , the former being located in the proximal part of the aneurysm sac with a relatively higher flow rate , and the latter being located in the distal part of the aneurysm sac with a relatively lower flow rate . from those ica patients , a cohort of 86 patients with ricabf who underwent endovascular coil embolization of aneurysm neck inclusion criteria were : 1 ) age < 80 years ; 2 ) angiography - confirmed ricabf ; 3 ) aneurysm of 515 mm in diameter ; 4 ) subarachnoid hemorrhage ( sah ) ( figure 1 ) identified on computed tomography to arise from the ricabf in the case of multiple aneurysms ; and 5 ) glasgow coma scale ( gcs ) score > 7 points . in each patient , standard preoperative assessment included angiography , chest radiography , electrocardiography , liver and kidney biochemistry , routine hematology , coagulation function , hepatitis b serology , blood typing , and plasma glucose . beijing , china ) was continuously perfused at a dose of 0.81.0 mg / h to prevent cerebral vasospasm . all included patients underwent endovascular coil embolization under general anaesthesia with intubation , and target - controlled perfusion of nimodipine ( 0.81.0 mg / h ) was maintained throughout the interventional treatment . out of these 86 patients , 25 patients ( 28.7% ) received systematic heparinization , whereas 61 patients received no heparinization due to the presence of a medium - size narrow - neck aneurysm , a favorable vascular condition , and an expected operative time of less than 1 hour . the seldinger technique was used to access the femoral artery , and the aneurysm was accessed using a prowler 10 ( cordis neurovascular , inc . , florida , usa ) or an echelon 10 or 14 ( micro therapeutics inc . dba ev3 neurovascular , ca , usa ) microcatheter equipped with a silverspeed 10 or 14 microguidewire ( micro therapeutics inc . the specific technique used for aneurysm neck embolization depended upon the width of the aneurysm neck . narrow - neck aneurysms ( body / neck ratio > 2 or diameter < 4 mm ) were coil - embolized in 59 patients , using a 3-dimensional compass / complex / cosmos or a 2-dimensional helical microplex coiling system ( microvention , inc . , tustin , ca ) , a hydrocoil embolic system ( microvention , inc . , 75a columbia , usa ) , or a hydrosoft / hypersoft / supersoft / cosmos embolic system ( microvention , inc . , 75a columbia , usa ) . only the sac ( the true aneurysm cavity ) connecting the parent artery , especially the aneurysm neck , was compactly embolized , while the distal sac ( the ricabf ) was not embolized or was loosely embolized . in the case of a large - orifice opening ( > 3 cm ) between the true aneurysm and the ricabf , the first coil might have been deployed into the ricabf , which required more cautious loose embolization . the delivery of the microcatheter tip and microcoils was more cautious if a high delivery friction was present or if the microcatheter tip projected into the aneurysm . if the ricabf ruptured incidentally upon endovascular coil embolization , the rupture opening was immediately coiled without withdrawing the microcatheter until the true aneurysm was completely compacted . upon achieving adequate support for the microcoil , the tip of the microcatheter was withdrawn into the proximal true aneurysm cavity for further compact embolization of the true aneurysm and aneurysm neck ( figure 2 ) . wide - neck aneurysms ( body / neck ratio < 2 or diameter > 4 mm ) , involving the ocular artery ( n=4 ) , posterior communicating artery ( n=17 ) , anterior communicating ( n=1 ) , superior cerebellar artery ( n=1 ) , middle cerebral artery ( n=1 ) , and basilar artery ( n=3 ) , were embolized using microcoils assisted with stenting ( neuroform , boston scientific corporation , natick , ma ; enterprise , cordis corporation , bridgewater township , new jersey ; solitaire ab , ev3 neurovascular , irvine , ca ) , double - microcatheter , semi - deploying stenting , or endovascular ballooning technique . the stent was delivered into the parent artery but not deployed , and the microcatheter was advanced into the first one - third of the aneurysm with the assistance of the guidewire . the stent was cautiously deployed or semi - deployed , or the endovascular balloon was dilated , to fix the microcatheter between the stent and the artery wall . using a double - microcatheter technique , a microcatheter was advanced into the aneurysm , and the coil was delivered but not released ; a second microcatheter was advanced into the aneurysm , and more microcoils were successively delivered until the aneurysm neck was compacted . briefly , at least 7 ml of contrast media ( yangtze river pharmaceutical group co. ltd . , taizhou , china ) , at a concentration of more than 250 mg / l , was injected at a rate of 4 ml / sec . angiographies were continuously captured for more than 8 sec , at a frame rate of 6 frames per second . compact aneurysm embolization was successful if the aneurysm was not visualized on at least 2 projections during dynamic angiography . intravenous perfusion of nimodipine was continued , and blood pressure was maintained at 110120/7080 mmhg . prophylactic antimicrobial agents , sedatives , and neurotrophic or cerebroprotective medication were given , as indicated . aspirin ( bayer healthcare , morristown , nj ) or clopidogrel ( sanofi winthrop industrie s.a . , france ) was prescribed for patients undergoing stent - assisted embolization to inhibit platelet aggregation and consequent endovascular thrombosis . following the intervention , the patients received a loading dose of 150 mg of clopidogrel and 200 mg aspirin on day 1 , followed by a dose of 75 mg clopidogrel or 100 mg aspirin per day . all patients were followed - up using the glasgow outcome scale ( extended ) ( gos - e ) and by cerebral angiography within 12 months after the operation . a recurrence was defined as any increase in the size of the aneurysm or its neck , or as a new hemorrhagic event of the embolized aneurysm . when a recurrence was found in a patient who was considered protected from rebleeding , or when retreatment was judged to be inappropriate , the patient did not undergo additional surgery , and another follow - up angiography was done within 12 months . in each patient , baseline characteristics were assessed , including demography , symptoms , and angiographic characteristics at presentation . treatment outcomes included secondary rupture / bleeding rate , rate of aneurysm neck embolization , aneurysm recurrence / residual , intraoperative incidents , and post - embolization complications , as well as gos - e improvement . continuous variables are presented as means standard deviation ( sd ) if the data were normally distributed . table 1 shows the baseline clinical characteristics of the 86 ricabf patients included in the present study . most of them were women ( 64/86 , 74.4% ) , and had sah ( 66/86 , 76.7% ) . patients were aged 50.710.6 years . on admission , according to the gcs , only 1 patient ( 1.2% ) had moderate brain damage ( gcs of 912 ) , while the remaining 85 ( 98.8% ) patients had minor brain damage ( gcs 13 ) . most of them had the classical clinical manifestations of sah ( 76.7% ) , while 12.8% had oculomotor nerve palsy , and 2.3% had frontal or orbital pain . according to the hunt - hess scale , 73 patients ( 84.9% ) were grade i , 12 ( 14.0% ) were grade ii , and 1 ( 1.2% ) was grade iii . table 2 shows the angiographic characteristics at presentation among the 86 patients with a total of 86 ricabf lesions included in the present study . there were 86 patients with a total of 100 aneurysms and there were 86 ricabf lesions within the 94 aneurysms accepted for endovascular embolization . the other 6 aneurysms were not treated because of their low risk of hemorrhage due to their small size and regular saccular shape . the 3 most commonly affected arteries were the posterior communicating artery ( 46/86 , 53.5% ) , the anterior communicating artery ( 20/86 , 23.3% ) , and the ophthalmic artery ( 7/86 , 8.1% ) . aneurysm neck was wide in 31.4% ( 27/86 ) of patients and narrow in 68.6% . simple embolization was performed in 77.9% ( 67/86 ) of patients , stent - assisted in 19.8% ( 17/86 ) , balloon - assisted in 1.2% ( 1/86 ) , and dual micro - catheter in 1.2% ( 1/86 ) . contrast filling in the aneurysm was assessed by the method published by raymond et al . . complete occlusion was achieved in 72 aneurysms ( 72/86 , 83.7% ) , while 12 aneurysms ( 12/86 , 14.0% ) had a residual neck , and 2 aneurysms ( 2/86 , 2.3% ) had a residual aneurysm . intraoperative cerebrovascular spasms , manifesting as a 40% reduction in vessel diameter , was observed in 4 patients ( 4.6% ) . intracatheter nimodipine ( 0.41.0 mg ) was slowly injected , and the blood pressure was maintained at 110120/7080 mmhg . intraoperative aneurysm bleeding occurred in 2 patients ( 2.3% ) ; heparin was stopped . and gos - e score was 5 in 73 patients ( 84.9% ) , 4 in 10 ( 11.6% ) , and of 3 in only 3 patients ( 3.5% ) . however , 9 patients ( 10.3% ) had cerebral infarction , 5 ( 5.7% ) had cerebrovascular spasms , 4 ( 4.6% ) had aneurysm bleeding , and 1 ( 1.1% ) had a secondary hydrocephalus . all complications were treated using appropriate standard treatments , and these patients only exhibited minimal sequelae . oculomotor palsy observed in 11 patients at admission completely improved in 8 ( 72.7% ) patients by postoperative day 2 . all patients were postoperatively followed up using cerebral angiography within 12 months ( median of 9.0 , interquartile range of 9.0 months ) . among the 86 patients available for follow - up , 83 patients kept stable compared with the immediate angiography , including 71 complete occlusions ( 71/72 ) , 11 residue necks ( 11/12 ) , and 1 residue aneurysms ( 1/2 ) , while 3 patients had recurrent aneurysms . the first recurrent case was a narrow - neck aneurysm with no residual aneurysm neck after simple coil embolization , but the follow - up angiography revealed the aneurysm neck to be slightly filled with contrast media . the first underwent stent - assisted embolization with a residual aneurysm and the residual part was slightly increased at follow - up . the last case underwent simple coil embolization and showed a residual neck , and the neck was slightly increased at the follow - up angiography . no revision surgery or embolization was performed these 3 cases with minor recurrence , and the patients were considered protected from rebleeding , but further short - term follow - up angiography was performed . no patient with minimal residual or recurrent aneurysm had a secondary aneurysm rupture during follow - up . table 1 shows the baseline clinical characteristics of the 86 ricabf patients included in the present study . most of them were women ( 64/86 , 74.4% ) , and had sah ( 66/86 , 76.7% ) . patients were aged 50.710.6 years . on admission , according to the gcs , only 1 patient ( 1.2% ) had moderate brain damage ( gcs of 912 ) , while the remaining 85 ( 98.8% ) patients had minor brain damage ( gcs 13 ) . most of them had the classical clinical manifestations of sah ( 76.7% ) , while 12.8% had oculomotor nerve palsy , and 2.3% had frontal or orbital pain . according to the hunt - hess scale , 73 patients ( 84.9% ) were grade i , 12 ( 14.0% ) were grade ii , and 1 ( 1.2% ) was grade iii . table 2 shows the angiographic characteristics at presentation among the 86 patients with a total of 86 ricabf lesions included in the present study . there were 86 patients with a total of 100 aneurysms and there were 86 ricabf lesions within the 94 aneurysms accepted for endovascular embolization . the other 6 aneurysms were not treated because of their low risk of hemorrhage due to their small size and regular saccular shape . the 3 most commonly affected arteries were the posterior communicating artery ( 46/86 , 53.5% ) , the anterior communicating artery ( 20/86 , 23.3% ) , and the ophthalmic artery ( 7/86 , 8.1% ) . aneurysm neck was wide in 31.4% ( 27/86 ) of patients and narrow in 68.6% . simple embolization was performed in 77.9% ( 67/86 ) of patients , stent - assisted in 19.8% ( 17/86 ) , balloon - assisted in 1.2% ( 1/86 ) , and dual micro - catheter in 1.2% ( 1/86 ) . contrast filling in the aneurysm was assessed by the method published by raymond et al . . complete occlusion was achieved in 72 aneurysms ( 72/86 , 83.7% ) , while 12 aneurysms ( 12/86 , 14.0% ) had a residual neck , and 2 aneurysms ( 2/86 , 2.3% ) had a residual aneurysm . intraoperative cerebrovascular spasms , manifesting as a 40% reduction in vessel diameter , was observed in 4 patients ( 4.6% ) . intracatheter nimodipine ( 0.41.0 mg ) was slowly injected , and the blood pressure was maintained at 110120/7080 mmhg . intraoperative aneurysm bleeding occurred in 2 patients ( 2.3% ) ; heparin was stopped . and gos - e score was 5 in 73 patients ( 84.9% ) , 4 in 10 ( 11.6% ) , and of 3 in only 3 patients ( 3.5% ) . however , 9 patients ( 10.3% ) had cerebral infarction , 5 ( 5.7% ) had cerebrovascular spasms , 4 ( 4.6% ) had aneurysm bleeding , and 1 ( 1.1% ) had a secondary hydrocephalus . all complications were treated using appropriate standard treatments , and these patients only exhibited minimal sequelae . oculomotor palsy observed in 11 patients at admission completely improved in 8 ( 72.7% ) patients by postoperative day 2 . all patients were postoperatively followed up using cerebral angiography within 12 months ( median of 9.0 , interquartile range of 9.0 months ) . among the 86 patients available for follow - up , 83 patients kept stable compared with the immediate angiography , including 71 complete occlusions ( 71/72 ) , 11 residue necks ( 11/12 ) , and 1 residue aneurysms ( 1/2 ) , while 3 patients had recurrent aneurysms . the first recurrent case was a narrow - neck aneurysm with no residual aneurysm neck after simple coil embolization , but the follow - up angiography revealed the aneurysm neck to be slightly filled with contrast media . the first underwent stent - assisted embolization with a residual aneurysm and the residual part was slightly increased at follow - up . the last case underwent simple coil embolization and showed a residual neck , and the neck was slightly increased at the follow - up angiography . no revision surgery or embolization was performed these 3 cases with minor recurrence , and the patients were considered protected from rebleeding , but further short - term follow - up angiography was performed . no patient with minimal residual or recurrent aneurysm had a secondary aneurysm rupture during follow - up . using endovascular coil embolization , aneurysm necks achieved complete occlusion in most patients ( 72/86 , 72.3% ) and postoperative gos - e was high in most patients . neural impairment observed in patients with ricabf derives mainly from the space - occupying effect of a large aneurysm , compressing nerves in its vicinity , such as the optic and oculomotor nerves . the severity of neural impairment is associated with aneurysm size , growth orientation , and aneurysm blood flow rate . it is known that the embolization of the aneurysm - bearing artery will shrink the aneurysm by up to 30% within 1 year , and the embolization of the aneurysm itself will significantly prolong the time of aneurysm shrinkage . moreover , embolization of the true aneurysm will discontinue blood flow into the ricabf and eliminate the hammer effect caused by the aneurysm pulsation . consequently , our results showed that oculomotor palsy observed in 11 ricabf patients at admission completely improved in 8 ( 72.7% ) patients after ricabf neck embolization . thus , endovascular embolization allows the elimination of the aneurysm with minimal interruption of blood supply to the brain . however , endovascular embolization presents a relatively high risk of aneurysm recurrence compared with the more invasive surgical closure , due to the fact that vascular endothelium may not be fully anastomosed at the aneurysm neck [ 2426 ] . the primary intraoperative safety concern arising from ricabf coil embolization is a higher risk of incidental rupture and bleeding , which results from iatrogenic injuries in most cases , compared with the risks arising from true ica embolization . major risk factors for intraoperative complications include underlying poor vascular strength , misplacement of catheter , guidewire , and/or microcoils , and excessive aneurysm compactness . compact embolization of distal ricabf will increase the luminal tension and cause subsequent rupture and bleeding . however , compact embolization of the aneurysm neck is much safer because the neck is bordered by the relatively stronger true aneurysm wall instead of the fragile fibroconnective tissues confining ricabf . reported that endovascular coil embolization without or with loose compaction of ricabf resulted in occasional intraoperative rupture , minimal aneurysm recurrence , and no secondary bleeding within a follow - up period of up to 60 months . in the case of a dumbbell - shaped ( wide - neck ) aneurysm , coils are more likely to be mistakenly deployed in the ricabf cavity upon neck embolization , and consequently cause rupture and bleeding . therefore , endovascular coil embolization of ricabf should be limited to the true aneurysm cavity to minimize iatrogenic rupture and bleeding . the long - term efficacy of coil embolization is known to largely depend upon whether complete embolization has been achieved at the first attempt ; aneurysm neck residual and coil compression are the primary risk factors contributing to aneurysm recurrence . up to 49% of patients are likely to have a recurrent aneurysm during the follow - up period if a residual aneurysm neck is present following embolization . complete compactness of the aneurysm neck will allow the migration and coverage of proliferating vascular endothelial cells , while blocking blood stream to the pre - existing aneurysm . moreover , compact embolization of proximal true aneurysm and ricabf neck can offer an adequate support for coils , and avoid postoperative recurrence associated with coil compression . studies showed that an aneurismal sah recurrence is 1522 times more likely than the expected rate of a first sah . a study showed aneurysm recurrence in 8% of patients treated with coil embolization . in a large series finally , a large systematic review showed that aneurysm recurrence occurred in 21% of 8161 aneurysms . in contrast , our study showed a low rate of aneurysm recurrence with compact embolization of aneurysm neck alone . the great majority ( > 80% ) of our patients achieved absolute complete compact embolization of aneurysm neck , and a small percentage ( < 3% ) of our patients achieved residual aneurysm . however , our follow - up results suggest that a residual neck might not contribute to a higher risk of aneurysm recurrence . the completion rate of aneurysm embolization depends upon the body / neck ratio , aneurysm neck size , and the included angle between the aneurysm and the aneurysm - bearing artery [ 11,21,3640 ] . the failure to achieve a complete compact embolization can be attributed to the limitation of the technique ; a small residual aneurysm neck may be present due to the dislocation of the coil on deployment , even when using the smallest and the most flexible microcoils available . in a short - term follow - up study , yu et al . reported that compact embolization of the proximal true aneurysm ( neck ) without embolizing the distal ricabf was associated with a nearly zero recurrence rate , similar to the recurrence rate following compact embolization of both proximal true aneurysm and distal ricabf . in the present study , indeed , 3 wide - neck aneurysms exhibited residual aneurysm following coil embolization , all of which were found to recur on follow - up angiography , but resulted in no secondary rupture or re - bleeding . a larger residual aneurysm neck may also be associated with the radiologist s learning curve . we acknowledge that our study was non - randomized and retrospective , and suffered from the limitations that are inherent in this kind of study . in conclusion , endovascular coil embolization of aneurysm neck is an effective and safe treatment modality for ricabf , and is associated with few intraoperative and postoperative complications . mid - term follow - up suggests a minimal recurrence rate and that recurrence is more likely to happen in wide - neck aneurysms . the long - term effectiveness and safety of this interventional technique need to be investigated in prospective , comparative studies .

backgroundruptured intracranial aneurysm ( ica ) with bleb formation ( ricabf ) is a special type of ruptured ica . however , the exact role and effectiveness of endovascular coil embolization ( ece ) in ricabf is unknown.we aimed to investigate the effectiveness and safety of ece of aneurysm neck for ricabf treatment.material/methodswe retrospectively assessed consecutive patients who were hospitalized in our endovascular intervention center between october 2004 and may 2012 . overall , 86 patients underwent ece of aneurysm neck for 86 ricabf . treatments outcomes included secondary rupture / bleeding rate , aneurysm neck embolization rate , residual / recurrent aneurysm , intraoperative incidents , and post - embolization complications , as well as improvements in the glasgow outcome scale ( extended ) ( gos - e).resultscomplete occlusion was achieved in 72 aneurysms ( 72/86 , 83.7% ) , while 12 aneurysms ( 12/86 , 14.0% ) had a residual neck , and 2 aneurysms ( 2/86 , 2.3% ) had a residual aneurysm . the postoperative gos - e was 3 in 3 patients ( 3.5% ) , 4 in 10 patients ( 11.6% ) , and 5 in 73 patients ( 84.9% ) . follow - up angiography was performed in all patients ( mean 9.0 months , interquartile range of 9.0 ) . recurrence was found in 3 patients ( 3/86 , 3.5% ) . no aneurysm rupture or bleeding was reported.conclusionsour mid - term follow - up study showed that ece of aneurysm neck was an effective and safe treatment modality for ricabf . the long - term effectiveness and safety of this interventional radiology technique need to be investigated in prospective and comparative studies .

Background Material and Methods Subjects Endovascular coil embolization of aneurysm neck Postoperative care and follow-up Measurements and outcomes Statistical analysis Results Clinical data at presentation Angiographic characteristics at presentation Embolization outcomes and complications Follow-up result Discussion Conclusions

intracranial aneurysms ( ica ) are localized , pathological dilation or ballooning of a cerebral artery , with an incidence of 25% in the general population . ruptured ica with bleb formation ( ricabf ) is a special type of ruptured ica in which a hematoma arises from a ruptured ica and becomes liquefied at the center . nevertheless , it has been proposed that endovascular intervention is an effective and minimally invasive treatment modality with less frequent complications . however , endovascular coil embolization is associated with some issues , particularly in wide - neck aneurysms , due to high shear stress . therefore , the primary treatment effect of endovascular intervention should be to decelerate or eliminate the vortex in the aneurysm neck prior to embolization . there is a lack of studies regarding the mid - term effectiveness and safety of endovascular coil embolization of aneurysm neck for the treatment of ricabf . therefore , we performed a retrospective , single - arm study in 86 patients to investigate the effectiveness and safety of this interventional radiology approach . one hundred and eighty - three patients with 201 icas were consecutively hospitalized at our interventional radiology center between october 2004 and may 2012 . the angiographic diagnostic criteria of ricabf were : 1 ) lesion showing as calabash - like , dumbbell - like , or irregular on computed tomography angiography ( cta ) , magnetic resonance angiography ( mra ) , or digital subtraction angiography ( dsa ) ; and 2 ) dsa hemodynamic study revealed the coexistence of turbulence and vortex in the ricabf on dsa , the former being located in the proximal part of the aneurysm sac with a relatively higher flow rate , and the latter being located in the distal part of the aneurysm sac with a relatively lower flow rate . from those ica patients , a cohort of 86 patients with ricabf who underwent endovascular coil embolization of aneurysm neck inclusion criteria were : 1 ) age < 80 years ; 2 ) angiography - confirmed ricabf ; 3 ) aneurysm of 515 mm in diameter ; 4 ) subarachnoid hemorrhage ( sah ) ( figure 1 ) identified on computed tomography to arise from the ricabf in the case of multiple aneurysms ; and 5 ) glasgow coma scale ( gcs ) score > 7 points . all included patients underwent endovascular coil embolization under general anaesthesia with intubation , and target - controlled perfusion of nimodipine ( 0.81.0 mg / h ) was maintained throughout the interventional treatment . out of these 86 patients , 25 patients ( 28.7% ) received systematic heparinization , whereas 61 patients received no heparinization due to the presence of a medium - size narrow - neck aneurysm , a favorable vascular condition , and an expected operative time of less than 1 hour . only the sac ( the true aneurysm cavity ) connecting the parent artery , especially the aneurysm neck , was compactly embolized , while the distal sac ( the ricabf ) was not embolized or was loosely embolized . if the ricabf ruptured incidentally upon endovascular coil embolization , the rupture opening was immediately coiled without withdrawing the microcatheter until the true aneurysm was completely compacted . wide - neck aneurysms ( body / neck ratio < 2 or diameter > 4 mm ) , involving the ocular artery ( n=4 ) , posterior communicating artery ( n=17 ) , anterior communicating ( n=1 ) , superior cerebellar artery ( n=1 ) , middle cerebral artery ( n=1 ) , and basilar artery ( n=3 ) , were embolized using microcoils assisted with stenting ( neuroform , boston scientific corporation , natick , ma ; enterprise , cordis corporation , bridgewater township , new jersey ; solitaire ab , ev3 neurovascular , irvine , ca ) , double - microcatheter , semi - deploying stenting , or endovascular ballooning technique . using a double - microcatheter technique , a microcatheter was advanced into the aneurysm , and the coil was delivered but not released ; a second microcatheter was advanced into the aneurysm , and more microcoils were successively delivered until the aneurysm neck was compacted . all patients were followed - up using the glasgow outcome scale ( extended ) ( gos - e ) and by cerebral angiography within 12 months after the operation . when a recurrence was found in a patient who was considered protected from rebleeding , or when retreatment was judged to be inappropriate , the patient did not undergo additional surgery , and another follow - up angiography was done within 12 months . treatment outcomes included secondary rupture / bleeding rate , rate of aneurysm neck embolization , aneurysm recurrence / residual , intraoperative incidents , and post - embolization complications , as well as gos - e improvement . one hundred and eighty - three patients with 201 icas were consecutively hospitalized at our interventional radiology center between october 2004 and may 2012 . the angiographic diagnostic criteria of ricabf were : 1 ) lesion showing as calabash - like , dumbbell - like , or irregular on computed tomography angiography ( cta ) , magnetic resonance angiography ( mra ) , or digital subtraction angiography ( dsa ) ; and 2 ) dsa hemodynamic study revealed the coexistence of turbulence and vortex in the ricabf on dsa , the former being located in the proximal part of the aneurysm sac with a relatively higher flow rate , and the latter being located in the distal part of the aneurysm sac with a relatively lower flow rate . from those ica patients , a cohort of 86 patients with ricabf who underwent endovascular coil embolization of aneurysm neck inclusion criteria were : 1 ) age < 80 years ; 2 ) angiography - confirmed ricabf ; 3 ) aneurysm of 515 mm in diameter ; 4 ) subarachnoid hemorrhage ( sah ) ( figure 1 ) identified on computed tomography to arise from the ricabf in the case of multiple aneurysms ; and 5 ) glasgow coma scale ( gcs ) score > 7 points . all included patients underwent endovascular coil embolization under general anaesthesia with intubation , and target - controlled perfusion of nimodipine ( 0.81.0 mg / h ) was maintained throughout the interventional treatment . out of these 86 patients , 25 patients ( 28.7% ) received systematic heparinization , whereas 61 patients received no heparinization due to the presence of a medium - size narrow - neck aneurysm , a favorable vascular condition , and an expected operative time of less than 1 hour . only the sac ( the true aneurysm cavity ) connecting the parent artery , especially the aneurysm neck , was compactly embolized , while the distal sac ( the ricabf ) was not embolized or was loosely embolized . if the ricabf ruptured incidentally upon endovascular coil embolization , the rupture opening was immediately coiled without withdrawing the microcatheter until the true aneurysm was completely compacted . wide - neck aneurysms ( body / neck ratio < 2 or diameter > 4 mm ) , involving the ocular artery ( n=4 ) , posterior communicating artery ( n=17 ) , anterior communicating ( n=1 ) , superior cerebellar artery ( n=1 ) , middle cerebral artery ( n=1 ) , and basilar artery ( n=3 ) , were embolized using microcoils assisted with stenting ( neuroform , boston scientific corporation , natick , ma ; enterprise , cordis corporation , bridgewater township , new jersey ; solitaire ab , ev3 neurovascular , irvine , ca ) , double - microcatheter , semi - deploying stenting , or endovascular ballooning technique . using a double - microcatheter technique , a microcatheter was advanced into the aneurysm , and the coil was delivered but not released ; a second microcatheter was advanced into the aneurysm , and more microcoils were successively delivered until the aneurysm neck was compacted . all patients were followed - up using the glasgow outcome scale ( extended ) ( gos - e ) and by cerebral angiography within 12 months after the operation . a recurrence was defined as any increase in the size of the aneurysm or its neck , or as a new hemorrhagic event of the embolized aneurysm . when a recurrence was found in a patient who was considered protected from rebleeding , or when retreatment was judged to be inappropriate , the patient did not undergo additional surgery , and another follow - up angiography was done within 12 months . treatment outcomes included secondary rupture / bleeding rate , rate of aneurysm neck embolization , aneurysm recurrence / residual , intraoperative incidents , and post - embolization complications , as well as gos - e improvement . most of them were women ( 64/86 , 74.4% ) , and had sah ( 66/86 , 76.7% ) . on admission , according to the gcs , only 1 patient ( 1.2% ) had moderate brain damage ( gcs of 912 ) , while the remaining 85 ( 98.8% ) patients had minor brain damage ( gcs 13 ) . most of them had the classical clinical manifestations of sah ( 76.7% ) , while 12.8% had oculomotor nerve palsy , and 2.3% had frontal or orbital pain . according to the hunt - hess scale , 73 patients ( 84.9% ) were grade i , 12 ( 14.0% ) were grade ii , and 1 ( 1.2% ) was grade iii . table 2 shows the angiographic characteristics at presentation among the 86 patients with a total of 86 ricabf lesions included in the present study . the 3 most commonly affected arteries were the posterior communicating artery ( 46/86 , 53.5% ) , the anterior communicating artery ( 20/86 , 23.3% ) , and the ophthalmic artery ( 7/86 , 8.1% ) . simple embolization was performed in 77.9% ( 67/86 ) of patients , stent - assisted in 19.8% ( 17/86 ) , balloon - assisted in 1.2% ( 1/86 ) , and dual micro - catheter in 1.2% ( 1/86 ) . complete occlusion was achieved in 72 aneurysms ( 72/86 , 83.7% ) , while 12 aneurysms ( 12/86 , 14.0% ) had a residual neck , and 2 aneurysms ( 2/86 , 2.3% ) had a residual aneurysm . and gos - e score was 5 in 73 patients ( 84.9% ) , 4 in 10 ( 11.6% ) , and of 3 in only 3 patients ( 3.5% ) . however , 9 patients ( 10.3% ) had cerebral infarction , 5 ( 5.7% ) had cerebrovascular spasms , 4 ( 4.6% ) had aneurysm bleeding , and 1 ( 1.1% ) had a secondary hydrocephalus . all patients were postoperatively followed up using cerebral angiography within 12 months ( median of 9.0 , interquartile range of 9.0 months ) . among the 86 patients available for follow - up , 83 patients kept stable compared with the immediate angiography , including 71 complete occlusions ( 71/72 ) , 11 residue necks ( 11/12 ) , and 1 residue aneurysms ( 1/2 ) , while 3 patients had recurrent aneurysms . the first recurrent case was a narrow - neck aneurysm with no residual aneurysm neck after simple coil embolization , but the follow - up angiography revealed the aneurysm neck to be slightly filled with contrast media . the first underwent stent - assisted embolization with a residual aneurysm and the residual part was slightly increased at follow - up . the last case underwent simple coil embolization and showed a residual neck , and the neck was slightly increased at the follow - up angiography . no revision surgery or embolization was performed these 3 cases with minor recurrence , and the patients were considered protected from rebleeding , but further short - term follow - up angiography was performed . no patient with minimal residual or recurrent aneurysm had a secondary aneurysm rupture during follow - up . most of them were women ( 64/86 , 74.4% ) , and had sah ( 66/86 , 76.7% ) . most of them had the classical clinical manifestations of sah ( 76.7% ) , while 12.8% had oculomotor nerve palsy , and 2.3% had frontal or orbital pain . according to the hunt - hess scale , 73 patients ( 84.9% ) were grade i , 12 ( 14.0% ) were grade ii , and 1 ( 1.2% ) was grade iii . table 2 shows the angiographic characteristics at presentation among the 86 patients with a total of 86 ricabf lesions included in the present study . the 3 most commonly affected arteries were the posterior communicating artery ( 46/86 , 53.5% ) , the anterior communicating artery ( 20/86 , 23.3% ) , and the ophthalmic artery ( 7/86 , 8.1% ) . simple embolization was performed in 77.9% ( 67/86 ) of patients , stent - assisted in 19.8% ( 17/86 ) , balloon - assisted in 1.2% ( 1/86 ) , and dual micro - catheter in 1.2% ( 1/86 ) . complete occlusion was achieved in 72 aneurysms ( 72/86 , 83.7% ) , while 12 aneurysms ( 12/86 , 14.0% ) had a residual neck , and 2 aneurysms ( 2/86 , 2.3% ) had a residual aneurysm . and gos - e score was 5 in 73 patients ( 84.9% ) , 4 in 10 ( 11.6% ) , and of 3 in only 3 patients ( 3.5% ) . however , 9 patients ( 10.3% ) had cerebral infarction , 5 ( 5.7% ) had cerebrovascular spasms , 4 ( 4.6% ) had aneurysm bleeding , and 1 ( 1.1% ) had a secondary hydrocephalus . all patients were postoperatively followed up using cerebral angiography within 12 months ( median of 9.0 , interquartile range of 9.0 months ) . among the 86 patients available for follow - up , 83 patients kept stable compared with the immediate angiography , including 71 complete occlusions ( 71/72 ) , 11 residue necks ( 11/12 ) , and 1 residue aneurysms ( 1/2 ) , while 3 patients had recurrent aneurysms . the first recurrent case was a narrow - neck aneurysm with no residual aneurysm neck after simple coil embolization , but the follow - up angiography revealed the aneurysm neck to be slightly filled with contrast media . the first underwent stent - assisted embolization with a residual aneurysm and the residual part was slightly increased at follow - up . the last case underwent simple coil embolization and showed a residual neck , and the neck was slightly increased at the follow - up angiography . no revision surgery or embolization was performed these 3 cases with minor recurrence , and the patients were considered protected from rebleeding , but further short - term follow - up angiography was performed . no patient with minimal residual or recurrent aneurysm had a secondary aneurysm rupture during follow - up . using endovascular coil embolization , aneurysm necks achieved complete occlusion in most patients ( 72/86 , 72.3% ) and postoperative gos - e was high in most patients . consequently , our results showed that oculomotor palsy observed in 11 ricabf patients at admission completely improved in 8 ( 72.7% ) patients after ricabf neck embolization . however , endovascular embolization presents a relatively high risk of aneurysm recurrence compared with the more invasive surgical closure , due to the fact that vascular endothelium may not be fully anastomosed at the aneurysm neck [ 2426 ] . reported that endovascular coil embolization without or with loose compaction of ricabf resulted in occasional intraoperative rupture , minimal aneurysm recurrence , and no secondary bleeding within a follow - up period of up to 60 months . in the case of a dumbbell - shaped ( wide - neck ) aneurysm , coils are more likely to be mistakenly deployed in the ricabf cavity upon neck embolization , and consequently cause rupture and bleeding . the long - term efficacy of coil embolization is known to largely depend upon whether complete embolization has been achieved at the first attempt ; aneurysm neck residual and coil compression are the primary risk factors contributing to aneurysm recurrence . up to 49% of patients are likely to have a recurrent aneurysm during the follow - up period if a residual aneurysm neck is present following embolization . the great majority ( > 80% ) of our patients achieved absolute complete compact embolization of aneurysm neck , and a small percentage ( < 3% ) of our patients achieved residual aneurysm . however , our follow - up results suggest that a residual neck might not contribute to a higher risk of aneurysm recurrence . the completion rate of aneurysm embolization depends upon the body / neck ratio , aneurysm neck size , and the included angle between the aneurysm and the aneurysm - bearing artery [ 11,21,3640 ] . in a short - term follow - up study , yu et al . in the present study , indeed , 3 wide - neck aneurysms exhibited residual aneurysm following coil embolization , all of which were found to recur on follow - up angiography , but resulted in no secondary rupture or re - bleeding . in conclusion , endovascular coil embolization of aneurysm neck is an effective and safe treatment modality for ricabf , and is associated with few intraoperative and postoperative complications . mid - term follow - up suggests a minimal recurrence rate and that recurrence is more likely to happen in wide - neck aneurysms . the long - term effectiveness and safety of this interventional technique need to be investigated in prospective , comparative studies .

as a result of the rapid development of effective treatments ( e.g. targeted therapy ) , the chances of survival for various forms of cancer have increased considerably in the last decade . given that the disease is now often treatable and has become comparatively manageable , it is no longer considered a although oncologic safety , obviously , remains the primary focus , more and more attention is paid to quality of life related issues in the treatment and nursing care of cancer patients . besides a permanent ( latent ) fear of relapses , cancer survivors often have to deal with physical defects , as is very clear in cases of breast cancer and head and neck cancer . these may result in functional disturbances such as problems with speaking and swallowing ( in the case of head and neck cancer ) or limited motility of the upper limb ( in the case of breast cancer ) which , evidently , can be important for a person s quality of life . in addition to this , however , are the changes in appearance often brought about by these physical blemishes . these changes in appearance , which can not simply be reduced to physical function or dysfunction , may affect a person s valuation of his or her body and thus change a person s body image. it is now generally assumed that body image related issues are essential to a person s quality of life [ 27 , 41].1 in contemporary ( psycho-)oncology attention to body image issues is increasing [ 1 , 8 , 14 , 16 , 63 , 64 ] . in the last decade specific body image self - report scales have been developed [ 3 , 4 , 7 , 27 ] . these questionnaires are mainly used to evaluate treatment in clinical trials and sometimes to support psychotherapeutic interventions.2 in the context of the increasing interest for body image related issues in oncology , i would like to suggest that body experience evaluation is valuable not only from a psychological perspective . my hypothesis is that body experience evaluation can play an important role in health care practices , especially in these kinds of practices where patients have to decide ( together with the medical professionals involved ) about interventions which will affect their physical appearance . in this paper , i would like to make visible the practical and ethical dimension of body experience evaluation in oncology , mainly concentrating on breast cancer . i will do so by means of a conceptual and phenomenological analysis of body image and bodily integrity . this paper thus also amounts to a medico - ethical analysis from a phenomenological perspective which , as has been rehearsed recently , is still an uncommon , and perhaps underestimated , perspective in health care analysis as well as in medical ethics . first , i will briefly explain the meaning of what is called body image , drawing on philosophical and psychological literature.3 second , i will discuss how body image is currently evaluated in breast cancer patients . i will do so by focusing on the way questions are phrased on the body image scale ( bis ) questionnaire and pointing to its inherent limitations . third , i will introduce the notions of bodily integrity and identification and will explain how these may be useful for understanding the impact of body experience evaluation in health care practice . in the final section , i will argue in favor of the application of body experience evaluation in counseling prior to ( surgical ) interventions in oncology . since the 1990s , body image research has increased enormously within ( clinical ) psychology and psychiatry . having its origin in psychological , neurological , psycho - analytic , and phenomenological - existential studies of the way in which people experience their own body [ 20 , 36 , 45 , 47 ] , the concept very often the term body image is associated with the simple idea of how the body looks like. psychologists , on the other hand , claim that it is a complex term which refers to the multifaceted experience of embodiment . although my primary interest in this paper concerns the usage of body image in ( medical ) psychology , for the sake of clarity , i will first briefly rehearse some conceptual distinctions made in contemporary philosophy . from a philosophical perspective , body image refers to a certain form and level of consciousness , i.e. the way one s own body is an body image as a system of perceptions , attitudes , and beliefs pertaining to one s own body ( p. 24 ) . this system requires a certain degree of consciousness , albeit often implicit or pre - reflective . indeed , very often i am not fully aware of how i perceive my own body . body schema which , according to gallagher , refers to a system of sensory - motor capacities that function without awareness or the necessity of perceptual monitoring the body schema operates in a close - to - automatic way , e.g. if i reach out for a glass , my hand shapes itself for picking up the glass . the distinction between body image and body schema is helpful in specifying ( pre)conscious and unconscious aspects in the experience of one s own body . normally , body image and body schema work together and form one integrated system.4 the philosophical interest in the notion of body image is particularly related to questions of consciousness or , more specifically , the relation between unconscious processes and ( pre ) conscious experiences . it is primarily interested in dimensions of ( bodily ) experience , leaving to one side questions concerning the degree of consciousness involved in these experiences . medical psychologists that test body image , using standard questionnaires , presuppose that body image involves a totally conscious experience of one s own body . indeed , self - report scales presuppose that participants are aware of what is being tested . compared to its use in philosophical discussion , the concept of body image in psychology has thus been narrowed down to a simple conscious experience of one s body . also , because of its focus on body experience and behavior , and less attention on underlying sensory - motor capacities , psychology deals exclusively with body image , leaving aside the body schema system . in the psychological practice of clinical trials , this narrow concept of body image is considered to be sufficient when testing bodily experience after medical interventions . in the last section of this paper , however , where i return to the phenomenon of the body image s preconscious edges , i will maintain that medical practice will benefit from a broader concept of body image . as i see it , it is by means of a narrative approach which allows a quest for giving voice to both explicit and implicit bodily experiences instead of a pure cognitive - psychological one , that professionals are able to articulate and interpret one s body image , including its less conscious dimensions and its temporal course . before criticizing contemporary psychological usage of body image , i will first explain and summarize it : body image involves a conscious experience of one s own body , which , nevertheless , does not simply refer to one s physical body . , i will specify the meaning of both ( 1 ) physical self and ( 2 ) relation.in psychological literature , physical self refers to one s appearance , one s physical competence , and one s physical intactness . this seems to be a rather straightforward definition , yet it includes different aspects of embodiment : whereas appearance has to do with one s own body as a certain object of perception , competence is directly related to motor intentionality and capacity and thus involves one s body as an agent . the notion of physical intactness can refer to both biological and functional intactness of one s body , and can thus include one s body as both object and agent . as i will discuss in the remainder of this paper , it is with regard to the idea of physical intactness in particular that confusion can arise . psychologists , exploring quality of life and body image in cancer survivors , sometimes use the term integrity to refer to the body s functional or biological intactness . , they , perhaps unwittingly , follow the biomedical discourse which makes no distinction between physical intactness and bodily integrity. i find this usage of the term bodily integrity inadequate . in my view , bodily integrity implies a relation to one s physical body ( which i will explain as a relation through identification ) . if one omits the relational aspect in body integrity one overlooks its normative dimension.according to psychological literature , the relation to one s physical self in body image can be ( a ) perceptual or ( b ) attitudinal.the perceptual component of body image refers to awareness of one s own body . this aspect is often associated with the representations of somatic sensations in one s brain , which has indeed resulted in a more neuro - scientific connotation of , the perceptual aspect refers to the ability to evaluate accurately the size , shape and position of one s body . thus examination of eating disorders such as anorexia sometimes involves assessment of the perceptual body image [ 38 , 55 ] . explicit disturbances of the perceptual body image include the experience of a phantom limb [ 42 , 45 ] or experimentally evoked illusory experiences such as the rubber hand illusion [ 6 , 49 ] . although some ( rare ) cases of phantom breast sensations after mastectomy have been reported , the assessment of perceptual components is not a central issue in body image research in cancer survivors . it rather focuses on the attitudinal aspects involved in the phenomenon of body image.the attitudinal relation to one s physical self can imply cognitive , affective and behavioral components . the affective component refers to the way one feels about one s body ( ashamed , embarrassed , disappointed , satisfied , proud , etc . ) . the valuation of one s own body can also be related to typical behavior , such as avoiding certain situations . although these three components are related to one another , there can be a dissonance between them . people with negative thoughts ( cognitive ) and feelings ( affective ) about their body , for instance , do not necessarily avoid mirrors . in psychological literature , physical self refers to one s appearance , one s physical competence , and one s physical intactness . this seems to be a rather straightforward definition , yet it includes different aspects of embodiment : whereas appearance has to do with one s own body as a certain object of perception , competence is directly related to motor intentionality and capacity and thus involves one s body as an agent . the notion of physical intactness can refer to both biological and functional intactness of one s body , and can thus include one s body as both object and agent . as i will discuss in the remainder of this paper , it is with regard to the idea of physical intactness in particular that confusion can arise . psychologists , exploring quality of life and body image in cancer survivors , sometimes use the term integrity to refer to the body s functional or biological intactness . , they , perhaps unwittingly , follow the biomedical discourse which makes no distinction between physical intactness and bodily integrity. i find this usage of the term bodily integrity inadequate . in my view , bodily integrity involves more than biological and functional intactness . like body image , bodily integrity implies a relation to one s physical body ( which i will explain as a relation through identification ) . according to psychological literature , the relation to one s physical self in body image can be ( a ) perceptual or ( b ) attitudinal.the perceptual component of body image refers to awareness of one s own body . this aspect is often associated with the representations of somatic sensations in one s brain , which has indeed resulted in a more neuro - scientific connotation of body image . at a psychological level , the perceptual aspect refers to the ability to evaluate accurately the size , shape and position of one s body . thus examination of eating disorders such as anorexia sometimes involves assessment of the perceptual body image [ 38 , 55 ] . explicit disturbances of the perceptual body image include the experience of a phantom limb [ 42 , 45 ] or experimentally evoked illusory experiences such as the rubber hand illusion [ 6 , 49 ] . although some ( rare ) cases of phantom breast sensations after mastectomy have been reported , the assessment of perceptual components is not a central issue in body image research in cancer survivors . it rather focuses on the attitudinal aspects involved in the phenomenon of body image.the attitudinal relation to one s physical self can imply cognitive , affective and behavioral components . the affective component refers to the way one feels about one s body ( ashamed , embarrassed , disappointed , satisfied , proud , etc . ) . the valuation of one s own body can also be related to typical behavior , such as avoiding certain situations . although these three components are related to one another , there can be a dissonance between them . people with negative thoughts ( cognitive ) and feelings ( affective ) about their body , for instance , do not necessarily avoid mirrors . this aspect is often associated with the representations of somatic sensations in one s brain , which has indeed resulted in a more neuro - scientific connotation of , the perceptual aspect refers to the ability to evaluate accurately the size , shape and position of one s body . thus examination of eating disorders such as anorexia sometimes involves assessment of the perceptual body image [ 38 , 55 ] . explicit disturbances of the perceptual body image include the experience of a phantom limb [ 42 , 45 ] or experimentally evoked illusory experiences such as the rubber hand illusion [ 6 , 49 ] . although some ( rare ) cases of phantom breast sensations after mastectomy have been reported , the assessment of perceptual components is not a central issue in body image research in cancer survivors . the attitudinal relation to one s physical self can imply cognitive , affective and behavioral components . the affective component refers to the way one feels about one s body ( ashamed , embarrassed , disappointed , satisfied , proud , etc . ) . the valuation of one s own body can also be related to typical behavior , such as avoiding certain situations . although these three components are related to one another , there can be a dissonance between them . people with negative thoughts ( cognitive ) and feelings ( affective ) about their body , for instance , do not necessarily avoid mirrors . body image disturbances in cancer survivors are predominantly caused by actual changes in appearance , though it should be noted that the actual diagnosis cancer , and even the mere knowledge that one is the carrier of a gene mutation which increases the risk of breast cancer , may affect one s thoughts and feelings about one s body . in what follows , i will concentrate on the impact ( expected ) physical damage can have on the way one experiences one s own body , and will limit myself to cases of disfiguring breast cancer.5 the european organization for research and treatment of cancer ( eortc ) quality of life study group takes responsibility for the development and validation of specific qol self - report scales for various cancers . next to the qol - breast cancer questionnaire ( eortc - qlq - br23 ) , it has developed a body image scale for ( breast ) cancer patients ( bis ) . apart from literature review and discussions with health care professionals , this 10-item scale is , based upon extensive interviews ( and subsequent debriefing interviews ) with breast cancer patients . it is designed to provide a quick impression of the intensity of a patient s distress : ranging from 0 to 30 , with 0 representing no distress and higher scores representing increasing distress . it has often been claimed that quantitative evaluations of patients experiences are incomplete ( cf . [ 13 , 21 ] ) . although i share the view that quantitative tools might be limited , i have no intention to criticize quantitative research as such . the question whether experience in itself is quantifiable or not is not my concern here . for this purpose , i reproduce the bis here and , subsequently , i will single out some issues concerning language to judge its adequacy.bis : in this questionnaire you will be asked how you feel about your appearance , and about changes that may have resulted from your disease or treatment . please read each item carefully , and place a firm tick on the line alongside the reply which comes closest to the way you have been feeling about yourself , during the past week.1 . have you been feeling less feminine / masculine as a result of your disease or treatment?65 . have you been feeling less sexually attractive as a result of your disease or treatment?7 . have you been dissatisfied with the appearance of your scar?.reply ratings : not at all ( score 0)a little ( score 1)quite a bit ( score 2)very much ( score 3)hopwood et al . bis : in this questionnaire you will be asked how you feel about your appearance , and about changes that may have resulted from your disease or treatment . please read each item carefully , and place a firm tick on the line alongside the reply which comes closest to the way you have been feeling about yourself , during the past week . have you been feeling less feminine / masculine as a result of your disease or treatment?6 5 . have you been feeling less sexually attractive as a result of your disease or treatment ? 7 . quite a bit ( score 2 ) to begin with , it is notable that nearly all questions are phrased in a negative way . up to a certain extent , it is understandable that the questions are phrased in this way ; debriefing interviews have made clear that most patients normally do not describe themselves in terms like feeling sexually attractive [ 27 , p. 191 ] . this might be true , yet the restriction to negative phrased questions precludes a fully fledged account of body experiences , since it forces informants to negative formulations only . what does it mean if a woman replies not at all to the question do you find it difficult to look at yourself naked? of course , it means that she has no difficulty with her own appearance . but this reply gives no information about the possibility of this woman being content ( or perhaps surprised or fascinated ) while looking at herself . conversely , a negative reply to a positive phrased question does not simply correspond with a genuine negative experience . consequently , a body image scale which is limited to either negative or positive phrasings does not adequately assess the wide range of possible body experiences . if one really aims at gaining insight into the experience of changes of appearance in cancer survivors , it is advisable to complement quantitative questionnaires with some open question . my second objection with this efficient and concise body image scale body image is not the same as the physical body , rather it implies a relation to it ; it comprises the way one experiences and values one s body . if we look at item ( 8) from the bis , for instance , we see that it is rather difficult to take into account clearly the relation between physical self and the experience of this physical self in such a briefly formulated question . the issue is about the feeling of bodily wholeness : have you been feeling the treatment has left your body less whole? one can imagine that respondents might have difficulties in understanding what exactly the examiner wants to know . given the fact that most of the questioned population literally has lost a body part this item may seem redundant . however , this item is not about physical loss in itself . it rather interrogates how a physical defect is experienced . as such it gives the respondents the possibility to express their own feeling of loss . not everybody experiences the loss of a body part in the same way , and it is even possible to retain one s feeling of wholeness after a mastectomy . the emphasis should thus be put upon feeling. it is questionable , however , whether the relation and distinction between one s physical loss and one s feelings about it are adequately formulated here . these problems in formulation are brought out even clearer if we look at the french translation of this item : avez vous ressenti le traitement comme une atteinte votre corps ou une mutilation .7 literally translated back into english it reads as follows did you feel that the treatment has injured or mutilated your body? assuming that most breast surgery in actual fact results in a greater or lesser degree of mutilation , this question is rather ambiguous . indeed , it is hard to imagine that a breast amputee will reply not at all to this question . and yet , it is well possible that these patients may experience their ( amputated ) body as whole . we thus need another approach to make explicit the experience of wholeness ( or lost wholeness ) in people who have to live with a body that from a biological perspective is no longer whole . that body image does not simply coincide with one s physical appearance is clear in cases in which a person may have an extremely negative feeling or attitude towards her or his body despite an unchanged or such is the case in body dismorphic disorder , body integrity identity disorder , and some eating disorders . since body image disturbances in cancer survivors are caused by changes in one s physical appearance , intactness ( and sometimes one s competence ) , one might be tempted to believe that there is a direct correlation between the degree of physical mutilation and the degree of body image dissatisfaction . indeed , if we look at various studies on body image and breast cancer , we see that the tendency to find correlations between physical mutilation and negative body image is one that is inherent to the design of such studies , e.g. studies that suggest that patients who received breast conservation have a better body image than those who received a mastectomy [ 2 , 19 ] , or that reconstructive surgery may restore a woman s body image . i would like to stress here , once again , that body image does not simply imply a coincidence with the physical body , i.e. neither with the body as a physical object ( appearance , biological intactness ) , nor with the body as an agent ( competence , functional intactness).8 body image involves the valuation of these aspects of embodiment . the incautious equation of physical self with its valuation is even more apparent in medical practice . for physicians , who predominantly operate from a biomedical perspective , it is almost taken for granted that the restoration of physical wholeness results in a ( more ) positive valuation of one s physical self . it is therefore not surprising that some physicians even suggest that all women who have to undergo a breast amputation , should be offered the option of a breast reconstruction if their physical condition allows it . without denying that the actual state of one s physical body can be crucial for the way one experiences and valuates it , i would like to emphasize here that the restoration of physical wholeness does not automatically yield the experience of bodily wholeness . sometimes women even experienced regret concerning their choice for breast reconstruction , especially when they were not able to value the physical restoration of their body in a positive way . it is not my intention to trivialize the importance of a physical intact ( female ) body . in that sense my view differs from radical feminist conceptions which criticize the practice of breast reconstruction and insist that women should not allow themselves to be seduced by the idea of bodily wholeness which is a construction of male biology and medicine [ 35 , 52 ] . like the biomedical discourse , this feminist discourse also implies a limited and one - sided normative claim . to facilitate good decision making , and thus good care , one should suspend both these discourses to concentrate first of all on patients embodied experiences . since the fixing of damaged bodies does not always simply imply the restoration of a patient s positive valuation of her body , health care practices that focus solely on normalizing interventions , such as a breast reconstruction , do not always show respect for a patient s bodily integrity . as a matter of fact , these types of interventions , however benevolently intended , may even violate a patient s bodily integrity . to justify this view on how medical professionals should take care of being related to one s physical self presupposes a process of identification , and that the extent to which this process is successful is crucial for one s bodily integrity . no doubt , the notion of bodily integrity i would like to propose here differs considerably from current normative conceptions in bioethics . leaving aside the details of various studies on bodily integrity in health practice , i believe that my idea of integrity differs in at least two ways . first , in most current studies on bodily integrity , the emphasis is placed on the question of whether it is morally desirable to intrude , be it medically , cosmetically or ritually , upon an ( intact ) inviolable but vulnerable body . for that reason , bodily integrity is mainly discussed in issues like organ donation , circumcision , and self - determination in sexual reproduction . as far as i know , it has never been an issue in the care and treatment of bodies that are no longer intact , such as in oncology . second , very often bodily integrity is understood as a pre - conceived idea of wholeness , i.e. wholeness according to biology , or wholeness according to a religious or moral system , or according to a certain ideology . in contrast with this , i consider bodily integrity first of all as an experience of wholeness . as i have explained in - depth elsewhere , the experience of bodily wholeness , including its underlying process of identification , can be understood on the basis of a phenomenological understanding of embodiment . in that sense , drawing on merleau - ponty s philosophy , she maintains that not just any physical change will threaten the integrity of the body - subject . rather , a physical change affects one s integrity only if it perturbs one s being and engaging in the world . i concur with this merleau - pontian view , yet , i would like to add that one s integrity may still be disrupted even in cases where one s functional being in the world is not really affected . indeed , merleau - ponty s philosophy of the body concentrates on motor intentionality and one s functional engaging in the world the embodied subject as i can ( je peux ) and , accordingly , he analyzes ( pathological ) cases in which this bodily potentiality is impaired . but he does not teach us much about changes in physical appearance that do not affect one s physical functioning , but which may nonetheless disrupt one s experience of wholeness and integrity . instead of focusing on the phenomenological idea of being bodily engaged in the world this experience is so typical since it is double - sided or , to phrase it slightly differently , one can experience one s own body according to two different modes of experience : as an object ( or krper ) and as a subject ( or leib ) ; or as corps objectif and as corps propre or vcu , a lived body which is one s own . it is especially because of the leib - experience that the experience of one s own body differs from the perception of other things or objects . the leib - experience is a localized lived - through experience of oneself , for instance , when in touching my left hand i have the feeling that i am touched , that it is my body that is touched . it is often claimed that contemporary medicine considers patients bodies exclusively as objects , totally ignoring their leib - experiences [ 31 , 56 ] . as i see it , an experience of wholeness or integrity implies that both experiences of one s own body concur with one another . these two experiences of one s own body could also be seen as the experience of having ( krper ) and being ( leib ) one s body . in the experience of having a body , one is distanced form oneself and does not coincide with oneself . in this experience , it is especially through the sense of vision that one can perceive oneself as a thing . facing one s mirror image one perceives one s own objectivized body , inspecting its various qualities . thing nevertheless as one s own because one identifies with it . to explain the process of identification , it is useful to briefly discuss the phenomenon of one s mirror image . lacan has argued that the recognition of one s own mirror image immediately implies a misrecognition ( mconnaissance ) since the image is not the same as oneself ; it is an externalization or alienation of oneself . identification through mirror recognition therefore does not simply mean x = x , but rather x = y : i am my mirror image though i do not totally coincide with it . according to psychoanalysis , identification not only indicates an equation of myself and my image , but also involves assimilation into it ; one desires to be like one s image . this latter aspects gains ascendency if we realize that one s own mirror image is directly related to one s visibility for others.9 identification through mirror recognition , therefore , equally implies a desire to assimilate to dominant ideals in one s society and culture , to respond to the ( normative ) gaze of others . for that reason , the process of self - identification is not an isolated , individual psychological process . in this paper , i will limit myself , nonetheless , to a discussion of identification on a personal , subjective level , so as not to overcomplicate my argument.10 although visible and imaginary aspects are important in the process of identification , i prefer to describe this process more generally as the possibility of being the body one has . i do so because not all experiences of one s body as a thing ( or krper ) are directly related to the sense of vision . a lame or sleeping limb , for instance , is a clear krper experience in which no vision is involved . as previously mentioned , in most cases in which one experiences one s body ( or parts of it ) as krper , one is nevertheless capable of identifying with it . whereas the condition of a sleeping limb may temporarily and slightly interrupt this process , other conditions , such as paralysis and amputation , may disturb it in a more profound way . these sensations normally produce the experience of mine - ness and here - ness which are typical for the leib experience . the cancelation of this experience in these conditions is due to neurological problems . whether one is able to be the body one has is , however , not only a neurological question . an extreme example of not being able to be the body one has is body integrity identity disorder ( biid ) . this is a condition in which one experiences one s own limb as something that does not belong to one s body and therefore has to be removed . biid patients are not able to be the body they have , and therefore they can not experience their body as a whole or an integrum . only after amputation they regain an experience of wholeness . this extreme ( psychiatric ) example shows painfully what happens if the process of bodily identification fails . it is not for nothing that it is called a disorder of one s identity and integrity . negative feelings about one s own body after disfiguring breast cancer , obviously , can not be compared to this psychiatric disorder . i believe , however , that breast cancer survivors can also have problems in identifying with their modified body , though they clearly differ from identification failure due to neurological and/or psychiatric conditions . if breast cancer survivors fail to identify positively with their modified body , they value their own body in a negative way . if , conversely , they succeed in being the body they have , it implies that they are able to experience and say : this body i have , however changed ( and mutilated ) it is , is me. it is very unlikely that cancer survivors will express their embodied self - experiences in this way . experiences of wholeness or discrepancy come to light most explicitly in certain feelings and emotions . if a breast cancer survivor is ( extremely ) ashamed of her body , i would say that her experience of bodily integrity is affected . understood in this way , bodily integrity is directly related to body image , and especially to the latter s affective dimension ( although this can never be fully separated from its cognitive and behavioral components ) . indeed a negative body image ( i.e. a negative valuation of one s physical appearance and one s physical intactness ) refers to the lack of a positive identification with one s physical self and , thus , to the loss of bodily integrity . i thus believe that the use of certain body image items can contribute to the articulation of a person s bodily integrity . in contrast with the aim of most body image evaluation the reinforcement of a patient s psychic well - being i suggest that body experience evaluation should not be used for psychological purposes only . examining a patient s experience of bodily integrity , with the help of body image items , can have a practical and ethical purpose as well . the ethical dimension of body experience valuation becomes clear if we look at moral values that are implicated in the notion of bodily integrity . stemming from the latin word integrum and the verb in - tangere , integrity signifies both wholeness and not touching . taken together it thus refers to a wholeness that should not be touched , not be hurt a wholeness that should be respected . applied to medical practices , this means that a patient s body should be treated with respect and prudence . medical professionals who necessarily have to intervene in a patient s body still have to respect as much as possible the integrum of this person s body . from a liberal stance , according to which a person owns his or her own body , bodily integrity more or less converges with the principle of autonomy ; i.e. a health professional may intrude upon a patient s body only if this patient has consented to the intervention . in spite of this , respect for bodily integrity is not exactly the same as respect for autonomy , because one s body is never fully possessed , nor something of which one has total disposal . no matter whether patients have consented to certain interventions , respect for bodily integrity also prescribes that medical professionals should not treat patients bodies as if they were just objects or a collection of organs , tissues and limbs [ 32 , 57 , 68 ] . in this way , bodily integrity is closely linked to human dignity and , to a lesser degree , to the medical ethical principle of nonmaleficence . in accordance with these conceptions , violation of bodily integrity occurs in , for example , the commercialization of human organs , disrespectful nursing and an amputation of the wrong limb . returning to the example of breast cancer , i would like to add something here that enlarges the moral scope of bodily integrity , namely , that benevolent treatments can also violate a person s bodily integrity . indeed , if a breast cancer survivor has received a breast reconstruction which eventually turns out to make no contribution at all to the restoration of her experience of bodily wholeness , the treatment has been an unneeded ( and irreversible ) intrusion . thus far , i have argued that medically successful treatments , even if they have been performed with prudence and professional discretion after patients have consented to it , still may result in the violation of a person s bodily integrity . one could object here that it is not fair to judge treatments with the benefit of hindsight , and that if a physician would have known that a treatment would not have the expected result s / he probably would not have performed it . it is not at all my intention , however , to express disapproval of medical practices such as breast reconstruction . my , perhaps extreme , view on bodily integrity should rather be seen as an invitation to support oncology care practices given that the evaluation of bodily wholeness could , i believe , be employed in assessment prior to interventions . body experience evaluation , which aims at the articulation of a patient s actual experience of bodily wholeness , can help patients ( who have to decide about an intervention that will change their body ) to weigh up their situation more clearly . deliberately considering their own embodied experiences ( with the help of the professionals involved ) , may enable patients to gain a more complete idea of the impact of various interventions . as such it can also be seen as making an important contribution in helping those involved arrive at a more informed decision . one may again raise an objection here : how could the evaluation of a person s body experience prior to intervention envisage how this person will valuate her or his body after the intervention ? of course , one can never fully foresee how a change in physical appearance and intactness will be experienced but this , in itself , does not leave the suggestion i am making here that explicit awareness of the way one actually values one s body may be useful in weighing up different alternatives any less compelling . i find this so mainly because the valuation of one s body one s body image is not something isolated . rather , it is directly related to what is valuable and meaningful for this person in his or her life . a person s long - term body image , which does not necessarily stay the same all the time , parallels a person s life story . body experience evaluation which aims at envisaging the impact of future interventions should thus take place against the background of a patient s narrative . endorsing the hermeneutical idea that experiences express themselves in stories [ 44 , 65 ] , i maintain that the interpretation of lived experience necessarily involves an interpretation of stories . also , the narrative approach can take into account the temporal aspect of the way one experiences one s own body . this temporal and narrative aspect of body image is hard , if not impossible , to frame in a quantitative evaluation tool . indeed , measuring a specific bodily experience at a certain point in time does not provide much information about how valuation of one s body is formed over time . body image is not an instant experience of one s body , and this is the reason that the actual valuation of one s body is not detached from a person s future . given this , it is not totally impossible to estimate , on the basis of actual body experience valuation , how someone will eventually valuate a certain change in physical appearance , intactness and competence . it is too nave to maintain that a story or narrative is a simple representation of one s experiences . the way one experiences one s body one s body image is not always completely transparent and conscious for one self . expressing one s body experiences thus already implies a process of self - interpretation . in this sense , the telling of a story is not only about what was experienced , but equally about what becomes experience in the telling and its reception . in this process of interpretation and story telling , health care professionals can play an important role . in a practice of so - called joint narrative work , medical professionals should not only listen to stories , but also help to build them while interpreting them . this is especially so in stressful situations , such as the fearful period after having being diagnosed with a ( life - threatening ) cancer and during which one has to make far - reaching decisions in a short space of time . here patients may have totally lost track , and may have great difficulties in articulating how they experience and value their own body . following frank s distinction between chaos and quest narratives , i believe that aspects of quest narratives will be especially helpful here . quest narratives are not about triumphing over one s illness ( restitution narrative ) , nor simply about being sucked into one s illness ( chaos ) . in a quest narrative , the narrator is constantly searching for a way to deal with his or her condition . the quest narrative is indeed the story of the patient who is telling the story and not just about remedy ( restitution narrative ) or suffering in which the teller has lost his or her voice . a quest narrative , remaining open and never ending let alone happilyaffords the ill person a voice as teller of her own story . obviously , counseling prior to interventions is not the place to build extensive life stories . the only thing i would like to suggest here is that counseling could also include a short but joint orientation of what is important for a patient in his or her life with a special focus on how this patient values his or her physical appearance and intactness.11 needless to say , to develop ( and implement ) this type of assessment , further research is required . to increase health care professionals sensitivity to the impact of body experience in health care practice , and to provide them with some practical aids to thematize this experience , it should first of all be examined how patients , both before and after treatment , put into words their experience of ( loss of ) bodily wholeness . these specific expressions can serve as a basis for prospective joint narrative work and can support health care professionals to initiate the process of jointly constructing the patient s body story. future research , therefore , needs to aim at the development of a rich , yet workable , vocabulary of patients body experiences .

evaluation of quality of life , psychic and bodily well - being is becoming increasingly important in oncology aftercare . this type of assessment is mainly carried out by medical psychologists . in this paper i will seek to show that body experience valuation has , besides its psychological usefulness , a normative and practical dimension . body experience evaluation aims at establishing the way a person experiences and appreciates his or her physical appearance , intactness and competence . this valuation constitutes one s body image. while , first , interpreting the meaning of body image and , second , indicating the limitations of current psychological body image assessment , i argue that the normative aspect of body image is related to the experience of bodily wholeness or bodily integrity . since this experience is contextualized by a person s life story , evaluation should also focus on narrative aspects . i finally suggest that the interpretation of body experience is not only valuable to assess a person s quality of life after treatment , but that it can also be useful in counseling prior to interventions , since it can support patients in making decisions about interventions that will change their bodies . to apply this type of evaluation to oncology practice , a rich and tailored vocabulary of body experiences has to be developed .

Introduction Body Image: Being Related to Ones Physical Self Evaluation of Body Experience in Breast Cancer Survivors From Body Image to Bodily Integrity Body Experience Evaluation Prior to Intervention

given that the disease is now often treatable and has become comparatively manageable , it is no longer considered a although oncologic safety , obviously , remains the primary focus , more and more attention is paid to quality of life related issues in the treatment and nursing care of cancer patients . these may result in functional disturbances such as problems with speaking and swallowing ( in the case of head and neck cancer ) or limited motility of the upper limb ( in the case of breast cancer ) which , evidently , can be important for a person s quality of life . these changes in appearance , which can not simply be reduced to physical function or dysfunction , may affect a person s valuation of his or her body and thus change a person s body image. it is now generally assumed that body image related issues are essential to a person s quality of life [ 27 , 41].1 in contemporary ( psycho-)oncology attention to body image issues is increasing [ 1 , 8 , 14 , 16 , 63 , 64 ] . these questionnaires are mainly used to evaluate treatment in clinical trials and sometimes to support psychotherapeutic interventions.2 in the context of the increasing interest for body image related issues in oncology , i would like to suggest that body experience evaluation is valuable not only from a psychological perspective . my hypothesis is that body experience evaluation can play an important role in health care practices , especially in these kinds of practices where patients have to decide ( together with the medical professionals involved ) about interventions which will affect their physical appearance . in this paper , i would like to make visible the practical and ethical dimension of body experience evaluation in oncology , mainly concentrating on breast cancer . i will do so by means of a conceptual and phenomenological analysis of body image and bodily integrity . first , i will briefly explain the meaning of what is called body image , drawing on philosophical and psychological literature.3 second , i will discuss how body image is currently evaluated in breast cancer patients . i will do so by focusing on the way questions are phrased on the body image scale ( bis ) questionnaire and pointing to its inherent limitations . third , i will introduce the notions of bodily integrity and identification and will explain how these may be useful for understanding the impact of body experience evaluation in health care practice . in the final section , i will argue in favor of the application of body experience evaluation in counseling prior to ( surgical ) interventions in oncology . having its origin in psychological , neurological , psycho - analytic , and phenomenological - existential studies of the way in which people experience their own body [ 20 , 36 , 45 , 47 ] , the concept very often the term body image is associated with the simple idea of how the body looks like. psychologists , on the other hand , claim that it is a complex term which refers to the multifaceted experience of embodiment . although my primary interest in this paper concerns the usage of body image in ( medical ) psychology , for the sake of clarity , i will first briefly rehearse some conceptual distinctions made in contemporary philosophy . the way one s own body is an body image as a system of perceptions , attitudes , and beliefs pertaining to one s own body ( p. 24 ) . the distinction between body image and body schema is helpful in specifying ( pre)conscious and unconscious aspects in the experience of one s own body . normally , body image and body schema work together and form one integrated system.4 the philosophical interest in the notion of body image is particularly related to questions of consciousness or , more specifically , the relation between unconscious processes and ( pre ) conscious experiences . medical psychologists that test body image , using standard questionnaires , presuppose that body image involves a totally conscious experience of one s own body . compared to its use in philosophical discussion , the concept of body image in psychology has thus been narrowed down to a simple conscious experience of one s body . also , because of its focus on body experience and behavior , and less attention on underlying sensory - motor capacities , psychology deals exclusively with body image , leaving aside the body schema system . in the psychological practice of clinical trials , this narrow concept of body image is considered to be sufficient when testing bodily experience after medical interventions . in the last section of this paper , however , where i return to the phenomenon of the body image s preconscious edges , i will maintain that medical practice will benefit from a broader concept of body image . as i see it , it is by means of a narrative approach which allows a quest for giving voice to both explicit and implicit bodily experiences instead of a pure cognitive - psychological one , that professionals are able to articulate and interpret one s body image , including its less conscious dimensions and its temporal course . before criticizing contemporary psychological usage of body image , i will first explain and summarize it : body image involves a conscious experience of one s own body , which , nevertheless , does not simply refer to one s physical body . , i will specify the meaning of both ( 1 ) physical self and ( 2 ) relation.in psychological literature , physical self refers to one s appearance , one s physical competence , and one s physical intactness . this seems to be a rather straightforward definition , yet it includes different aspects of embodiment : whereas appearance has to do with one s own body as a certain object of perception , competence is directly related to motor intentionality and capacity and thus involves one s body as an agent . as i will discuss in the remainder of this paper , it is with regard to the idea of physical intactness in particular that confusion can arise . psychologists , exploring quality of life and body image in cancer survivors , sometimes use the term integrity to refer to the body s functional or biological intactness . , they , perhaps unwittingly , follow the biomedical discourse which makes no distinction between physical intactness and bodily integrity. it rather focuses on the attitudinal aspects involved in the phenomenon of body image.the attitudinal relation to one s physical self can imply cognitive , affective and behavioral components . the affective component refers to the way one feels about one s body ( ashamed , embarrassed , disappointed , satisfied , proud , etc . ) the valuation of one s own body can also be related to typical behavior , such as avoiding certain situations . this seems to be a rather straightforward definition , yet it includes different aspects of embodiment : whereas appearance has to do with one s own body as a certain object of perception , competence is directly related to motor intentionality and capacity and thus involves one s body as an agent . psychologists , exploring quality of life and body image in cancer survivors , sometimes use the term integrity to refer to the body s functional or biological intactness . , they , perhaps unwittingly , follow the biomedical discourse which makes no distinction between physical intactness and bodily integrity. like body image , bodily integrity implies a relation to one s physical body ( which i will explain as a relation through identification ) . according to psychological literature , the relation to one s physical self in body image can be ( a ) perceptual or ( b ) attitudinal.the perceptual component of body image refers to awareness of one s own body . this aspect is often associated with the representations of somatic sensations in one s brain , which has indeed resulted in a more neuro - scientific connotation of body image . at a psychological level , the perceptual aspect refers to the ability to evaluate accurately the size , shape and position of one s body . it rather focuses on the attitudinal aspects involved in the phenomenon of body image.the attitudinal relation to one s physical self can imply cognitive , affective and behavioral components . the affective component refers to the way one feels about one s body ( ashamed , embarrassed , disappointed , satisfied , proud , etc . ) the valuation of one s own body can also be related to typical behavior , such as avoiding certain situations . explicit disturbances of the perceptual body image include the experience of a phantom limb [ 42 , 45 ] or experimentally evoked illusory experiences such as the rubber hand illusion [ 6 , 49 ] . the affective component refers to the way one feels about one s body ( ashamed , embarrassed , disappointed , satisfied , proud , etc . ) the valuation of one s own body can also be related to typical behavior , such as avoiding certain situations . body image disturbances in cancer survivors are predominantly caused by actual changes in appearance , though it should be noted that the actual diagnosis cancer , and even the mere knowledge that one is the carrier of a gene mutation which increases the risk of breast cancer , may affect one s thoughts and feelings about one s body . in what follows , i will concentrate on the impact ( expected ) physical damage can have on the way one experiences one s own body , and will limit myself to cases of disfiguring breast cancer.5 the european organization for research and treatment of cancer ( eortc ) quality of life study group takes responsibility for the development and validation of specific qol self - report scales for various cancers . for this purpose , i reproduce the bis here and , subsequently , i will single out some issues concerning language to judge its adequacy.bis : in this questionnaire you will be asked how you feel about your appearance , and about changes that may have resulted from your disease or treatment . this might be true , yet the restriction to negative phrased questions precludes a fully fledged account of body experiences , since it forces informants to negative formulations only . consequently , a body image scale which is limited to either negative or positive phrasings does not adequately assess the wide range of possible body experiences . my second objection with this efficient and concise body image scale body image is not the same as the physical body , rather it implies a relation to it ; it comprises the way one experiences and values one s body . that body image does not simply coincide with one s physical appearance is clear in cases in which a person may have an extremely negative feeling or attitude towards her or his body despite an unchanged or such is the case in body dismorphic disorder , body integrity identity disorder , and some eating disorders . since body image disturbances in cancer survivors are caused by changes in one s physical appearance , intactness ( and sometimes one s competence ) , one might be tempted to believe that there is a direct correlation between the degree of physical mutilation and the degree of body image dissatisfaction . indeed , if we look at various studies on body image and breast cancer , we see that the tendency to find correlations between physical mutilation and negative body image is one that is inherent to the design of such studies , e.g. studies that suggest that patients who received breast conservation have a better body image than those who received a mastectomy [ 2 , 19 ] , or that reconstructive surgery may restore a woman s body image . i would like to stress here , once again , that body image does not simply imply a coincidence with the physical body , i.e. without denying that the actual state of one s physical body can be crucial for the way one experiences and valuates it , i would like to emphasize here that the restoration of physical wholeness does not automatically yield the experience of bodily wholeness . in that sense my view differs from radical feminist conceptions which criticize the practice of breast reconstruction and insist that women should not allow themselves to be seduced by the idea of bodily wholeness which is a construction of male biology and medicine [ 35 , 52 ] . to justify this view on how medical professionals should take care of being related to one s physical self presupposes a process of identification , and that the extent to which this process is successful is crucial for one s bodily integrity . as i have explained in - depth elsewhere , the experience of bodily wholeness , including its underlying process of identification , can be understood on the basis of a phenomenological understanding of embodiment . i concur with this merleau - pontian view , yet , i would like to add that one s integrity may still be disrupted even in cases where one s functional being in the world is not really affected . but he does not teach us much about changes in physical appearance that do not affect one s physical functioning , but which may nonetheless disrupt one s experience of wholeness and integrity . instead of focusing on the phenomenological idea of being bodily engaged in the world this experience is so typical since it is double - sided or , to phrase it slightly differently , one can experience one s own body according to two different modes of experience : as an object ( or krper ) and as a subject ( or leib ) ; or as corps objectif and as corps propre or vcu , a lived body which is one s own . it is especially because of the leib - experience that the experience of one s own body differs from the perception of other things or objects . the leib - experience is a localized lived - through experience of oneself , for instance , when in touching my left hand i have the feeling that i am touched , that it is my body that is touched . these two experiences of one s own body could also be seen as the experience of having ( krper ) and being ( leib ) one s body . lacan has argued that the recognition of one s own mirror image immediately implies a misrecognition ( mconnaissance ) since the image is not the same as oneself ; it is an externalization or alienation of oneself . according to psychoanalysis , identification not only indicates an equation of myself and my image , but also involves assimilation into it ; one desires to be like one s image . this latter aspects gains ascendency if we realize that one s own mirror image is directly related to one s visibility for others.9 identification through mirror recognition , therefore , equally implies a desire to assimilate to dominant ideals in one s society and culture , to respond to the ( normative ) gaze of others . in this paper , i will limit myself , nonetheless , to a discussion of identification on a personal , subjective level , so as not to overcomplicate my argument.10 although visible and imaginary aspects are important in the process of identification , i prefer to describe this process more generally as the possibility of being the body one has . i do so because not all experiences of one s body as a thing ( or krper ) are directly related to the sense of vision . this is a condition in which one experiences one s own limb as something that does not belong to one s body and therefore has to be removed . it is not for nothing that it is called a disorder of one s identity and integrity . if a breast cancer survivor is ( extremely ) ashamed of her body , i would say that her experience of bodily integrity is affected . understood in this way , bodily integrity is directly related to body image , and especially to the latter s affective dimension ( although this can never be fully separated from its cognitive and behavioral components ) . a negative valuation of one s physical appearance and one s physical intactness ) refers to the lack of a positive identification with one s physical self and , thus , to the loss of bodily integrity . i thus believe that the use of certain body image items can contribute to the articulation of a person s bodily integrity . in contrast with the aim of most body image evaluation the reinforcement of a patient s psychic well - being i suggest that body experience evaluation should not be used for psychological purposes only . examining a patient s experience of bodily integrity , with the help of body image items , can have a practical and ethical purpose as well . the ethical dimension of body experience valuation becomes clear if we look at moral values that are implicated in the notion of bodily integrity . from a liberal stance , according to which a person owns his or her own body , bodily integrity more or less converges with the principle of autonomy ; i.e. in spite of this , respect for bodily integrity is not exactly the same as respect for autonomy , because one s body is never fully possessed , nor something of which one has total disposal . in this way , bodily integrity is closely linked to human dignity and , to a lesser degree , to the medical ethical principle of nonmaleficence . returning to the example of breast cancer , i would like to add something here that enlarges the moral scope of bodily integrity , namely , that benevolent treatments can also violate a person s bodily integrity . indeed , if a breast cancer survivor has received a breast reconstruction which eventually turns out to make no contribution at all to the restoration of her experience of bodily wholeness , the treatment has been an unneeded ( and irreversible ) intrusion . thus far , i have argued that medically successful treatments , even if they have been performed with prudence and professional discretion after patients have consented to it , still may result in the violation of a person s bodily integrity . my , perhaps extreme , view on bodily integrity should rather be seen as an invitation to support oncology care practices given that the evaluation of bodily wholeness could , i believe , be employed in assessment prior to interventions . body experience evaluation , which aims at the articulation of a patient s actual experience of bodily wholeness , can help patients ( who have to decide about an intervention that will change their body ) to weigh up their situation more clearly . as such it can also be seen as making an important contribution in helping those involved arrive at a more informed decision . one may again raise an objection here : how could the evaluation of a person s body experience prior to intervention envisage how this person will valuate her or his body after the intervention ? of course , one can never fully foresee how a change in physical appearance and intactness will be experienced but this , in itself , does not leave the suggestion i am making here that explicit awareness of the way one actually values one s body may be useful in weighing up different alternatives any less compelling . i find this so mainly because the valuation of one s body one s body image is not something isolated . a person s long - term body image , which does not necessarily stay the same all the time , parallels a person s life story . body experience evaluation which aims at envisaging the impact of future interventions should thus take place against the background of a patient s narrative . endorsing the hermeneutical idea that experiences express themselves in stories [ 44 , 65 ] , i maintain that the interpretation of lived experience necessarily involves an interpretation of stories . this temporal and narrative aspect of body image is hard , if not impossible , to frame in a quantitative evaluation tool . body image is not an instant experience of one s body , and this is the reason that the actual valuation of one s body is not detached from a person s future . given this , it is not totally impossible to estimate , on the basis of actual body experience valuation , how someone will eventually valuate a certain change in physical appearance , intactness and competence . the way one experiences one s body one s body image is not always completely transparent and conscious for one self . expressing one s body experiences thus already implies a process of self - interpretation . in this sense , the telling of a story is not only about what was experienced , but equally about what becomes experience in the telling and its reception . obviously , counseling prior to interventions is not the place to build extensive life stories . the only thing i would like to suggest here is that counseling could also include a short but joint orientation of what is important for a patient in his or her life with a special focus on how this patient values his or her physical appearance and intactness.11 needless to say , to develop ( and implement ) this type of assessment , further research is required . to increase health care professionals sensitivity to the impact of body experience in health care practice , and to provide them with some practical aids to thematize this experience , it should first of all be examined how patients , both before and after treatment , put into words their experience of ( loss of ) bodily wholeness . future research , therefore , needs to aim at the development of a rich , yet workable , vocabulary of patients body experiences .

primary care evidence - based guidelines for non - specific back pain highlight the importance of identifying indicators of poor prognosis in order that treatment can be targeted appropriately ( chou et al . , 2007 ; van tulder et al . , 2006 ) investigators increasingly advocate that better identification of potentially modifiable prognostic indicators may lead to more effective early secondary prevention of back pain in primary care ( boersma and linton , 2005 ; morley and vlaeyen , 2005 ; jellema et al . , 2006 ; koes et al . , 2006 ) . several back pain clinical tools exist to aid clinicians in identifying patients either at risk of chronicity or to improve targeting of treatment ( childs et al . , 2004 ; hicks et al . , 2005 ; dionne et al . , 2005 ; truchon and cote , 2005 ; duijts et al . , 2006 ; the start back tool ( sbt ) is a recently validated tool developed to identify subgroups of patients to guide the provision of early secondary prevention in primary care ( hill et al . , 2008 ) . the conceptual purposes of the sbt ( hill et al . , 2008 ) were to identify patients with potentially treatment modifiable prognostic indicators using a brief , user - friendly tool and to validate cut - off scores for subgrouping patients into 1 of 3 a priori initial treatment options in primary care ( low , medium and high risk groups ) . the development and validation of the start back tool ( sbt ) was recently reported ( hill et al . , 2008 ) , but a head to head comparison with a the rebro musculoskeletal pain screening questionnaire ( mpsq : linton and hallden , 1998 ) was considered the most appropriate reference standard against which to compare the sbt , as it is one of the most widely used tools in clinical practice to similarly differentiate primary care back pain patients , and it has a common conceptual purpose with the sbt of identifying high risk patients requiring targeted treatment . as well as being a popular instrument , , 2008 ; hough et al . , 2007 ; margison and french , 2007 ; nordeman et al . , 2006 ; jellema et al . , 2007 ; gabel et al . , 2008 ; hurley et al . , 2001 ; grotle et al . , 2006 ; linton and boersma , 2003 ; boersma and linton , 2005 ; dunstan et al . , 2005 ) . clinicians wanting to follow evidence - based practice guidelines to assess prognostic indicators for chronicity ( chou et al . , 2007 ) , require brief and practical tools to help them identify subgroups of patients that may require early , targeted secondary prevention pathways . the publication of the sbt ( hill et al . , 2008 ) provides clinicians with a choice , in addition to an existing reference standard instrument , the mpsq . however , in order to decide which clinical tool to use in practice , clinicians need to know how these instruments compare , including the baseline clinical characteristics of the subgroups identified by each tool . the overall aim of this study was to therefore provide a head to head comparison of the sbt s concurrent validity against a best available reference standard , the mpsq . our objectives were to test the correlation between the sbt and mpsq scores and agreement of patients allocated to medium and high risk subgroups ; and to compare the abilities of the sbt and mpsq scales to discriminate patients according to validated reference standard measures . the mpsq consists of 24 self - report items ( 21 items are scored ) , selected following a literature review to identify strong independent risk factors for work absence . poor prognosis as accumulated sick leave of 30 days or more at six months follow up . the mpsq s 21 scored items use an 11-point response format , apart from item 1 ( pain sites ) , which has five descriptive components that are double weighted . the reliability of the mpsq has been reported with a kappa of 0.83 ( linton and hallden , 1998 ) and external validity , with a number of different high risk cut - off scores , has been established in a variety of patient populations and settings ( heneweer et al . , 2007 ; , 2008 ; hough et al . , 2007 ; margison and french , 2007 ; nordeman et al . , 2006 ; jellema et al . , 2007 ; gabel et al . , 2008 ; hurley et al . , 2001 ; grotle et al . , 2006 ; linton and boersma , 2003 ; boersma and linton , 2005 ; dunstan et al . , the predictive validity of the mpsq has also been investigated in a number of studies , as summarised by hockings et al . other authors have used it to classify patients into low , medium and high at risk groups ( nordeman et al . , 2006 ) . poor prognosis following a literature review and secondary analysis to identify strong independent predictors for persistent disabling back pain . disagree ) , apart from one bothersomeness item , which uses a likert scale . overall sbt scores range from 0 - 9 and are produced by summing all positive items ; a psychosocial subscale score ranging from 0 to 5 is produced by summing bothersomeness , fear , catastrophising , anxiety , and depression items ( items 1 , 4 , 7 , 8 , and 9 ) . the predictive validity and external validity of the start back tool has been reported , as well as the sbt s reliability , with a kappa of 0.79 ( hill et al . , 2008 ) . prognostic constructs included in both tools as single screening items include disability , fear avoidance , anxiety , depression and also an item on the patient s perceived chance that current pain may become persistent or that the back pain is never going to get any better. both instruments allocate at risk patients based on the presence or absence of known indicators of poor prognosis . the sbt also discriminates patients using single items for bothersomeness , referred leg pain , and comorbid pain , while the mpsq discriminates on the basis of additional items for pain intensity , coping and work - related factors . potential differences between the mpsq and sbt were explored using data from a cross - sectional survey of consecutive adult patients ( n = 131 ) who consulted their gp with low back pain . the methods of patient recruitment have already been published elsewhere ( hill et al . , 2008 ) . in brief , 244 participants were invited to complete a questionnaire containing both the mpsq and the sbt from 8 general practices in north staffordshire and central cheshire , uk using computerised read codes to identify recent back pain consulters . the sbt and mpsq were scored according to the methods specified by the instrument developers ( hill et al . , 2008 medium and high risk groups using derived cut - off scores for each instrument . for the sbt , cut - offs recommended in the original article were used ( hill et al . , 2008 ) ; for the mpsq , we replicated the high risk cut - off score determined within a uk primary care population by hurley et al . ( 2001 ) of 112 ; and a cut - off of 90 to separate low , from medium risk group patients , as used by nordeman et al . ( 2006 ) . to score the mpsq among patients who were not workers , ( four work - related items are only relevant to those patients in work ) , the procedure used by jellema et al . ( 2007 ) was followed , where the mean score of the remaining 17 items was imputed , on the condition that at least 75% of all items were completed . correlations between the sbt and mpsq , and reference standard constructs included in both instruments : disability ( roland and morris disability questionnaire , rmdq : roland and morris , 1983 ) fear avoidance ( tampa scale of kinesiaphobia , tsk : kori et al . , 1990 ) and catastrophising ( pain catastrophising scale , pcs : sullivan et al . , 1995 ) were calculated using spearman s rank correlations . the magnitude of the reported correlation coefficient was evaluated with a correlation of 0.10.3 was small , > 0.30.5 was moderate , and greater than 0.5 was large ( cohen , 1998 ) . in addition , box and whisker plot graphs were produced to visually present the correlation between the sbt and mpsq.1 observed agreement of the two tool s allocation to medium and high risk subgroups was examined by calculating absolute agreement , and agreement beyond chance statistically evaluated using a weighted cohen s kappa test . kappa values were classified for reference as follows : less than 0.00 showed poor agreement , 0.000.20 , slight agreement ; 0.210.40 , fair agreement ; 0.410.60 , moderate agreement ; 0.610.80 , substantial agreement ; and greater than 0.80 , near perfect agreement ( landis and koch , 1977 ) . the mcnemar bowker test was used to determine whether disagreement observed was evenly balanced or significantly skewed towards the lower or higher group . the characteristics of patients selected for high subgroups using the sbt and mpsq were compared using median scores for age , gender , episode duration , bothersomeness , pain intensity , days off work , fear ( tsk ) , catastrophising ( pcs ) , mpsq scores and sbt scores ( mean values were used for age ) . the mann whitney test was used to test for statistical differences between continuous data and the chi - squared test used for categorical data among the characteristics for discordant individuals ( patients for whom group allocation differed ) . finally , the ability of the two instruments total ( and subscale ) scores to discriminate patients was compared by plotting receiver operator characteristic ( roc ) curves for various different reference standard case definitions provided in table 3 ) and calculating aucs ( 95% cis ) , with statistical differences tested using the wilcoxon statistic ( hanley and mcneil , 1982 ) . the mpsq consists of 24 self - report items ( 21 items are scored ) , selected following a literature review to identify strong independent risk factors for work absence . poor prognosis as accumulated sick leave of 30 days or more at six months follow up . the mpsq s 21 scored items use an 11-point response format , apart from item 1 ( pain sites ) , which has five descriptive components that are double weighted . the reliability of the mpsq has been reported with a kappa of 0.83 ( linton and hallden , 1998 ) and external validity , with a number of different high risk cut - off scores , has been established in a variety of patient populations and settings ( heneweer et al . , 2007 ; , 2008 ; hough et al . , 2007 ; margison and french , 2007 ; nordeman et al . , 2006 ; jellema et al . , 2007 ; gabel et al . , 2008 ; hurley et al . , 2001 ; grotle et al . , 2006 ; linton and boersma , 2003 ; boersma and linton , 2005 ; dunstan et al . , the predictive validity of the mpsq has also been investigated in a number of studies , as summarised by hockings et al . other authors have used it to classify patients into low , medium and high at risk groups ( nordeman et al . , 2006 ) . poor prognosis following a literature review and secondary analysis to identify strong independent predictors for persistent disabling back pain . disagree ) , apart from one bothersomeness item , which uses a likert scale . overall sbt scores range from 0 - 9 and are produced by summing all positive items ; a psychosocial subscale score ranging from 0 to 5 is produced by summing bothersomeness , fear , catastrophising , anxiety , and depression items ( items 1 , 4 , 7 , 8 , and 9 ) . the predictive validity and external validity of the start back tool has been reported , as well as the sbt s reliability , with a kappa of 0.79 ( hill et al . , 2008 ) . prognostic constructs included in both tools as single screening items include disability , fear avoidance , anxiety , depression and also an item on the patient s perceived chance that current pain may become persistent or that the back pain is never going to get any better. both instruments allocate at risk patients based on the presence or absence of known indicators of poor prognosis . the sbt also discriminates patients using single items for bothersomeness , referred leg pain , and comorbid pain , while the mpsq discriminates on the basis of additional items for pain intensity , coping and work - related factors . potential differences between the mpsq and sbt were explored using data from a cross - sectional survey of consecutive adult patients ( n = 131 ) who consulted their gp with low back pain . the methods of patient recruitment have already been published elsewhere ( hill et al . , 2008 ) . in brief , 244 participants were invited to complete a questionnaire containing both the mpsq and the sbt from 8 general practices in north staffordshire and central cheshire , uk using computerised read codes to identify recent back pain consulters . the sbt and mpsq were scored according to the methods specified by the instrument developers ( hill et al . , 2008 ; medium and high risk groups using derived cut - off scores for each instrument . for the sbt , cut - offs recommended in the original article were used ( hill et al . , 2008 ) ; for the mpsq , we replicated the high risk cut - off score determined within a uk primary care population by hurley et al . ( 2001 ) of 112 ; and a cut - off of 90 to separate low , from medium risk group patients , as used by nordeman et al . ( 2006 ) . to score the mpsq among patients who were not workers , ( four work - related items are only relevant to those patients in work ) , the procedure used by jellema et al . ( 2007 ) was followed , where the mean score of the remaining 17 items was imputed , on the condition that at least 75% of all items were completed . correlations between the sbt and mpsq , and reference standard constructs included in both instruments : disability ( roland and morris disability questionnaire , rmdq : roland and morris , 1983 ) fear avoidance ( tampa scale of kinesiaphobia , tsk : kori et al . , 1990 ) and catastrophising ( pain catastrophising scale , pcs : sullivan et al . the magnitude of the reported correlation coefficient was evaluated with a correlation of 0.10.3 was small , > 0.30.5 was moderate , and greater than 0.5 was large ( cohen , 1998 ) . in addition , box and whisker plot graphs were produced to visually present the correlation between the sbt and mpsq.1 observed agreement of the two tool s allocation to medium and high risk subgroups was examined by calculating absolute agreement , and agreement beyond chance statistically evaluated using a weighted cohen s kappa test . kappa values were classified for reference as follows : less than 0.00 showed poor agreement , 0.000.20 , slight agreement ; 0.210.40 , fair agreement ; 0.410.60 , moderate agreement ; 0.610.80 , substantial agreement ; and greater than 0.80 , near perfect agreement ( landis and koch , 1977 ) . the mcnemar bowker test was used to determine whether disagreement observed was evenly balanced or significantly skewed towards the lower or higher group . high subgroups using the sbt and mpsq were compared using median scores for age , gender , episode duration , bothersomeness , pain intensity , days off work , fear ( tsk ) , catastrophising ( pcs ) , mpsq scores and sbt scores ( mean values were used for age ) . the mann whitney test was used to test for statistical differences between continuous data and the chi - squared test used for categorical data among the characteristics for discordant individuals ( patients for whom group allocation differed ) . finally , the ability of the two instruments total ( and subscale ) scores to discriminate patients was compared by plotting receiver operator characteristic ( roc ) curves for various different reference standard case definitions provided in table 3 ) and calculating aucs ( 95% cis ) , with statistical differences tested using the wilcoxon statistic ( hanley and mcneil , 1982 ) . complete data on the sbt and mpsq was available for 130/244 ( 53% ) patients . the sample was 60% female , with a mean ( sd ) age of 44 ( 10.0 ) and disability score ( rmdq ) of 8.6 ( 6.6 ) . back pain episode durations were varied with 25% < 1 month , 27% 16 months , and 48% > 6 months , and 43% reported being the median scores ( range ) for sbt and mpsq were 4.5 ( 09 ) and 98.8 ( 26.3189.0 ) , respectively . the spearman s rank correlation coefficients for the sbt total scores and psychosocial subscale scores with the mpsq scores were 0.802 and 0.769 respectively . large and was similar to the correlation between disability ( rmdq ) and the subgroup tools ( sbt = 0.813 ; mpsq = 0.830 ) . figures for fear ( tsk ) ( sbt psychological subscale = 0.659 ; mpsq = 0.683 ) and catastrophising ( pcs ) ( sbt = 0.671 ; mpsq = 0.656 ) were lower but also classified as 1 presents box plot graphs of the mpsq total score against the sbt ( a ) total score and ( b ) psychosocial subscale and demonstrate that increasing mpsq scores , correlated with higher sbt scores across the full range of both instrument scales . medium and high risk groups by the sbt and mpsq were 40% cf 40% , 35% cf 22% , and 25% cf 38% , respectively . low risk proportion was therefore the same , but the sbt allocated considerably fewer patients to the medium risk group than the mpsq ( mcnemar bowker test p = 0.022 ) . observed agreement between the two instruments for allocation to medium and high risk groups , is presented in table 1 , with absolute agreement in 62% of patients . weighted cohen s kappa for agreement in allocation to the three subgroups beyond chance was the characteristics of the low risk groups were almost identical ( e.g. both had an rmdq median of 2 and a pain nrs of 1.7 ) . medium risk groups differed only slightly with higher pain and disability scores among those allocated by the sbt and the high risk groups were also very similar although the mpsq high risk group had slightly more females and had longer episode durations . however , these differences between the clinical characteristics in the medium and high risk groups produced by both tools were not statistically significant ( p > 0.05 ) . the ability of the two instruments total scores ( and sbt subscale scores ) to discriminate patients defined as cases on reference standards is given in fig . there were no significant differences in the discriminative abilities of the sbt and mpsq scales for cases of disability , catastrophising , fear , comorbid pain , time off work or episode duration reference standards . this study has provided a comparative analysis of the measurement properties of the sbt and the mpsq for patients with low back pain , consulting in primary care . the mpsq ( 21 items ) has been available for nearly a decade and is a popular clinical tool with numerous external validation studies in comparison to the start back tool ( 9-items ) , which has been more recently validated and has potential advantages in terms of its length and scoring simplicity . both share broadly similar purposes , although subtle conceptual differences have become apparent through conducting this investigation , which should be highlighted for clinicians seeking to choose which tool is most appropriate for their context . firstly , the mpsq was conceived as a prognostic screening tool , although in practice it has adopted a subgrouping function with established cut - offs across a variety of settings to allocate patients to low and high risk groups . in contrast , the sbt was primarily conceived as a subgrouping tool , with cut - offs based on baseline factors and not on future outcome , and for specifically defined low , medium and high risk treatment subgroups . this study found that the completion rate for both instruments was high ( n = 130/131 for both ) and that their three subgroups clinical characteristics were broadly similar . substantial , suggesting that both tools have a similar ability to differentiate patients based on their baseline characteristics . although the characteristics of patients in the three subgroups allocated by each tool were not significantly different , significant differences were observed in the proportions of patients allocated to the high and medium risk groups , with a smaller proportion of patients allocated to the high risk group by the sbt than the mpsq . one factor in the choice of instrument used by clinicians may therefore be the availability of services for the management of a high risk group of patients . an analysis of both instrument scales abilities to discriminate a number of relevant reference standards was investigated to determine whether similarities in the subgroup clinical characteristics were due to tool cut - off points used , or due to similar discriminative abilities of the instrument scales . the results demonstrated that the two instrument scales similarly discriminated the majority of clinical characteristics including disability , catastrophising , fear , comorbid pain , time off work or episode duration . the mpsq total scale was significantly better at discriminating patients baseline pain intensity , while the sbt was better for discriminating baseline bothersomeness of back pain and referred leg pain . one substantial limitation of this study is that only cross - sectional data was available for directly comparing the performance of both instruments , preventing a comparison of the instrument s predictive validity . however , guidelines for the management of non - specific back pain highlight the importance of identifying indicators of poor prognosis , rather than predicting individual patient s outcomes , in order that treatment can be targeted appropriately ( chou et al . , 2007 ; van tulder et al . , 2006 ) . traditionally , validation of the utility of such instrument scales has predominantly focused on establishing an instrument s predictive validity , although the sbt subgroup cut - offs were specifically validated to optimally differentiate patients with specific baseline clinical characteristics . to date , there have been no studies that have compared the predictive abilities of both instruments within a single sample , but published data on the predictive validity of the mpsq ( hockings et al . , 2008 ) reports aucs for persisting disability from 0.68 to 0.83 , which compares similarly to reported aucs of 0.80 for the sbt ( hill phd thesis , 2008 ) . a further limitation is the potential for non - response bias influencing generalizability as only 53% of the sample responded , which may have inflated the proportion classified by the two instruments to the medium and high risk subgroups , as people with longer term or more severe problems are more likely to respond to the questionnaire . however , non - response bias is likely to have equally affected both instruments and is unlikely to have influenced the tool comparisons made in this study . this study found that the completion rate for both instruments was high ( n = 130/131 for both ) and that their three subgroups clinical characteristics were broadly similar . substantial , suggesting that both tools have a similar ability to differentiate patients based on their baseline characteristics . although the characteristics of patients in the three subgroups allocated by each tool were not significantly different , significant differences were observed in the proportions of patients allocated to the high and medium risk groups , with a smaller proportion of patients allocated to the high risk group by the sbt than the mpsq . one factor in the choice of instrument used by clinicians may therefore be the availability of services for the management of a high risk group of patients . an analysis of both instrument scales abilities to discriminate a number of relevant reference standards was investigated to determine whether similarities in the subgroup clinical characteristics were due to tool cut - off points used , or due to similar discriminative abilities of the instrument scales . the results demonstrated that the two instrument scales similarly discriminated the majority of clinical characteristics including disability , catastrophising , fear , comorbid pain , time off work or episode duration . the mpsq total scale was significantly better at discriminating patients baseline pain intensity , while the sbt was better for discriminating baseline bothersomeness of back pain and referred leg pain . one substantial limitation of this study is that only cross - sectional data was available for directly comparing the performance of both instruments , preventing a comparison of the instrument s predictive validity . however , guidelines for the management of non - specific back pain highlight the importance of identifying indicators of poor prognosis , rather than predicting individual patient s outcomes , in order that treatment can be targeted appropriately ( chou et al . , 2007 ; van tulder et al . , 2006 ) . traditionally , validation of the utility of such instrument scales has predominantly focused on establishing an instrument s predictive validity , although the sbt subgroup cut - offs were specifically validated to optimally differentiate patients with specific baseline clinical characteristics . to date , there have been no studies that have compared the predictive abilities of both instruments within a single sample , but published data on the predictive validity of the mpsq ( hockings et al . , 2008 ) reports aucs for persisting disability from 0.68 to 0.83 , which compares similarly to reported aucs of 0.80 for the sbt ( hill phd thesis , 2008 ) . a further limitation is the potential for non - response bias influencing generalizability as only 53% of the sample responded , which may have inflated the proportion classified by the two instruments to the medium and high risk subgroups , as people with longer term or more severe problems are more likely to respond to the questionnaire . however , non - response bias is likely to have equally affected both instruments and is unlikely to have influenced the tool comparisons made in this study . this study has compared measurement properties of the sbt to those of the mpsq , and found them to be similar in respect to the subgroup patient characteristics and abilities of the scales to differentiate according to validated reference standard measures . the sbt is quicker for patients to complete and easier for clinicians to score , although is able to identify a high risk subgroup with similar clinical characteristics to the mpsq . apart from the differences in instrument length , the results of this study suggest that the main difference between the two instruments was the proportion of patients each tool allocated to the high risk group , with the sbt identifying 25% as high risk , and the mpsq 38% as high risk . this might be of relevance to clinicians working within services that have resource limitations for treating high risk patients . recommendations for future research include ; a direct comparison of the predictive validity of both instruments , and also an investigation of possible instrument item redundancy . clinicians need to be aware of the relative strengths and weakness of the two clinical tools before selecting which tool is most appropriate to help decision making in their specific clinical context and setting . this head to head comparison has demonstrated that the sbt s discriminative abilities appear to be at least equivalent , and preferable in some criteria , to the mpsq , which is the most widely used current tool in clinical practice .

introductionclinicians require brief , practical tools to help identify low back pain ( lbp ) subgroups requiring early , targeted secondary prevention . the start back tool ( sbt ) was recently validated to subgroup lbp patients into early treatment pathways.aimto test the sbt s concurrent validity against an existing , popular lbp subgrouping tool , the rebro musculoskeletal pain screening questionnaire ( mpsq ) , and to compare the clinical characteristics of subgroups identified by each tool.methodstwo hundred and forty - four consecutive non - specific lbp consulters at 8 uk gp practices aged 1859 years were invited to complete a questionnaire . measures included the mpsq and sbt , disability , fear , catastrophising , pain intensity , episode duration and demographics . instruments were compared using spearman s correlations , tests for subgroup agreement and discriminant analysis of subgroup characteristics according to reference standards.resultscompleted sbt ( 9-items ) and mpsq ( 24-items ) data was available for 130/244 patients ( 53% ) . the correlation of sbt and mpsq scores was excellent ( rs = 0.80 ) . subgroup characteristics were similar across the low , medium and high subgroups , but , the proportions allocated to low , medium and high risk groups were different , with fewer patients in the sbt s high risk group . both instruments similarly discriminated for reference standards such as disability , catastrophising , fear , comorbid pain and time off work . the mpsq was better at discriminating pain intensity , while the sbt was better for discriminating bothersomeness of back pain and referred leg pain.conclusionsthe sbt baseline psychometrics performed similarly to the mpsq , but the sbt is shorter and easier to score and is an appropriate alternative for identifying high risk lbp patients in primary care .

Introduction Methods Properties of the MPSQ and SBT Data collection Analysis of data Results Discussion Differences in subgroup allocation Limitations Conclusion

primary care evidence - based guidelines for non - specific back pain highlight the importance of identifying indicators of poor prognosis in order that treatment can be targeted appropriately ( chou et al . , 2006 ) investigators increasingly advocate that better identification of potentially modifiable prognostic indicators may lead to more effective early secondary prevention of back pain in primary care ( boersma and linton , 2005 ; morley and vlaeyen , 2005 ; jellema et al . , 2006 ; the start back tool ( sbt ) is a recently validated tool developed to identify subgroups of patients to guide the provision of early secondary prevention in primary care ( hill et al . , 2008 ) were to identify patients with potentially treatment modifiable prognostic indicators using a brief , user - friendly tool and to validate cut - off scores for subgrouping patients into 1 of 3 a priori initial treatment options in primary care ( low , medium and high risk groups ) . the development and validation of the start back tool ( sbt ) was recently reported ( hill et al . , 2008 ) , but a head to head comparison with a the rebro musculoskeletal pain screening questionnaire ( mpsq : linton and hallden , 1998 ) was considered the most appropriate reference standard against which to compare the sbt , as it is one of the most widely used tools in clinical practice to similarly differentiate primary care back pain patients , and it has a common conceptual purpose with the sbt of identifying high risk patients requiring targeted treatment . , 2007 ) , require brief and practical tools to help them identify subgroups of patients that may require early , targeted secondary prevention pathways . , 2008 ) provides clinicians with a choice , in addition to an existing reference standard instrument , the mpsq . however , in order to decide which clinical tool to use in practice , clinicians need to know how these instruments compare , including the baseline clinical characteristics of the subgroups identified by each tool . the overall aim of this study was to therefore provide a head to head comparison of the sbt s concurrent validity against a best available reference standard , the mpsq . our objectives were to test the correlation between the sbt and mpsq scores and agreement of patients allocated to medium and high risk subgroups ; and to compare the abilities of the sbt and mpsq scales to discriminate patients according to validated reference standard measures . the reliability of the mpsq has been reported with a kappa of 0.83 ( linton and hallden , 1998 ) and external validity , with a number of different high risk cut - off scores , has been established in a variety of patient populations and settings ( heneweer et al . other authors have used it to classify patients into low , medium and high at risk groups ( nordeman et al . overall sbt scores range from 0 - 9 and are produced by summing all positive items ; a psychosocial subscale score ranging from 0 to 5 is produced by summing bothersomeness , fear , catastrophising , anxiety , and depression items ( items 1 , 4 , 7 , 8 , and 9 ) . the predictive validity and external validity of the start back tool has been reported , as well as the sbt s reliability , with a kappa of 0.79 ( hill et al . prognostic constructs included in both tools as single screening items include disability , fear avoidance , anxiety , depression and also an item on the patient s perceived chance that current pain may become persistent or that the back pain is never going to get any better. the sbt also discriminates patients using single items for bothersomeness , referred leg pain , and comorbid pain , while the mpsq discriminates on the basis of additional items for pain intensity , coping and work - related factors . potential differences between the mpsq and sbt were explored using data from a cross - sectional survey of consecutive adult patients ( n = 131 ) who consulted their gp with low back pain . in brief , 244 participants were invited to complete a questionnaire containing both the mpsq and the sbt from 8 general practices in north staffordshire and central cheshire , uk using computerised read codes to identify recent back pain consulters . the sbt and mpsq were scored according to the methods specified by the instrument developers ( hill et al . , 2008 medium and high risk groups using derived cut - off scores for each instrument . for the sbt , cut - offs recommended in the original article were used ( hill et al . , 2008 ) ; for the mpsq , we replicated the high risk cut - off score determined within a uk primary care population by hurley et al . to score the mpsq among patients who were not workers , ( four work - related items are only relevant to those patients in work ) , the procedure used by jellema et al . correlations between the sbt and mpsq , and reference standard constructs included in both instruments : disability ( roland and morris disability questionnaire , rmdq : roland and morris , 1983 ) fear avoidance ( tampa scale of kinesiaphobia , tsk : kori et al . in addition , box and whisker plot graphs were produced to visually present the correlation between the sbt and mpsq.1 observed agreement of the two tool s allocation to medium and high risk subgroups was examined by calculating absolute agreement , and agreement beyond chance statistically evaluated using a weighted cohen s kappa test . the characteristics of patients selected for high subgroups using the sbt and mpsq were compared using median scores for age , gender , episode duration , bothersomeness , pain intensity , days off work , fear ( tsk ) , catastrophising ( pcs ) , mpsq scores and sbt scores ( mean values were used for age ) . finally , the ability of the two instruments total ( and subscale ) scores to discriminate patients was compared by plotting receiver operator characteristic ( roc ) curves for various different reference standard case definitions provided in table 3 ) and calculating aucs ( 95% cis ) , with statistical differences tested using the wilcoxon statistic ( hanley and mcneil , 1982 ) . the reliability of the mpsq has been reported with a kappa of 0.83 ( linton and hallden , 1998 ) and external validity , with a number of different high risk cut - off scores , has been established in a variety of patient populations and settings ( heneweer et al . other authors have used it to classify patients into low , medium and high at risk groups ( nordeman et al . overall sbt scores range from 0 - 9 and are produced by summing all positive items ; a psychosocial subscale score ranging from 0 to 5 is produced by summing bothersomeness , fear , catastrophising , anxiety , and depression items ( items 1 , 4 , 7 , 8 , and 9 ) . the predictive validity and external validity of the start back tool has been reported , as well as the sbt s reliability , with a kappa of 0.79 ( hill et al . prognostic constructs included in both tools as single screening items include disability , fear avoidance , anxiety , depression and also an item on the patient s perceived chance that current pain may become persistent or that the back pain is never going to get any better. the sbt also discriminates patients using single items for bothersomeness , referred leg pain , and comorbid pain , while the mpsq discriminates on the basis of additional items for pain intensity , coping and work - related factors . potential differences between the mpsq and sbt were explored using data from a cross - sectional survey of consecutive adult patients ( n = 131 ) who consulted their gp with low back pain . in brief , 244 participants were invited to complete a questionnaire containing both the mpsq and the sbt from 8 general practices in north staffordshire and central cheshire , uk using computerised read codes to identify recent back pain consulters . the sbt and mpsq were scored according to the methods specified by the instrument developers ( hill et al . , 2008 ; medium and high risk groups using derived cut - off scores for each instrument . for the sbt , cut - offs recommended in the original article were used ( hill et al . , 2008 ) ; for the mpsq , we replicated the high risk cut - off score determined within a uk primary care population by hurley et al . to score the mpsq among patients who were not workers , ( four work - related items are only relevant to those patients in work ) , the procedure used by jellema et al . correlations between the sbt and mpsq , and reference standard constructs included in both instruments : disability ( roland and morris disability questionnaire , rmdq : roland and morris , 1983 ) fear avoidance ( tampa scale of kinesiaphobia , tsk : kori et al . in addition , box and whisker plot graphs were produced to visually present the correlation between the sbt and mpsq.1 observed agreement of the two tool s allocation to medium and high risk subgroups was examined by calculating absolute agreement , and agreement beyond chance statistically evaluated using a weighted cohen s kappa test . high subgroups using the sbt and mpsq were compared using median scores for age , gender , episode duration , bothersomeness , pain intensity , days off work , fear ( tsk ) , catastrophising ( pcs ) , mpsq scores and sbt scores ( mean values were used for age ) . finally , the ability of the two instruments total ( and subscale ) scores to discriminate patients was compared by plotting receiver operator characteristic ( roc ) curves for various different reference standard case definitions provided in table 3 ) and calculating aucs ( 95% cis ) , with statistical differences tested using the wilcoxon statistic ( hanley and mcneil , 1982 ) . complete data on the sbt and mpsq was available for 130/244 ( 53% ) patients . back pain episode durations were varied with 25% < 1 month , 27% 16 months , and 48% > 6 months , and 43% reported being the median scores ( range ) for sbt and mpsq were 4.5 ( 09 ) and 98.8 ( 26.3189.0 ) , respectively . the spearman s rank correlation coefficients for the sbt total scores and psychosocial subscale scores with the mpsq scores were 0.802 and 0.769 respectively . large and was similar to the correlation between disability ( rmdq ) and the subgroup tools ( sbt = 0.813 ; mpsq = 0.830 ) . figures for fear ( tsk ) ( sbt psychological subscale = 0.659 ; mpsq = 0.683 ) and catastrophising ( pcs ) ( sbt = 0.671 ; mpsq = 0.656 ) were lower but also classified as 1 presents box plot graphs of the mpsq total score against the sbt ( a ) total score and ( b ) psychosocial subscale and demonstrate that increasing mpsq scores , correlated with higher sbt scores across the full range of both instrument scales . medium and high risk groups by the sbt and mpsq were 40% cf 40% , 35% cf 22% , and 25% cf 38% , respectively . low risk proportion was therefore the same , but the sbt allocated considerably fewer patients to the medium risk group than the mpsq ( mcnemar bowker test p = 0.022 ) . observed agreement between the two instruments for allocation to medium and high risk groups , is presented in table 1 , with absolute agreement in 62% of patients . weighted cohen s kappa for agreement in allocation to the three subgroups beyond chance was the characteristics of the low risk groups were almost identical ( e.g. medium risk groups differed only slightly with higher pain and disability scores among those allocated by the sbt and the high risk groups were also very similar although the mpsq high risk group had slightly more females and had longer episode durations . however , these differences between the clinical characteristics in the medium and high risk groups produced by both tools were not statistically significant ( p > 0.05 ) . there were no significant differences in the discriminative abilities of the sbt and mpsq scales for cases of disability , catastrophising , fear , comorbid pain , time off work or episode duration reference standards . this study has provided a comparative analysis of the measurement properties of the sbt and the mpsq for patients with low back pain , consulting in primary care . the mpsq ( 21 items ) has been available for nearly a decade and is a popular clinical tool with numerous external validation studies in comparison to the start back tool ( 9-items ) , which has been more recently validated and has potential advantages in terms of its length and scoring simplicity . firstly , the mpsq was conceived as a prognostic screening tool , although in practice it has adopted a subgrouping function with established cut - offs across a variety of settings to allocate patients to low and high risk groups . in contrast , the sbt was primarily conceived as a subgrouping tool , with cut - offs based on baseline factors and not on future outcome , and for specifically defined low , medium and high risk treatment subgroups . this study found that the completion rate for both instruments was high ( n = 130/131 for both ) and that their three subgroups clinical characteristics were broadly similar . although the characteristics of patients in the three subgroups allocated by each tool were not significantly different , significant differences were observed in the proportions of patients allocated to the high and medium risk groups , with a smaller proportion of patients allocated to the high risk group by the sbt than the mpsq . one factor in the choice of instrument used by clinicians may therefore be the availability of services for the management of a high risk group of patients . an analysis of both instrument scales abilities to discriminate a number of relevant reference standards was investigated to determine whether similarities in the subgroup clinical characteristics were due to tool cut - off points used , or due to similar discriminative abilities of the instrument scales . the results demonstrated that the two instrument scales similarly discriminated the majority of clinical characteristics including disability , catastrophising , fear , comorbid pain , time off work or episode duration . the mpsq total scale was significantly better at discriminating patients baseline pain intensity , while the sbt was better for discriminating baseline bothersomeness of back pain and referred leg pain . one substantial limitation of this study is that only cross - sectional data was available for directly comparing the performance of both instruments , preventing a comparison of the instrument s predictive validity . however , guidelines for the management of non - specific back pain highlight the importance of identifying indicators of poor prognosis , rather than predicting individual patient s outcomes , in order that treatment can be targeted appropriately ( chou et al . traditionally , validation of the utility of such instrument scales has predominantly focused on establishing an instrument s predictive validity , although the sbt subgroup cut - offs were specifically validated to optimally differentiate patients with specific baseline clinical characteristics . to date , there have been no studies that have compared the predictive abilities of both instruments within a single sample , but published data on the predictive validity of the mpsq ( hockings et al . , 2008 ) reports aucs for persisting disability from 0.68 to 0.83 , which compares similarly to reported aucs of 0.80 for the sbt ( hill phd thesis , 2008 ) . a further limitation is the potential for non - response bias influencing generalizability as only 53% of the sample responded , which may have inflated the proportion classified by the two instruments to the medium and high risk subgroups , as people with longer term or more severe problems are more likely to respond to the questionnaire . however , non - response bias is likely to have equally affected both instruments and is unlikely to have influenced the tool comparisons made in this study . this study found that the completion rate for both instruments was high ( n = 130/131 for both ) and that their three subgroups clinical characteristics were broadly similar . although the characteristics of patients in the three subgroups allocated by each tool were not significantly different , significant differences were observed in the proportions of patients allocated to the high and medium risk groups , with a smaller proportion of patients allocated to the high risk group by the sbt than the mpsq . one factor in the choice of instrument used by clinicians may therefore be the availability of services for the management of a high risk group of patients . an analysis of both instrument scales abilities to discriminate a number of relevant reference standards was investigated to determine whether similarities in the subgroup clinical characteristics were due to tool cut - off points used , or due to similar discriminative abilities of the instrument scales . the results demonstrated that the two instrument scales similarly discriminated the majority of clinical characteristics including disability , catastrophising , fear , comorbid pain , time off work or episode duration . the mpsq total scale was significantly better at discriminating patients baseline pain intensity , while the sbt was better for discriminating baseline bothersomeness of back pain and referred leg pain . one substantial limitation of this study is that only cross - sectional data was available for directly comparing the performance of both instruments , preventing a comparison of the instrument s predictive validity . however , guidelines for the management of non - specific back pain highlight the importance of identifying indicators of poor prognosis , rather than predicting individual patient s outcomes , in order that treatment can be targeted appropriately ( chou et al . traditionally , validation of the utility of such instrument scales has predominantly focused on establishing an instrument s predictive validity , although the sbt subgroup cut - offs were specifically validated to optimally differentiate patients with specific baseline clinical characteristics . to date , there have been no studies that have compared the predictive abilities of both instruments within a single sample , but published data on the predictive validity of the mpsq ( hockings et al . , 2008 ) reports aucs for persisting disability from 0.68 to 0.83 , which compares similarly to reported aucs of 0.80 for the sbt ( hill phd thesis , 2008 ) . a further limitation is the potential for non - response bias influencing generalizability as only 53% of the sample responded , which may have inflated the proportion classified by the two instruments to the medium and high risk subgroups , as people with longer term or more severe problems are more likely to respond to the questionnaire . however , non - response bias is likely to have equally affected both instruments and is unlikely to have influenced the tool comparisons made in this study . this study has compared measurement properties of the sbt to those of the mpsq , and found them to be similar in respect to the subgroup patient characteristics and abilities of the scales to differentiate according to validated reference standard measures . the sbt is quicker for patients to complete and easier for clinicians to score , although is able to identify a high risk subgroup with similar clinical characteristics to the mpsq . apart from the differences in instrument length , the results of this study suggest that the main difference between the two instruments was the proportion of patients each tool allocated to the high risk group , with the sbt identifying 25% as high risk , and the mpsq 38% as high risk . this head to head comparison has demonstrated that the sbt s discriminative abilities appear to be at least equivalent , and preferable in some criteria , to the mpsq , which is the most widely used current tool in clinical practice .

early - onset scoliosis ( eos ) is characterized by curvature of the spine > 10 , appearing before the age of 10 years.1 eos can be defined as idiopathic , congenital , neuromuscular , or associated with various syndromes.24 if untreated , eos can progress to cause significant disfigurement and chest wall deformity , with the potential for worsening pulmonary insufficiency and poor quality of life.3 treatment aims to monitor and assist thoracic development , focusing on controlling the structural deformity by supporting normal spinal and chest wall growth . treatment options include casting or bracing , spinal fusion , and growth - friendly surgical techniques.3 the traditional growing rod ( tgr ) technique requires periodic surgical lengthenings of the device to maintain curve correction while allowing for continued spinal growth . this technique has proven useful for patients with progressive and severe curves , or for growing children for whom a delay to fusion is required to allow spine and thoracic development.5 the primary disadvantages of tgr are the risks associated with general anesthetic and wound complications due to repeated invasive surgical procedures.6 tgr surgery is also associated with substantial socioeconomic , psychological , and health - related quality - of - life ( hrqol ) disadvantages for patients and their caregivers . this is due to substantial reductions in patients normal activity levels and the indirect costs associated with the time taken from school and work for each invasive procedure.7 recent technical advances have aimed to overcome the drawbacks associated with tgr . in particular , the magnetically controlled growing rod ( mcgr ) technique is a new technology in which a growing rod , once implanted during an invasive surgical procedure ( similar to tgr ) , is lengthened noninvasively using a hand - held magnetic external remote controller.8 using the hand - held remote controller , elongation is performed in millimeters in two ways : continuous and incremental . continuous mode maximizes lengthening until a set force is achieved ( the device s safety feature stops distraction at ~42 lbs of axial force ) . the incremental setting allows programmable / custom lengthening based on patient growth chart targets . the preclinical and clinical findings of treatment with mcgr have demonstrated mcgr as safe and effective over a followup period of up to 2 years.710 the primary advantage of the mcgr technology , compared with tgr , is that the device can be lengthened noninvasively in the physician office without anesthesia . this eliminates the need for periodic surgeries for lengthening , necessary with tgr , and has potential to reduce long - term medical resource consumption and costs in comparison to tgr and improve the hrqol for affected children.11 the health - related costs associated with the treatment of eos , using mcgr , have recently been shown to be lower compared with tgr in the european setting , specifically in france and the uk . charroin et al11 reported the cumulative cost savings over 4 years to be 6,135 from the french sickness fund perspective . these savings were attributed to a reduction in hospital stays , periodic invasive procedures , spinal cord monitoring , anesthesia , and pharmaceutical use . in a second economic study , conducted for the uk by rolton et al,12 the estimated cumulative cost savings per child after 5 years of treatment with mcgr compared with tgr was reported to be 8,888 . one further economic study , conducted from the us payer perspective , also reported the cumulative cost savings ( $ 39,632 per patient at year 5 ) for mcgr compared with tgr.13 the objective of this research was to perform a cost analysis of mcgr compared with tgr for eos from the us integrated health care delivery system perspective over the complete 6-year episode of care from initial implantation until final fusion . to our knowledge , this is the first cost analysis of mcgr compared to tgr performed from the provider , as opposed to the payer perspective , and the first such cost analysis that accounts for the complete episode of care . we hypothesized that over time , the higher mcgr implant cost will be offset by eliminating repeated tgr surgical lengthenings , as seen in previous research . an economic model was developed using microsoft excel to compare the cost of mcgr and tgr , from the perspective of the us integrated health care delivery system . for both tgr and mcgr , the model estimated the cumulative costs associated with initial implantation , surgical revisions , device exchange , rod lengthenings , and final spinal fusion over a 6-year episode of care , from initial implantation to final fusion . the model also accounted for the occurrence of surgical - site infection ( ssi ; deep and superficial ) and device failure . model parameters were estimated on the basis of published literature , analyses of a multicenter eos database comprising the us institutions , and a series of one - on - one interviews conducted with six pediatric spine surgeons , two private payers , and two hospital purchasers . confidential data of patients of the clinicians / hospital purchasers or of beneficiaries from payers were not obtained or used for any aspect of the cost analysis . furthermore , interviewees themselves were not being evaluated ( ie , clinician and hospital performance were not being evaluated ) and , therefore , the interviewees are not considered subjects requiring ethics committee approval and subject consent . rather , the interviewees provided feedback on the model framework , content ( assumptions ) , and relevance . all costs represent 2015 us dollars , and were discounted at an annual rate of 3.0% , as recommended by the congressional budget office.14 the model framework and clinical parameters are reported in table 1 , and medical resource use is reported in table 2 . of note , the tgr and mcgr device failure rates were obtained from a previously conducted economic evaluation commissioned by the national institute for health and care excellence , as reported in the external assessment center report15 and journal publication.16 these rates were calculated based on the best available evidence . assumptions made in the development of this model include the following : clinical effectiveness ( curve correction and increase in thoracic height ) is equivalent between mcgr and tgr;10 resource use between tgr and mcgr with respect to initial implantation , revision , exchange , and final fusion is similar ( with the exception of the device cost , where appropriate ) ; one x - ray is required per insertion , lengthening procedure , exchange , revision , deep ssi , and final fusion ; all patients for mcgr and tgr require one device exchange surgery at 3.8 years , based on the average spine growth observed in a child aged 6 years;17 and deep ssi requires a complete replacement of implants , and superficial infection requires oral antibiotics . the components replaced during a partial revision procedure ( table 2 ) were based on the study of bess et al18 and expert clinical advice . among patients with partial removal , pedicle screw / hooks were assumed to be replaced 95% of the time , rod set screws 61% of the time , and all other components ( including rods and connectors ) 100% of the time . it was further assumed that mcgr costs were not incurred for partial or complete revisions due to mcgr failure within 1 year following an mcgr implantation or mcgr exchange ( in the unlikely event of a manufacturing defect ) ; other costs for the revisions were incurred ( eg , hospital facility costs and professional fees ) . for revisions due to anchor or foundation issues within 1 year following an mcgr implantation or mcgr exchange , hospital inpatient facility costs were based on medicare payments ( diagnosis - related group [ drg ] data ) , physician professional fees were based on current procedural terminology data , and hospital outpatient facility costs were based on ambulatory payment classification data . because bundled drg payments include the tgr device cost , for mcgr hospital inpatient procedures , the tgr device costs were subtracted from the drg payment , and the mcgr device costs were added to account for the difference in device cost . the total costs for procedures included in the model are shown in table 3 . unit costs for these procedures , including all drg , current procedural terminology , and ambulatory payment classification codes and costs , as well as x - ray , intraoperative neurophysiological monitoring , and anesthesia codes and costs , can be viewed in the supplementary materials . extensive one - way sensitivity analyses were conducted on a range of model variables to establish how robust the cumulative cost savings were to realistic changes in the boundaries of the key parameters and assumptions of the model . the lower and upper bounds for these parameters are outlined in tables 13 and the supplementary materials . two - way sensitivity analyses were also conducted for device failure rate and the months between lengthenings . an economic model was developed using microsoft excel to compare the cost of mcgr and tgr , from the perspective of the us integrated health care delivery system . for both tgr and mcgr , the model estimated the cumulative costs associated with initial implantation , surgical revisions , device exchange , rod lengthenings , and final spinal fusion over a 6-year episode of care , from initial implantation to final fusion . the model also accounted for the occurrence of surgical - site infection ( ssi ; deep and superficial ) and device failure . model parameters were estimated on the basis of published literature , analyses of a multicenter eos database comprising the us institutions , and a series of one - on - one interviews conducted with six pediatric spine surgeons , two private payers , and two hospital purchasers . confidential data of patients of the clinicians / hospital purchasers or of beneficiaries from payers were not obtained or used for any aspect of the cost analysis . furthermore , interviewees themselves were not being evaluated ( ie , clinician and hospital performance were not being evaluated ) and , therefore , the interviewees are not considered subjects requiring ethics committee approval and subject consent . rather , the interviewees provided feedback on the model framework , content ( assumptions ) , and relevance . all costs represent 2015 us dollars , and were discounted at an annual rate of 3.0% , as recommended by the congressional budget office.14 the model framework and clinical parameters are reported in table 1 , and medical resource use is reported in table 2 . of note , the tgr and mcgr device failure rates were obtained from a previously conducted economic evaluation commissioned by the national institute for health and care excellence , as reported in the external assessment center report15 and journal publication.16 these rates were calculated based on the best available evidence . assumptions made in the development of this model include the following : clinical effectiveness ( curve correction and increase in thoracic height ) is equivalent between mcgr and tgr;10 resource use between tgr and mcgr with respect to initial implantation , revision , exchange , and final fusion is similar ( with the exception of the device cost , where appropriate ) ; one x - ray is required per insertion , lengthening procedure , exchange , revision , deep ssi , and final fusion ; all patients for mcgr and tgr require one device exchange surgery at 3.8 years , based on the average spine growth observed in a child aged 6 years;17 and deep ssi requires a complete replacement of implants , and superficial infection requires oral antibiotics . the components replaced during a partial revision procedure ( table 2 ) were based on the study of bess et al18 and expert clinical advice . among patients with partial removal , pedicle screw / hooks were assumed to be replaced 95% of the time , rod set screws 61% of the time , and all other components ( including rods and connectors ) 100% of the time . it was further assumed that mcgr costs were not incurred for partial or complete revisions due to mcgr failure within 1 year following an mcgr implantation or mcgr exchange ( in the unlikely event of a manufacturing defect ) ; other costs for the revisions were incurred ( eg , hospital facility costs and professional fees ) . for revisions due to anchor or foundation issues within 1 year following an mcgr implantation or mcgr exchange , hospital inpatient facility costs were based on medicare payments ( diagnosis - related group [ drg ] data ) , physician professional fees were based on current procedural terminology data , and hospital outpatient facility costs were based on ambulatory payment classification data . because bundled drg payments include the tgr device cost , for mcgr hospital inpatient procedures , the tgr device costs were subtracted from the drg payment , and the mcgr device costs were added to account for the difference in device cost . unit costs for these procedures , including all drg , current procedural terminology , and ambulatory payment classification codes and costs , as well as x - ray , intraoperative neurophysiological monitoring , and anesthesia codes and costs , can be viewed in the supplementary materials . extensive one - way sensitivity analyses were conducted on a range of model variables to establish how robust the cumulative cost savings were to realistic changes in the boundaries of the key parameters and assumptions of the model . the lower and upper bounds for these parameters are outlined in tables 13 and the supplementary materials . two - way sensitivity analyses were also conducted for device failure rate and the months between lengthenings . over the 6-year episode of care , mcgr was associated with an estimated 11.2 fewer invasive procedures per patient compared with tgr ( mcgr : 3.33 ; tgr : 14.53 ) . when simulating 1,000 eos patients over the 6-year episode of care , mcgr was associated with an estimated 270 fewer deep ssis ( mcgr : 80 ; tgr : 350 ) and 197 fewer revisions due to device failure compared with tgr ( mcgr : 332 ; tgr : 529 ) . the cumulative costs for treatment of eos with mcgr compared with tgr are illustrated in figure 1 . as shown in figure 1 , the higher cost associated with the initial insertion and exchange ( at 3.8 years ) of mcgr was offset by the repeated tgr surgical lengthenings and associated complications . the cumulative cost for mcgr vs tgr over the 6-year episode of care was cost neutral ( $ 61 ; mcgr minus tgr ) from the us integrated health care delivery system perspective . extensive sensitivity analyses were conducted . as shown in the one - way sensitivity analysis ( figure 2 ) , the cumulative cost savings of mcgr was most sensitive to mcgr dual rod use , months between tgr lengthenings , tgr lengthenings that are inpatient ( vs outpatient ) , mcgr cost , and time horizon . the results were less sensitive to the percent of ssis that are deep infections and years to exchange surgery . when less certain model parameters were varied in the two - way sensitivity analyses , specifically the mcgr device failure rate ( figure 3 ) and months between mcgr lengthenings ( figure 4 ) , the costs fell within a relatively narrow range when based on realistic clinical scenarios , suggesting that the economic model is robust to plausible parameter values . in the base - case analysis that evaluated cumulative 6-year costs , the differential was $ 61 ; that is , cost neutrality was achieved by the conclusion of the 6-year episode of care . the results were sensitive , however , to the time horizon due to the timing of certain procedures in the process of care , most notably the exchange surgery at 3.8 years . as a result , ~73% of the cumulative costs for mcgr were accrued in year 1 ( initial implantation ) and year 4 ( exchange surgery ) ; these costs were then offset over time . over the 6-year episode of care , mcgr was associated with an estimated 11.2 fewer invasive procedures per patient compared with tgr ( mcgr : 3.33 ; tgr : 14.53 ) . when simulating 1,000 eos patients over the 6-year episode of care , mcgr was associated with an estimated 270 fewer deep ssis ( mcgr : 80 ; tgr : 350 ) and 197 fewer revisions due to device failure compared with tgr ( mcgr : 332 ; tgr : 529 ) . the cumulative costs for treatment of eos with mcgr compared with tgr are illustrated in figure 1 . as shown in figure 1 , the higher cost associated with the initial insertion and exchange ( at 3.8 years ) of mcgr was offset by the repeated tgr surgical lengthenings and associated complications . the cumulative cost for mcgr vs tgr over the 6-year episode of care was cost neutral ( $ 61 ; mcgr minus tgr ) from the us integrated health care delivery system perspective . extensive sensitivity analyses were conducted . as shown in the one - way sensitivity analysis ( figure 2 ) , the cumulative cost savings of mcgr was most sensitive to mcgr dual rod use , months between tgr lengthenings , tgr lengthenings that are inpatient ( vs outpatient ) , mcgr cost , and time horizon . the results were less sensitive to the percent of ssis that are deep infections and years to exchange surgery . when less certain model parameters were varied in the two - way sensitivity analyses , specifically the mcgr device failure rate ( figure 3 ) and months between mcgr lengthenings ( figure 4 ) , the costs fell within a relatively narrow range when based on realistic clinical scenarios , suggesting that the economic model is robust to plausible parameter values . in the base - case analysis that evaluated cumulative 6-year costs , the differential was $ 61 ; that is , cost neutrality was achieved by the conclusion of the 6-year episode of care . the results were sensitive , however , to the time horizon due to the timing of certain procedures in the process of care , most notably the exchange surgery at 3.8 years . as a result , ~73% of the cumulative costs for mcgr were accrued in year 1 ( initial implantation ) and year 4 ( exchange surgery ) ; these costs were then offset over time . economic models facilitate exploring the extent to which costs of a new intervention may be offset by reductions in other medical costs.19 the economic model presented in this study demonstrates that the cost impact of mcgr is offset by eliminating repeated tgr surgical lengthenings and associated complications . the cost offsets for mcgr were driven by fewer deep ssis , less device failures resulting in fewer revisions , and absence of a hospital inpatient stay , anesthesia , and intraoperative neurophysiological monitoring during mcgr lengthenings . in the us , providers are becoming increasingly at financial risk as bundled payments shift the risk of financial accountability from the payer to the provider.20 with the trend toward bundled payments in orthopedics , providers will be at risk for a defined episode of care , including hospital stays , complications , and implant costs.21 this cost analysis demonstrates , from a provider perspective , the efficient provision of care with mcgr . our analysis differs in several ways from previous analyses of mcgr conducted for the european and the us settings of care.1113 most notably , our analysis evaluated cumulative costs over the 6-year episode of care ( from initial implantation through final fusion ) inclusive of the exchange surgery at 3.8 years for both tgr and mcgr . in addition , our analysis reflected the us practice patterns where the majority ( 85% ) of cases use dual - rod rather than single - rod constructs , and tgr lengthenings are performed in both the hospital inpatient and outpatient settings ( with more frail children requiring greater resources such as an intensive care unit stay).22 on the other hand , the uk study by rolton et al,12 the french study by charroin et al,11 and the us payer perspective study by su et al13 all demonstrated cumulative cost savings without accounting for the exchange surgery and assumed that tgr lengthenings are performed entirely in the hospital inpatient setting . these differences in modeling approaches may represent variations in either practice patterns or modeling assumptions . charroin et al11 and su et al13 explain that their time horizons ( 4 and 5 years , respectively ) were selected to accommodate the complete lengthening capacity of mcgr ( 4.8 cm ) , not necessarily a full episode of care . after achieving mcgr maximal range of lengthening at 4.8 cm , su et al13 acknowledged that mcgr is likely to require an exchange to accommodate further growth . in the present study at 3 years following initial implantation and prior to the exchange surgery at 3.8 years , mcgr resulted in a cumulative cost savings of $ 2,943 compared to tgr ( figure 1 ) , which is consistent with the prior research studies that did not account for the exchange surgery . this finding of cumulative cost savings at 3 years following initial implantation of mcgr may also apply to surgeries involving conversions of tgr to mcgr where an mcgr exchange procedure is not anticipated.23 as noted previously , we performed a cost analysis of mcgr compared with tgr for eos from the us provider perspective , whereas su et al13 performed a similar analysis but from the us payer perspective . we used medicare payments as a proxy for provider costs , a well - accepted methodology in economic analyses.24 on the other hand , su et al13 determined costs using the kids inpatient database in the nis healthcare cost and utilization project database . of note , the database contains hospital charge data ( as reflected on a hospital bill ) , not insurer payments or the actual cost to the hospital for the provision of care to the patient . given that insurers generally pay less than hospital charges,25 it remains unclear to what degree the use of hospital charges may have affected the difference in cumulative costs between tgr and mcgr reported by su et al.13 further , in our study , we incorporated a distribution of practice settings ( roughly half hospital inpatient and half hospital outpatient ) for performing tgr lengthenings based on a multicenter eos database comprising us institutions,22 whereas su et al13 assumed that tgr lengthenings were performed entirely in the hospital inpatient setting in the us . the approximate medicare reimbursement for tgr lengthenings is $ 6,000 ( when considering both practice settings ) , compared to the $ 10,000 in hospital inpatient charges noted by su et al.13 if one takes 60% of the cumulative cost of tgr lengthenings estimated by su et al,13 the cost savings of mcgr reported by su et al13 are diminished ; nevertheless , it appears that mcgr would be cost neutral in year 5 from the us payer perspective , which is similar to our results at year 6 from the us provider perspective . best case analysis of the potential cost savings associated with mcgr for the treatment of eos . the uk study by rolton et al12 and the french study by charroin et al11 are perhaps slightly less so but still show cost savings . nonetheless , the trend in all of these studies is that while there is an added expense from the new technology , the cost offsets appear to economically justify the adoption of mcgr . with use of mcgr , there is substantial value from obviating repeat anesthetic and surgical events , as well as avoiding family disruption . the significant yet unquantified clinical benefit and associated financial benefit from avoiding repeated surgical tgr lengthenings warrants further investigation . at present , the mcgr literature on device failure is limited to small numbers of patients with a follow - up period of 2 years . sensitivity analyses were performed on the mcgr device failure rate in light of this limitation . where data for mcgr were not available from the published literature , conservative assumptions were made by using the same parameter values as for tgr based on tgr clinical studies , in particular the study by bess et al.18 when these less certain mcgr clinical parameters were varied within a plausible range , the inferences of the model did not change suggesting that the model is robust . while not a limitation per se , it is worth noting that the model presented is a cost analysis and not a cost - effectiveness analysis therefore , similar to previous economic research of mcgr , it has not accounted for , 1 ) the pain , psychological distress , and compromised hrqol associated with invasive tgr lengthening surgeries , or 2 ) the shorter recovery time with mcgr lengthenings , which results in less time away from usual activities for young patients and their families . therefore , further analyses developed specifically to measure and evaluate hrqol should be considered for future research . cost offsets for mcgr accrue over time and reflect the elimination of repeated invasive tgr surgical lengthenings and their associated complications . despite the higher up - front cost for insertion of the mcgr implant and later for exchange of the mcgr implant , the cumulative cost of mcgr is offset over the 6-year episode of care from the perspective of the us integrated health care delivery system . the hrqol gained by mcgr patients and their caregivers is likely to be substantial and warrants further investigation .

purposetraditional growing rod ( tgr ) for early - onset scoliosis ( eos ) is effective but requires repeated invasive surgical lengthenings under general anesthesia . magnetically controlled growing rod ( mcgr ) is lengthened noninvasively using a hand - held magnetic external remote controller in a physician office ; however , the mcgr implant is expensive , and the cumulative cost savings have not been well studied . we compared direct medical costs of mcgr and tgr for eos from the us integrated health care delivery system perspective . we hypothesized that over time , the mcgr implant cost will be offset by eliminating repeated tgr surgical lengthenings.methodsfor both tgr and mcgr , the economic model estimated the cumulative costs for initial implantation , lengthenings , revisions due to device failure , surgical - site infections , device exchanges ( at 3.8 years ) , and final fusion , over a 6-year episode of care . model parameters were estimated from published literature , a multicenter eos database of us institutions , and interviews . costs were discounted at 3.0% annually and represent 2015 us dollars.resultsof 1,000 simulated patients over 6 years , mcgr was associated with an estimated 270 fewer deep surgical - site infections and 197 fewer revisions due to device failure compared with tgr . mcgr was projected to cost an additional $ 61 per patient over the 6-year episode of care compared with tgr . sensitivity analyses indicated that the results were sensitive to changes in the percentage of mcgr dual rod use , months between tgr lengthenings , percentage of hospital inpatient ( vs outpatient ) tgr lengthenings , and mcgr implant cost.conclusioncost neutrality of mcgr to tgr was achieved over the 6-year episode of care by eliminating repeated tgr surgical lengthenings . to our knowledge , this is the first cost analysis comparing mcgr to tgr from the us provider perspective which demonstrates the efficient provision of care with mcgr .

Introduction Materials and methods Model overview Model assumptions and data sources Sensitivity analysis Results Base-case results Sensitivity analysis Discussion Limitations Conclusion

early - onset scoliosis ( eos ) is characterized by curvature of the spine > 10 , appearing before the age of 10 years.1 eos can be defined as idiopathic , congenital , neuromuscular , or associated with various syndromes.24 if untreated , eos can progress to cause significant disfigurement and chest wall deformity , with the potential for worsening pulmonary insufficiency and poor quality of life.3 treatment aims to monitor and assist thoracic development , focusing on controlling the structural deformity by supporting normal spinal and chest wall growth . treatment options include casting or bracing , spinal fusion , and growth - friendly surgical techniques.3 the traditional growing rod ( tgr ) technique requires periodic surgical lengthenings of the device to maintain curve correction while allowing for continued spinal growth . this technique has proven useful for patients with progressive and severe curves , or for growing children for whom a delay to fusion is required to allow spine and thoracic development.5 the primary disadvantages of tgr are the risks associated with general anesthetic and wound complications due to repeated invasive surgical procedures.6 tgr surgery is also associated with substantial socioeconomic , psychological , and health - related quality - of - life ( hrqol ) disadvantages for patients and their caregivers . this is due to substantial reductions in patients normal activity levels and the indirect costs associated with the time taken from school and work for each invasive procedure.7 recent technical advances have aimed to overcome the drawbacks associated with tgr . in particular , the magnetically controlled growing rod ( mcgr ) technique is a new technology in which a growing rod , once implanted during an invasive surgical procedure ( similar to tgr ) , is lengthened noninvasively using a hand - held magnetic external remote controller.8 using the hand - held remote controller , elongation is performed in millimeters in two ways : continuous and incremental . the preclinical and clinical findings of treatment with mcgr have demonstrated mcgr as safe and effective over a followup period of up to 2 years.710 the primary advantage of the mcgr technology , compared with tgr , is that the device can be lengthened noninvasively in the physician office without anesthesia . this eliminates the need for periodic surgeries for lengthening , necessary with tgr , and has potential to reduce long - term medical resource consumption and costs in comparison to tgr and improve the hrqol for affected children.11 the health - related costs associated with the treatment of eos , using mcgr , have recently been shown to be lower compared with tgr in the european setting , specifically in france and the uk . charroin et al11 reported the cumulative cost savings over 4 years to be 6,135 from the french sickness fund perspective . in a second economic study , conducted for the uk by rolton et al,12 the estimated cumulative cost savings per child after 5 years of treatment with mcgr compared with tgr was reported to be 8,888 . one further economic study , conducted from the us payer perspective , also reported the cumulative cost savings ( $ 39,632 per patient at year 5 ) for mcgr compared with tgr.13 the objective of this research was to perform a cost analysis of mcgr compared with tgr for eos from the us integrated health care delivery system perspective over the complete 6-year episode of care from initial implantation until final fusion . to our knowledge , this is the first cost analysis of mcgr compared to tgr performed from the provider , as opposed to the payer perspective , and the first such cost analysis that accounts for the complete episode of care . we hypothesized that over time , the higher mcgr implant cost will be offset by eliminating repeated tgr surgical lengthenings , as seen in previous research . an economic model was developed using microsoft excel to compare the cost of mcgr and tgr , from the perspective of the us integrated health care delivery system . for both tgr and mcgr , the model estimated the cumulative costs associated with initial implantation , surgical revisions , device exchange , rod lengthenings , and final spinal fusion over a 6-year episode of care , from initial implantation to final fusion . model parameters were estimated on the basis of published literature , analyses of a multicenter eos database comprising the us institutions , and a series of one - on - one interviews conducted with six pediatric spine surgeons , two private payers , and two hospital purchasers . all costs represent 2015 us dollars , and were discounted at an annual rate of 3.0% , as recommended by the congressional budget office.14 the model framework and clinical parameters are reported in table 1 , and medical resource use is reported in table 2 . of note , the tgr and mcgr device failure rates were obtained from a previously conducted economic evaluation commissioned by the national institute for health and care excellence , as reported in the external assessment center report15 and journal publication.16 these rates were calculated based on the best available evidence . assumptions made in the development of this model include the following : clinical effectiveness ( curve correction and increase in thoracic height ) is equivalent between mcgr and tgr;10 resource use between tgr and mcgr with respect to initial implantation , revision , exchange , and final fusion is similar ( with the exception of the device cost , where appropriate ) ; one x - ray is required per insertion , lengthening procedure , exchange , revision , deep ssi , and final fusion ; all patients for mcgr and tgr require one device exchange surgery at 3.8 years , based on the average spine growth observed in a child aged 6 years;17 and deep ssi requires a complete replacement of implants , and superficial infection requires oral antibiotics . it was further assumed that mcgr costs were not incurred for partial or complete revisions due to mcgr failure within 1 year following an mcgr implantation or mcgr exchange ( in the unlikely event of a manufacturing defect ) ; other costs for the revisions were incurred ( eg , hospital facility costs and professional fees ) . for revisions due to anchor or foundation issues within 1 year following an mcgr implantation or mcgr exchange , hospital inpatient facility costs were based on medicare payments ( diagnosis - related group [ drg ] data ) , physician professional fees were based on current procedural terminology data , and hospital outpatient facility costs were based on ambulatory payment classification data . because bundled drg payments include the tgr device cost , for mcgr hospital inpatient procedures , the tgr device costs were subtracted from the drg payment , and the mcgr device costs were added to account for the difference in device cost . extensive one - way sensitivity analyses were conducted on a range of model variables to establish how robust the cumulative cost savings were to realistic changes in the boundaries of the key parameters and assumptions of the model . two - way sensitivity analyses were also conducted for device failure rate and the months between lengthenings . an economic model was developed using microsoft excel to compare the cost of mcgr and tgr , from the perspective of the us integrated health care delivery system . for both tgr and mcgr , the model estimated the cumulative costs associated with initial implantation , surgical revisions , device exchange , rod lengthenings , and final spinal fusion over a 6-year episode of care , from initial implantation to final fusion . model parameters were estimated on the basis of published literature , analyses of a multicenter eos database comprising the us institutions , and a series of one - on - one interviews conducted with six pediatric spine surgeons , two private payers , and two hospital purchasers . all costs represent 2015 us dollars , and were discounted at an annual rate of 3.0% , as recommended by the congressional budget office.14 the model framework and clinical parameters are reported in table 1 , and medical resource use is reported in table 2 . of note , the tgr and mcgr device failure rates were obtained from a previously conducted economic evaluation commissioned by the national institute for health and care excellence , as reported in the external assessment center report15 and journal publication.16 these rates were calculated based on the best available evidence . assumptions made in the development of this model include the following : clinical effectiveness ( curve correction and increase in thoracic height ) is equivalent between mcgr and tgr;10 resource use between tgr and mcgr with respect to initial implantation , revision , exchange , and final fusion is similar ( with the exception of the device cost , where appropriate ) ; one x - ray is required per insertion , lengthening procedure , exchange , revision , deep ssi , and final fusion ; all patients for mcgr and tgr require one device exchange surgery at 3.8 years , based on the average spine growth observed in a child aged 6 years;17 and deep ssi requires a complete replacement of implants , and superficial infection requires oral antibiotics . it was further assumed that mcgr costs were not incurred for partial or complete revisions due to mcgr failure within 1 year following an mcgr implantation or mcgr exchange ( in the unlikely event of a manufacturing defect ) ; other costs for the revisions were incurred ( eg , hospital facility costs and professional fees ) . for revisions due to anchor or foundation issues within 1 year following an mcgr implantation or mcgr exchange , hospital inpatient facility costs were based on medicare payments ( diagnosis - related group [ drg ] data ) , physician professional fees were based on current procedural terminology data , and hospital outpatient facility costs were based on ambulatory payment classification data . because bundled drg payments include the tgr device cost , for mcgr hospital inpatient procedures , the tgr device costs were subtracted from the drg payment , and the mcgr device costs were added to account for the difference in device cost . extensive one - way sensitivity analyses were conducted on a range of model variables to establish how robust the cumulative cost savings were to realistic changes in the boundaries of the key parameters and assumptions of the model . two - way sensitivity analyses were also conducted for device failure rate and the months between lengthenings . over the 6-year episode of care , mcgr was associated with an estimated 11.2 fewer invasive procedures per patient compared with tgr ( mcgr : 3.33 ; tgr : 14.53 ) . when simulating 1,000 eos patients over the 6-year episode of care , mcgr was associated with an estimated 270 fewer deep ssis ( mcgr : 80 ; tgr : 350 ) and 197 fewer revisions due to device failure compared with tgr ( mcgr : 332 ; tgr : 529 ) . the cumulative costs for treatment of eos with mcgr compared with tgr are illustrated in figure 1 . as shown in figure 1 , the higher cost associated with the initial insertion and exchange ( at 3.8 years ) of mcgr was offset by the repeated tgr surgical lengthenings and associated complications . the cumulative cost for mcgr vs tgr over the 6-year episode of care was cost neutral ( $ 61 ; mcgr minus tgr ) from the us integrated health care delivery system perspective . as shown in the one - way sensitivity analysis ( figure 2 ) , the cumulative cost savings of mcgr was most sensitive to mcgr dual rod use , months between tgr lengthenings , tgr lengthenings that are inpatient ( vs outpatient ) , mcgr cost , and time horizon . the results were less sensitive to the percent of ssis that are deep infections and years to exchange surgery . when less certain model parameters were varied in the two - way sensitivity analyses , specifically the mcgr device failure rate ( figure 3 ) and months between mcgr lengthenings ( figure 4 ) , the costs fell within a relatively narrow range when based on realistic clinical scenarios , suggesting that the economic model is robust to plausible parameter values . in the base - case analysis that evaluated cumulative 6-year costs , the differential was $ 61 ; that is , cost neutrality was achieved by the conclusion of the 6-year episode of care . the results were sensitive , however , to the time horizon due to the timing of certain procedures in the process of care , most notably the exchange surgery at 3.8 years . as a result , ~73% of the cumulative costs for mcgr were accrued in year 1 ( initial implantation ) and year 4 ( exchange surgery ) ; these costs were then offset over time . over the 6-year episode of care , mcgr was associated with an estimated 11.2 fewer invasive procedures per patient compared with tgr ( mcgr : 3.33 ; tgr : 14.53 ) . when simulating 1,000 eos patients over the 6-year episode of care , mcgr was associated with an estimated 270 fewer deep ssis ( mcgr : 80 ; tgr : 350 ) and 197 fewer revisions due to device failure compared with tgr ( mcgr : 332 ; tgr : 529 ) . the cumulative costs for treatment of eos with mcgr compared with tgr are illustrated in figure 1 . as shown in figure 1 , the higher cost associated with the initial insertion and exchange ( at 3.8 years ) of mcgr was offset by the repeated tgr surgical lengthenings and associated complications . the cumulative cost for mcgr vs tgr over the 6-year episode of care was cost neutral ( $ 61 ; mcgr minus tgr ) from the us integrated health care delivery system perspective . as shown in the one - way sensitivity analysis ( figure 2 ) , the cumulative cost savings of mcgr was most sensitive to mcgr dual rod use , months between tgr lengthenings , tgr lengthenings that are inpatient ( vs outpatient ) , mcgr cost , and time horizon . the results were less sensitive to the percent of ssis that are deep infections and years to exchange surgery . when less certain model parameters were varied in the two - way sensitivity analyses , specifically the mcgr device failure rate ( figure 3 ) and months between mcgr lengthenings ( figure 4 ) , the costs fell within a relatively narrow range when based on realistic clinical scenarios , suggesting that the economic model is robust to plausible parameter values . in the base - case analysis that evaluated cumulative 6-year costs , the differential was $ 61 ; that is , cost neutrality was achieved by the conclusion of the 6-year episode of care . the results were sensitive , however , to the time horizon due to the timing of certain procedures in the process of care , most notably the exchange surgery at 3.8 years . as a result , ~73% of the cumulative costs for mcgr were accrued in year 1 ( initial implantation ) and year 4 ( exchange surgery ) ; these costs were then offset over time . economic models facilitate exploring the extent to which costs of a new intervention may be offset by reductions in other medical costs.19 the economic model presented in this study demonstrates that the cost impact of mcgr is offset by eliminating repeated tgr surgical lengthenings and associated complications . the cost offsets for mcgr were driven by fewer deep ssis , less device failures resulting in fewer revisions , and absence of a hospital inpatient stay , anesthesia , and intraoperative neurophysiological monitoring during mcgr lengthenings . in the us , providers are becoming increasingly at financial risk as bundled payments shift the risk of financial accountability from the payer to the provider.20 with the trend toward bundled payments in orthopedics , providers will be at risk for a defined episode of care , including hospital stays , complications , and implant costs.21 this cost analysis demonstrates , from a provider perspective , the efficient provision of care with mcgr . our analysis differs in several ways from previous analyses of mcgr conducted for the european and the us settings of care.1113 most notably , our analysis evaluated cumulative costs over the 6-year episode of care ( from initial implantation through final fusion ) inclusive of the exchange surgery at 3.8 years for both tgr and mcgr . in addition , our analysis reflected the us practice patterns where the majority ( 85% ) of cases use dual - rod rather than single - rod constructs , and tgr lengthenings are performed in both the hospital inpatient and outpatient settings ( with more frail children requiring greater resources such as an intensive care unit stay).22 on the other hand , the uk study by rolton et al,12 the french study by charroin et al,11 and the us payer perspective study by su et al13 all demonstrated cumulative cost savings without accounting for the exchange surgery and assumed that tgr lengthenings are performed entirely in the hospital inpatient setting . charroin et al11 and su et al13 explain that their time horizons ( 4 and 5 years , respectively ) were selected to accommodate the complete lengthening capacity of mcgr ( 4.8 cm ) , not necessarily a full episode of care . in the present study at 3 years following initial implantation and prior to the exchange surgery at 3.8 years , mcgr resulted in a cumulative cost savings of $ 2,943 compared to tgr ( figure 1 ) , which is consistent with the prior research studies that did not account for the exchange surgery . this finding of cumulative cost savings at 3 years following initial implantation of mcgr may also apply to surgeries involving conversions of tgr to mcgr where an mcgr exchange procedure is not anticipated.23 as noted previously , we performed a cost analysis of mcgr compared with tgr for eos from the us provider perspective , whereas su et al13 performed a similar analysis but from the us payer perspective . of note , the database contains hospital charge data ( as reflected on a hospital bill ) , not insurer payments or the actual cost to the hospital for the provision of care to the patient . given that insurers generally pay less than hospital charges,25 it remains unclear to what degree the use of hospital charges may have affected the difference in cumulative costs between tgr and mcgr reported by su et al.13 further , in our study , we incorporated a distribution of practice settings ( roughly half hospital inpatient and half hospital outpatient ) for performing tgr lengthenings based on a multicenter eos database comprising us institutions,22 whereas su et al13 assumed that tgr lengthenings were performed entirely in the hospital inpatient setting in the us . the approximate medicare reimbursement for tgr lengthenings is $ 6,000 ( when considering both practice settings ) , compared to the $ 10,000 in hospital inpatient charges noted by su et al.13 if one takes 60% of the cumulative cost of tgr lengthenings estimated by su et al,13 the cost savings of mcgr reported by su et al13 are diminished ; nevertheless , it appears that mcgr would be cost neutral in year 5 from the us payer perspective , which is similar to our results at year 6 from the us provider perspective . where data for mcgr were not available from the published literature , conservative assumptions were made by using the same parameter values as for tgr based on tgr clinical studies , in particular the study by bess et al.18 when these less certain mcgr clinical parameters were varied within a plausible range , the inferences of the model did not change suggesting that the model is robust . while not a limitation per se , it is worth noting that the model presented is a cost analysis and not a cost - effectiveness analysis therefore , similar to previous economic research of mcgr , it has not accounted for , 1 ) the pain , psychological distress , and compromised hrqol associated with invasive tgr lengthening surgeries , or 2 ) the shorter recovery time with mcgr lengthenings , which results in less time away from usual activities for young patients and their families . cost offsets for mcgr accrue over time and reflect the elimination of repeated invasive tgr surgical lengthenings and their associated complications . despite the higher up - front cost for insertion of the mcgr implant and later for exchange of the mcgr implant , the cumulative cost of mcgr is offset over the 6-year episode of care from the perspective of the us integrated health care delivery system .

throughout the history of canada , first nations interests have not been represented with respect to laws , regulations and enforcement towards the environmental protection and management of their lands.1,2 in canada , a series of municipal , provincial and federal laws and regulations have been developed to govern land use for the environmental protection of off - reserve communities . although these laws and regulations are in place , enforced , and utilized as tools to control an extensive assortment of environmental risks in off - reserve communities , no such laws of environmental protection are currently in place for first nations communities.24 for over a century ( from 1876 to the early 1980 s ) , the indian act has been the main mechanism employed to govern the use of first nations lands).5 under this act , indian and northern affairs canada ( inac ) is the authority responsible for regulating first nations land use , environmental management and protection . in the early 1980 s , through the development of land management and administrative programs , inac provided some options to first nations to enable communities to assume more land management responsibilities . the regional lands administration program ( rlap ) and the delegated lands management programs ( dlmp ) , introduced in the 1980 s , but then replaced in 2005 by the reserve land and environment management program ( rlemp ) , enabled the transfer of various land use planning , environmental and natural resource management and administrative land transaction responsibilities from inac to first nations3,6 the only current alternative , under the indian act , is the first nations land management act ( fnlma ) , a federal law enacted in 1999 which provided signatory first nations law - making authority over reserve lands , resources and the environment.7 the fnlma sanctioned a framework agreement on first nations land management , negotiated and signed , in 1996 , between 14 first nations and the minister of indian affairs and northern development . in 2002 , the land management regime was opened to other interested first nations , however , policy decisions were made in 2008 to close the regime to new entrants due to the lack of sustainable long - term funding and thus the majority of first nations communities are governed by inac under the indian act.3,6 during negotiations of the framework agreement , solid waste was identified as one of 4 major environmental threats to first nations communities.3,8 in addition , discharge of household sewage and industrial and commercial wastewater into surface water , fuel storage tanks and environmental emergencies , such as chemical spills , were considered essential for environmental protection and recognized to pose significant risks on reserves . in 2007 , environment canada also recognized landfills , solid and hazardous wastes , as well as the air emissions from incineration and open burning of garbage as significant on - reserve risks requiring immediate attention.3 the minister of indian and northern affairs canada , under the indian reserve waste disposal regulations , is responsible for the operation of landfills and waste dumps in first nations communities.9 to date , inac neither promotes nor conducts significant surveillance of dumping sites and is not equipped to monitor compliance , conduct inspections or enforce waste disposal regulations.3,10 thus the majority of waste sites in first nations communities across canada remain unregulated.1,2 under current national and provincial legislation , the location of landfills on off - reserve communities requires a geological site selection process ; however in most first nations communities , derivation of landfill ( more appropriately referred to as community dump site ) location does not consider this process . as a result , community dump sites are often developed in geologically unacceptable sites and are not equipped with modern engineered liners and leachate collection systems . community dump sites are known to be located in areas with silty sandy soils , near surface water features , rather than in more geologically acceptable areas composed of less permeable soils and distant from major water sources.1,4 additionally , abandoned dump sites are not monitored for gas emissions or appropriately sealed with a clay cap , to prevent vertical penetration of water into wastes.1,4,11 at present there are no central records documenting past as well as current waste disposal practices in saskatchewan first nations and the number and location of active and inactive dump sites are not known.1,11 inadequately managed solid waste disposal sites are common sources of pollution in first nations communities12 and the detrimental effects of ineffective waste management practices on human and environmental health is well documented.13 first nations communities are highly dependent on the health of the environment and the concerns of first nations people with respect to the adverse effects of inadequate dump site locations , garbage burning , and poor waste management practices have increased over the years.1,4,11,14 the historical poor management , monitoring and remediation of solid waste facilities across canada s first nations communities and the lack of current resolve over this issue has left many first nations people feeling the consequences of environmental pollution . one of the first well - documented cases , to the present issue includes mercury poisoning in the asubpeeschoseewagong netum anishnabek community in the 1960 s as a result of the dryden chemical company discharging chemical waste directly into the english - wabigoon river system , a main water source for the asubpeeschoseewagong netum anishnabeck , or grassy narrows first nations community located north of kenora , ontario . the anishnabek suffered economically , socially and culturally as a result of the mismanagement and illegal disposal of industrial chemical waste . some thirty years later , there are still severe restrictions on game fish consumption,1517 unemployment is at a high of 80% , social problems have arisen as a result of the loss of the communities self - sufficiency,15 community members are more reliant on market foods due to the growing lack of confidence in traditional foods due to the pollution of their river system and today , still suffer the effects of mercury poisoning.18 the disruption and decay of cultural practices and participation in traditional economy ( hunting , fishing trapping ) , as a result of perceived , or documented , cases of environmental contamination , has had a negative impact on the health and well - being of individuals , their families , and communities and has contributed to the current inequity in the health status of first nations in canada.19,20 in 1976 the united states environmental protection agency ( usepa ) stated : 90 percent of municipal and industrial wastes are disposed of on land in environmentally questionable ways . the results are potential public health problems , groundwater contamination by leachate , surface water pollution by runoff , air pollution from open burning , fires , and explosions at dumps , and risks to ecological systems.21 in fact , cases have been documented where leachate from municipal solid waste disposal sites have contaminated groundwater that supplied residential wells in sayvill , long island new york,22 and rockford illinois.23 hazardous substances associated with waste management have been identified as persistent environmental pollutants and known or suspected human carcinogens.13 waste incineration is known to produce a variety of pollutants from the combustion of chemical , industrial and household wastes . these can be grouped as particles and gases , metals and organic compounds.24 metals such as cadmium , mercury , arsenic , chromium , nickel ; organics that include , dioxins , polychlorinated biphenyls ( pcb ) , polyaromatic hydrocarbons ( pahs ) ; particulate matter and sulphur dioxide , are waste associated pollutants that have been classified as having the greatest potential impact on human health based on their environmental persistence , bioaccumulation , amount emitted and their inherent toxicity.13 the impact of current and historical waste disposal practices of the environment and human health of first nations communities across canada has yet to be adequately addressed or investigated . ( stc ) and the health canada regional environmental health officer , for the saskatchewan region , identified the potential for contamination of surface and groundwater by historical and current dump sites as a top priority . this issue was identified as critical since most first nations rely on local surface or groundwater for their drinking water supplies , and the potential impacts to health and environment associated with waste disposal practices is potentially significant through contamination of drinking water supplies in first nations communities . groundwater analysis , in areas of active and inactive community dumps , as well as backyard waste disposal pits indicated that health- and aesthetic- based water quality guidelines for canadian drinking water quality ( gcdwq ) were exceeded in water samples collected for analysis . parameters found to exceed gcdwq25 included : chloride , sulphate , ph , arsenic , barium , iron , manganese , lead , total dissolved solids and total coliform bacteria.1 the results of the water analysis demonstrated a risk to groundwater quality in the areas of waste disposal . chemical analysis of soil and ash samples collected from one active community dump site indicated the presence of dioxins and furans at concentrations well above the canadian council of ministers of the environment ( ccme)26 soil quality guidelines for the protection of human and environmental health . these results suggest a risk for the community to dioxin and furan exposures at levels above the safety guidelines . a community - based participatory research project ( cbpr ) was initiated to describe waste disposal practices and to explore health and environmental concerns related to these practices in three saskatchewan first nations communities . this research project serves to present the views of first nations elders on past and present waste disposal practices in their communities . this article is a presentation and analysis of the knowledge presented on waste management practices during sharing circles and one to one interviews with participating elders from three saskatchewan first nations communities . the community - based participatory research project was initiated by the saskatoon tribal council health and family services to investigate concerns related to waste disposal and possible groundwater contamination in first nations communities in saskatchewan . the research reported here was part of the larger cbpr project that examined the hydrological , microbiological , and toxicological aspects of active and inactive waste sites in three saskatchewan first nations communities . community - based participatory research strives to be community situated , collaborative and action oriented.2729 this specific project was based on a respectful collaboration built between researchers and communities for the purpose of creating new knowledge and understanding of waste disposal that was of practical relevance to the involved communities . with the inclusion of a qualitative approach , we aimed to gain an understanding of the past and present waste disposal practices in three first nations communities and the potential human and environmental health concerns related to these practices of waste disposal . all participating communities are located north of saskatoon , saskatchewan , canada , an urban center with approximately 250,000 residents located in the center of the province of saskatchewan . mistawasis first nation is located northwest of saskatoon , has approximately 2,254 registered members , and is situated on 12,431 hectares of land , the majority of which is forest and lakes , with some agriculture activity.1 the muskeg lake cree nations is located north of saskatoon , has approximately 1950 registered members , a land mass of 7536 hectares and agricultural is a major economic activity . the muskoday first nation is located northeast of saskatoon , spans approximately 9687 hectares and has 1583 registered members.1 depending on community preference , information was gathered utilizing one of two approaches ; a one to one interview or a sharing circle.30,31 two one to one interviews were conducted with two female elders from one of the participating communities . three sharing circles , consisting of both men and women were conducted in each of the participating communities . interviews and sharing circles were held at the health centre s of the participating communities . significant topics of inquiry directed discussions however opportunities for communication between participants as well as participants and interviewer took place during these sessions . they were invited to share their experiences of waste disposal as they remembered it when they were younger ( late 1950 s ) . elders were also asked to share their perspectives on present waste management practices and on the possible impacts of waste practices on community and environmental health . in general , one to one interviews took place with female participants and ranged in duration from seven to twenty five minutes . sharing circles generally had equal gender representation , were composed of at least three men and three women and ranged in duration from thirteen to sixty minutes . interviews and sharing circles were videotaped and recorded into dvd format and conducted in the summer of 2004 . in keeping with the principles of ownership , control , access , possession ( ocap ) and community - based participatory research,32 a consultative and approval process was undertaken with elders along with chief and council prior to the engagement of research and dissemination activities.28 thematic content analysis of transcribed interview and sharing circles was conducted to identify main themes arising from participants responses.33 video recordings were digitally transcribed verbatim . utilizing atlas.ti software , transcribed data were managed , organized , and coded by use of topic and descriptive categories , into emerging themes.34,35 major emergent themes were identified using an iterative process of comparison and evaluation across interviews and sharing circles . comparative data analyses36 were used to uncover similar and dissimilar relationships among emergent themes within and between communities . the analysis revealed that historical waste disposal practices among the three communities were fairly consistent . in the past options for waste disposal were limited and the homeowner was exclusively responsible for disposing waste . typically , households generated small volumes of garbage and a backyard pit or personal waste site was the most common past practice of waste disposal . larger waste sites , designated for the disposal and eventual burial of metals , iron scrap , construction materials and appliances , were also utilized in the past by community residents . the number of larger waste sites , in operation , at any one time , in a single community , was not determined . however it was assumed that a single large waste site was in operation until capacity , at which time , the contents at the site were buried and a new site was created . elders did not refer to these large waste sites as public dumping grounds or landfills but considered these sites as places to store junk . overtime , the responsibility for waste disposal transferred from the homeowner to the community . waste disposal practices evolved from backyard pits and larger waste sites to weekly pick - up of un - segregated garbage with waste disposal into larger community dump sites . this present practice , a convention on many first nations communities across canada today , includes open - air dumping coupled with trash burning . like many communities across the nation , community dumps , are built without engineered liners , leachate collection systems , and a site selection process inclusive of sound geological information.1 open - air dumping coupled with trash burning was the current practice in two communities . weekly garbage truck pick - up with off - reserve disposal in an appropriately engineered landfill , governed by a local municipality , was currently practiced in the third community . elders expressed that community dumpsite locations as well as the practice of open trash burning were significant health issues related to waste disposal practices in their communities . waste segregation and recycling were recommended as future waste management practices by elders from all communities . overall , discussions with elders were centered on the description of past and present practices of waste disposal , the health and environmental issues related to these waste practices and the options for future waste management . all participants indicated that historically very little household waste was generated and most waste consisted of glass and old cans . for example one elder explained : we did nt create much garbage , you know . a few tomato cans and usually a pile of moss that my mother used for moss bags . and that s all i knew of a few tomato cans and usually a pile of moss that my mother used for moss bags . and that s all i knew of . elders recalled some disposal of old furniture , vehicle scraps , oil cans and wood . these items were in general , personally disposed in an area within an individual s backyard or in a larger waste sited located in a designated area within the community . these sites of waste disposal were not considered public dump sites and were often referred to as places to store junk . elders recollected that these junk storage sites were also utilized for the disposal of metals and iron scrap such as fridges and stoves , as well as the disposal of construction materials during the period when reserve housing was built . for example one elder recalled : i do nt remember any landfills or anything like that . we may have had some place to store junk like iron and stuff like that . we may have had some place to store junk like iron and stuff like that . elders described that community members had very few options for waste disposal , there was no central management and methods were limited . public garbage dumps were non - existent and in general community residents were responsible for their own waste disposal . primarily due to convenience and as a result of the limitations in waste disposal methodologies , historically , a small pit dug in the backyard was the most common past practice for disposing waste . briefly a household member would simply dig a pit in the backyard , dispose their garbage into the pit , ignite the garbage and eventually cover the ashes with dirt thus refilling the pit . an alternate site in the backyard the methods of past waste disposal were expressed by three elders as follows : they used to dig a hole . and burn it . then cover it.when we used to throw our garbage , you know , in the garbage dump , we used to burn it right away . so when it got full , just buried it and then make another little hole . just a little hole.we did nt have much choice in my younger days garbage was nt just thrown around . it was always kept , you know , stuff that got burnt was burnt and the stuff that could be buried was buried . they used to dig a hole . and burn it . go in there and light a match . and then cover it . when we used to throw our garbage , you know , in the garbage dump , we used to burn it right away . so when it got full , just buried it and then make another little hole . we did nt have much choice in my younger days garbage was nt just thrown around . it was always kept , you know , stuff that got burnt was burnt and the stuff that could be buried was buried . all elders recalled that numerous unfenced backyard pits and larger dump sites were sporadically located across reserve land . these past dump sites were generally placed in locations away from homes however as the community expanded , houses were built in close proximity to covered waste sites . backyard pits and larger dump sites , designated for metal waste , were not considered public waste sites and central records documenting the location or quantity of these sites were not available or in existence . elders commented that it was likely that existing residences are located in close proximity to covered waste sites as a result of sporadic placement and absence of any central waste site records . three elder s commented on this topic as follows : there used to be one by [ name ] there , like , before the house was built.[name ] was saying there was one over here where her place is now too . you know , [ name ] was telling us that right where our garden is was a dump . and when [ name ] works that up , he brings up old tin and stuff . there used to be one by [ name ] there , like , before the house was built . [ name ] was saying there was one over here where her place is now too . you know , [ name ] was telling us that right where our garden is was a dump . and when [ name ] works that up , he brings up old tin and stuff . in summary , past waste disposal practices were similar among communities , and homeowners were primarily responsible for disposing waste . historically , households generated small volumes of garbage and a backyard pit was the most convenient , practical and common practice for waste disposal . a small number of larger waste sites were utilized for the disposal and eventual burial of metals , iron scrap , construction materials and appliances and a small number are still in operation today . past waste sites were sporadic and not formally documented and as communities grew , houses were built in close proximity to covered waste sites perhaps posing some health risks . the total number of backyard or personal waste disposal pits could not be determined in each community . however it was estimated , based on the information gathered , that at least 100 nonoperational pits , could exist on community land . these pits were mainly utilized for the disposal of household , some agricultural , and construction wastes . although backyard pits were primarily utilized for household waste disposal in the past , it was established that in one community , a backyard pit was utilized for the preservation of wooden fence posts and still contained traces of chromium , copper and arsenic . like the backyard pits , elders also indicated that a few larger waste sites were still in operation today . the past locations and total number of the nonoperational sites was not known or recorded . elders stated that there was a strong possibility homes were built in areas where these former waste sites were once in operation . open - air dumping , coupled with trash burning was the current waste management practice employed in two of the three communities involved in the project . resident s of these communities disposed all household and industrial waste into residential bins which were emptied weekly by use of a garbage truck . waste was transported to , and then disposed of , in the community s waste site . waste disposed was un - segregated and included : plastics , household products , paper , industrial wastes such as paint and oil containers as well as tires . one participant describes how tires , oil and paint cans , and old oil , items that generally require separate disposal , are simply disposed of along with other materials . i think it s one big hole , yeah . all refuse brought to the community waste site was burned . in one community garbage was burned twice a week , every monday and thursday . the details regarding the frequency of garbage burning in the other community was not explained . segregation and recycling of waste materials was not currently practiced in these communities . with the exception of two concerns ; burning garbage and waste site location , the elders of these communities were generally satisfied with the current waste disposal practices in their communities . two elders commented on the current waste practices as follows : looking at it from past years and now , it s much cleaner.im proud of our reserve , you know ? very proud . i m proud of our reserve , you know ? very proud . cause it looks clean all the time . weekly garbage truck pick - up with off - reserve disposal , to a neighbouring city landfill , was the current practice in one of the three communities involved in the project . household garbage was disposed into residential bins , emptied weekly by garbage truck and then transported to the landfill . to some extent waste segregation for example ; paint cans and other paint products were separated from household garbage and taken to a depot in the city . various metals and household appliances ( washers , dryers , refrigerators , freezers , stoves , bed frames etc . ) were disposed in a designated site on reserve . how these items were dealt with following their disposal to the site elders , from this community also indicated that a community waste site utilized prior to off - reserve disposal was cleaned out with the majority of its contents hauled to the neighboring city s landfill . a non - resident would travel from the nearby city and haul the cars off reserve however this is not a current practice and abandoned cars litter the community . with the exception of the abandoned car issue the elders indicated that they are pleased with the current waste disposal methods practiced in their community . as one elder stated : we re sort of ah , fairly happy with what we got with dealing with our waste right now . we re sort of ah , fairly happy with what we got with dealing with our waste right now . elders noted that overtime , the volume of household waste increased and the responsibility of waste disposal on reserve transferred from the individual to the community . as a result the practice of waste disposal evolved from the backyard pit to the community dump site . however , the modification in disposal methods may have occurred in time with , and as a consequence of , population growth and the increased availability of over - packaged commercial commodities within these communities . present waste disposal methods practiced encompassed either open - air dumping coupled with trash burning or off - reserve disposal . on the whole , all elders raised concerns over the potential effects of past and present waste disposal practices on human and environmental health . health related discussions were centered on dump site locations and the current practice of open air trash burning at the community dump sites . methods of dump and burn are commonly utilized in first nations across saskatchewan , yet these methods came to a halt in non - reserve communities with the enforcement of the provincial new clean air regulations act in 1989.37 the community waste sites contain a variety of waste materials including ; plastics , wood , paper , cardboard , tires , and electronics . open air trash burning is known to create conditions for incomplete combustion26 and , as a result , a complex mixture of substances can be released to air for potential inhalation . carbon monoxide , carbon dioxide , and nitrogen oxides represent the largest portion of pollutants emitted from open trash burning.26,38 however , other chemicals such as ; benzene , styrene , formaldehyde , polychlorinated dibenzodioxins ( pcdds ; also known as dioxins ) , polychlorinated biphenyls ( pcbs ) , polychlorinated dibenzofurans ( pcdfs ; also known as furans ) , and heavy metals ( lead , mercury and arsenic ) have been detected in smoke produced through the incineration of waste materials.3840 open air burning was conducted in two of the three communities . this practice was carried out mainly due to the small size and limited capacity of the current community dump sites . all elders were dissatisfied with the practice of open air trash burning in their communities . elders demonstrated concern for community members living in close proximity to the community dump and spoke of potential risks to their health , the health of their children and the air quality in their communities in relation to this practice of waste disposal . the worst part of it is they burn that and all that smoke gets into our little village . the worst part of it is they burn that and all that smoke gets into our little village . another elder expressed great concern over the impacts to the ambient air quality in their community as a result of burning tires at the community dump site . the elder commented : no [ i do nt notice any smells from the garbage dump ] , but the only thing i do nt like seeing burnt is tires . there s a thing in those tires , they come and it goes all over . and maybe there is a lot of other things there that we do nt like [ being burnt ] . no [ i do nt notice any smells from the garbage dump ] , but the only thing i do nt like seeing burnt is tires . there s a thing in those tires , they come and it goes all over . and maybe there is a lot of other things there that we do nt like [ being burnt ] . the burning of refuse helps to minimize the build - up of materials in the community dump sites ; however , as a number of respondents pointed out , the smoke is a nuisance , and they are concerned with the possible impacts to ambient air quality , exposures to airborne contaminants and the impacts to the health of their children and community members as a result of these airborne exposures . all participating elders noted that past and present dump site locations were an environmental issue . elders expressed concern over the potential contamination of local lakes and rivers in their communities through run - off from the community dump site . for example as two elder s acknowledged : there s a lake by the garbage dump too . and some of these lakes , you know , they have a stream going from lake to lake or creek and that can carry that disease right down to where we get our water.so what would be stopping that there after they burn that , melts the plastic , big rain comes . there s a lake by the garbage dump too . and some of these lakes , you know , they have a stream going from lake to lake or creek and that can carry that disease right down to where we get our water . so what would be stopping that there after they burn that , melts the plastic , big rain comes . it is not surprising that ground and surface water pollution was an environmental concern for the elders of these communities . first nations waste sites in general , are not built with engineered liners , leachate collection systems , and a site selection process , based on sound geological information , is not completed . for example ; one of the current operational community dumpsites , was located in an area of silty sandy soils close to a lake.1 elders from all three communities recalled that in the past , a major staple of their diet was fish . however , elder s indicated that members in the community , including themselves , no longer eat fish locally caught in their communities . this change in fish consumption behaviour was mainly attributed to the perception that the lakes , rivers and fish are contaminated . elders noted , through visual observations , that fish health , appearance and meat quality have declined over the years . not like they used to anyway.you could go down to the river anytime and go and catch a few fish , you know , for a meal . you could go down to the river anytime and go and catch a few fish , you know , for a meal . elder s comments indicate that point source contamination of community water and fish supplies are considerable concerns in their communities . throughout discussions on waste disposal practices they pointed out for example that , separating garbage at home into recyclable , compostable , and toxic materials would be important steps in decreasing the amount of garbage sent to the community waste site . there is all kinds of ways that we would cut down on the amount that s going into the landfill . one thing we could do to help is ah , i know i would have a hard time with it , but we get used to things and you start doing them . the garbage should be sorted at home and then plastics could be recycled papers , those can all be recycled . the only thing i can think of right now is what we talked about is some of our disposal , i guess , garbage bins , we call it here . cardboard boxes , stuff like that . that stuff can be recycled you see- certainly take a lot less room on [ name ] truck there . elders indicated that the recycling of plastics , papers and batteries could be introduced as a first step for waste reduction . they indicated that special recycling bins could be distributed to individual homes in the community or could be located in designated locations where community members could then drop off their recyclables . several elders noted : i think maybe that s what we could use on this reserve here is a place to , maybe even in the village , maybe three or four places where a person could take these recyclable materials , just like a mesh bin or something . take these paint cans and stuff and throw them in there instead of throwing them anyplace else . and they could be picked up there once in a while.but you see , if we had a recycling bin , all that stuff would be put in there , whatever we do nt use , like magazines ... i still say we could use a recycling bin for paper and also for cans . like paint cans and all that stuff i think maybe that s what we could use on this reserve here is a place to , maybe even in the village , maybe three or four places where a person could take these recyclable materials , just like a mesh bin or something . take these paint cans and stuff and throw them in there instead of throwing them anyplace else . and but you see , if we had a recycling bin , all that stuff would be put in there , whatever we do nt use , like magazines ... i still say we could use a recycling bin for paper and also for cans . like paint cans and all that stuff elders recognized that it would take some time to implement segregation and recycling in their communities . however they optimistically indicated that overtime these methods would eventually be accepted and practiced within their communities . as one elder briefly commented : but it would catch on . it would work . the elders from these communities provided several relevant solutions to remedy some of the issues with current waste disposal practices in their communities . introducing bins for recyclable materials and teaching residents to separate garbage at home are achievable targets that would reduce waste entering the community dump site . all participants indicated that historically very little household waste was generated and most waste consisted of glass and old cans . for example one elder explained : we did nt create much garbage , you know . a few tomato cans and usually a pile of moss that my mother used for moss bags . and that s all i knew of a few tomato cans and usually a pile of moss that my mother used for moss bags . and that s all i knew of . elders recalled some disposal of old furniture , vehicle scraps , oil cans and wood . these items were in general , personally disposed in an area within an individual s backyard or in a larger waste sited located in a designated area within the community . these sites of waste disposal were not considered public dump sites and were often referred to as places to store junk . elders recollected that these junk storage sites were also utilized for the disposal of metals and iron scrap such as fridges and stoves , as well as the disposal of construction materials during the period when reserve housing was built . for example one elder recalled : i do nt remember any landfills or anything like that . we may have had some place to store junk like iron and stuff like that . we may have had some place to store junk like iron and stuff like that . elders described that community members had very few options for waste disposal , there was no central management and methods were limited . public garbage dumps were non - existent and in general community residents were responsible for their own waste disposal . primarily due to convenience and as a result of the limitations in waste disposal methodologies , historically , a small pit dug in the backyard was the most common past practice for disposing waste . briefly a household member would simply dig a pit in the backyard , dispose their garbage into the pit , ignite the garbage and eventually cover the ashes with dirt thus refilling the pit . an alternate site in the backyard the methods of past waste disposal were expressed by three elders as follows : they used to dig a hole . and burn it . then cover it.when we used to throw our garbage , you know , in the garbage dump , we used to burn it right away . so when it got full , just buried it and then make another little hole . just a little hole.we did nt have much choice in my younger days garbage was nt just thrown around . it was always kept , you know , stuff that got burnt was burnt and the stuff that could be buried was buried . when we used to throw our garbage , you know , in the garbage dump , we used to burn it right away . so when it got full , just buried it and then make another little hole . we did nt have much choice in my younger days garbage was nt just thrown around . it was always kept , you know , stuff that got burnt was burnt and the stuff that could be buried was buried . all elders recalled that numerous unfenced backyard pits and larger dump sites were sporadically located across reserve land . these past dump sites were generally placed in locations away from homes however as the community expanded , houses were built in close proximity to covered waste sites . backyard pits and larger dump sites , designated for metal waste , were not considered public waste sites and central records documenting the location or quantity of these sites were not available or in existence . elders commented that it was likely that existing residences are located in close proximity to covered waste sites as a result of sporadic placement and absence of any central waste site records . three elder s commented on this topic as follows : there used to be one by [ name ] there , like , before the house was built.[name ] was saying there was one over here where her place is now too . you know , [ name ] was telling us that right where our garden is was a dump . and when [ name ] works that up , he brings up old tin and stuff . there used to be one by [ name ] there , like , before the house was built . [ name ] was saying there was one over here where her place is now too . you know , [ name ] was telling us that right where our garden is was a dump . and when [ name ] works that up , he brings up old tin and stuff . in summary , past waste disposal practices were similar among communities , and homeowners were primarily responsible for disposing waste . historically , households generated small volumes of garbage and a backyard pit was the most convenient , practical and common practice for waste disposal . a small number of larger waste sites were utilized for the disposal and eventual burial of metals , iron scrap , construction materials and appliances and a small number are still in operation today . past waste sites were sporadic and not formally documented and as communities grew , houses were built in close proximity to covered waste sites perhaps posing some health risks . the total number of backyard or personal waste disposal pits could not be determined in each community . however it was estimated , based on the information gathered , that at least 100 nonoperational pits , could exist on community land . these pits were mainly utilized for the disposal of household , some agricultural , and construction wastes . although backyard pits were primarily utilized for household waste disposal in the past , it was established that in one community , a backyard pit was utilized for the preservation of wooden fence posts and still contained traces of chromium , copper and arsenic . like the backyard pits , elders also indicated that a few larger waste sites were still in operation today . the past locations and total number of the nonoperational sites was not known or recorded . elders stated that there was a strong possibility homes were built in areas where these former waste sites were once in operation . open - air dumping , coupled with trash burning was the current waste management practice employed in two of the three communities involved in the project . resident s of these communities disposed all household and industrial waste into residential bins which were emptied weekly by use of a garbage truck . waste was transported to , and then disposed of , in the community s waste site . waste disposed was un - segregated and included : plastics , household products , paper , industrial wastes such as paint and oil containers as well as tires . one participant describes how tires , oil and paint cans , and old oil , items that generally require separate disposal , are simply disposed of along with other materials . i think it s one big hole , yeah . all refuse brought to the community waste site was burned . in one community garbage was burned twice a week , every monday and thursday . the details regarding the frequency of garbage burning in the other community was not explained . segregation and recycling of waste materials was not currently practiced in these communities . with the exception of two concerns ; burning garbage and waste site location , the elders of these communities were generally satisfied with the current waste disposal practices in their communities . two elders commented on the current waste practices as follows : looking at it from past years and now , it s much cleaner.im proud of our reserve , you know ? very proud . cause it looks clean all the time . looking at it from past years and now , it s much cleaner weekly garbage truck pick - up with off - reserve disposal , to a neighbouring city landfill , was the current practice in one of the three communities involved in the project . household garbage was disposed into residential bins , emptied weekly by garbage truck and then transported to the landfill . to some extent waste segregation for example ; paint cans and other paint products were separated from household garbage and taken to a depot in the city . various metals and household appliances ( washers , dryers , refrigerators , freezers , stoves , bed frames etc . ) were disposed in a designated site on reserve . how these items were dealt with following their disposal to the site elders , from this community also indicated that a community waste site utilized prior to off - reserve disposal was cleaned out with the majority of its contents hauled to the neighboring city s landfill . a non - resident would travel from the nearby city and haul the cars off reserve however this is not a current practice and abandoned cars litter the community . with the exception of the abandoned car issue the elders indicated that they are pleased with the current waste disposal methods practiced in their community . as one elder stated : we re sort of ah , fairly happy with what we got with dealing with our waste right now . we re sort of ah , fairly happy with what we got with dealing with our waste right now . elders noted that overtime , the volume of household waste increased and the responsibility of waste disposal on reserve transferred from the individual to the community . as a result the practice of waste disposal evolved from the backyard pit to the community dump site . however , the modification in disposal methods may have occurred in time with , and as a consequence of , population growth and the increased availability of over - packaged commercial commodities within these communities . present waste disposal methods practiced encompassed either open - air dumping coupled with trash burning or off - reserve disposal . on the whole , all elders raised concerns over the potential effects of past and present waste disposal practices on human and environmental health . health related discussions were centered on dump site locations and the current practice of open air trash burning at the community dump sites . methods of dump and burn are commonly utilized in first nations across saskatchewan , yet these methods came to a halt in non - reserve communities with the enforcement of the provincial new clean air regulations act in 1989.37 the community waste sites contain a variety of waste materials including ; plastics , wood , paper , cardboard , tires , and electronics . open air trash burning is known to create conditions for incomplete combustion26 and , as a result , a complex mixture of substances can be released to air for potential inhalation . carbon monoxide , carbon dioxide , and nitrogen oxides represent the largest portion of pollutants emitted from open trash burning.26,38 however , other chemicals such as ; benzene , styrene , formaldehyde , polychlorinated dibenzodioxins ( pcdds ; also known as dioxins ) , polychlorinated biphenyls ( pcbs ) , polychlorinated dibenzofurans ( pcdfs ; also known as furans ) , and heavy metals ( lead , mercury and arsenic ) have been detected in smoke produced through the incineration of waste materials.3840 open air burning was conducted in two of the three communities . this practice was carried out mainly due to the small size and limited capacity of the current community dump sites . all elders were dissatisfied with the practice of open air trash burning in their communities . elders demonstrated concern for community members living in close proximity to the community dump and spoke of potential risks to their health , the health of their children and the air quality in their communities in relation to this practice of waste disposal . the worst part of it is they burn that and all that smoke gets into our little village . the worst part of it is they burn that and all that smoke gets into our little village . another elder expressed great concern over the impacts to the ambient air quality in their community as a result of burning tires at the community dump site . the elder commented : no [ i do nt notice any smells from the garbage dump ] , but the only thing i do nt like seeing burnt is tires . there s a thing in those tires , they come and it goes all over . and maybe there is a lot of other things there that we do nt like [ being burnt ] . no [ i do nt notice any smells from the garbage dump ] , but the only thing i do nt like seeing burnt is tires . there s a thing in those tires , they come and it goes all over . and maybe there is a lot of other things there that we do nt like [ being burnt ] . the burning of refuse helps to minimize the build - up of materials in the community dump sites ; however , as a number of respondents pointed out , the smoke is a nuisance , and they are concerned with the possible impacts to ambient air quality , exposures to airborne contaminants and the impacts to the health of their children and community members as a result of these airborne exposures . all participating elders noted that past and present dump site locations were an environmental issue . elders expressed concern over the potential contamination of local lakes and rivers in their communities through run - off from the community dump site . for example as two elder s acknowledged : there s a lake by the garbage dump too . and some of these lakes , you know , they have a stream going from lake to lake or creek and that can carry that disease right down to where we get our water.so what would be stopping that there after they burn that , melts the plastic , big rain comes . there s a lake by the garbage dump too . and some of these lakes , you know , they have a stream going from lake to lake or creek and that can carry that disease right down to where we get our water . so what would be stopping that there after they burn that , melts the plastic , big rain comes . it is not surprising that ground and surface water pollution was an environmental concern for the elders of these communities . first nations waste sites in general , are not built with engineered liners , leachate collection systems , and a site selection process , based on sound geological information , is not completed . for example ; one of the current operational community dumpsites , was located in an area of silty sandy soils close to a lake.1 elders from all three communities recalled that in the past , a major staple of their diet was fish . however , elder s indicated that members in the community , including themselves , no longer eat fish locally caught in their communities . this change in fish consumption behaviour was mainly attributed to the perception that the lakes , rivers and fish are contaminated . elders noted , through visual observations , that fish health , appearance and meat quality have declined over the years . not like they used to anyway.you could go down to the river anytime and go and catch a few fish , you know , for a meal . well people do fish around here but i do nt think anyone eats them . there the fish do nt even look good to me . not like they used to anyway . you could go down to the river anytime and go and catch a few fish , you know , for a meal . elder s comments indicate that point source contamination of community water and fish supplies are considerable concerns in their communities . throughout discussions on waste disposal practices , elders provided recommendations to improve current waste management practices in their communities . they pointed out for example that , separating garbage at home into recyclable , compostable , and toxic materials would be important steps in decreasing the amount of garbage sent to the community waste site . there is all kinds of ways that we would cut down on the amount that s going into the landfill . one thing we could do to help is ah , i know i would have a hard time with it , but we get used to things and you start doing them . the garbage should be sorted at home and then plastics could be recycled the only thing i can think of right now is what we talked about is some of our disposal , i guess , garbage bins , we call it here . that stuff can be recycled you see- certainly take a lot less room on [ name ] truck there . elders indicated that the recycling of plastics , papers and batteries could be introduced as a first step for waste reduction . they indicated that special recycling bins could be distributed to individual homes in the community or could be located in designated locations where community members could then drop off their recyclables . several elders noted : i think maybe that s what we could use on this reserve here is a place to , maybe even in the village , maybe three or four places where a person could take these recyclable materials , just like a mesh bin or something . take these paint cans and stuff and throw them in there instead of throwing them anyplace else . and they could be picked up there once in a while.but you see , if we had a recycling bin , all that stuff would be put in there , whatever we do nt use , like magazines ... i still say we could use a recycling bin for paper and also for cans . like paint cans and all that stuff i think maybe that s what we could use on this reserve here is a place to , maybe even in the village , maybe three or four places where a person could take these recyclable materials , just like a mesh bin or something . take these paint cans and stuff and throw them in there instead of throwing them anyplace else . and they could be picked up there once in a while . but you see , if we had a recycling bin , all that stuff would be put in there , whatever we do nt use , like magazines ... i still say we could use a recycling bin for paper and also for cans . like paint cans and all that stuff elders recognized that it would take some time to implement segregation and recycling in their communities . however they optimistically indicated that overtime these methods would eventually be accepted and practiced within their communities . as one elder briefly commented : but it would catch on . it would work . the elders from these communities provided several relevant solutions to remedy some of the issues with current waste disposal practices in their communities . introducing bins for recyclable materials and teaching residents to separate garbage at home are achievable targets that would reduce waste entering the community dump site . where and how to begin waste management programs is a critical issue for first nations communities with limited resources . the fundamental problem that faces the management of virtually all solid wastes is they comprise complex mixtures . comprehensive solid waste management incorporates a diverse range of activities including reduction , recycling , segregation ( separation ) , modification , treatment and disposal all of which have varying levels of sophistication . first nations communities across canada are not homogenous , have various waste disposal requirements and perhaps in some communities , due to their geographic locations may not have the opportunities to partner with municipalities . thus , community - based strategies for solid waste disposal and management may be the best option for first nations communities across canada . possible strategies of addressing immediate waste management issues could include the adoption of waste reduction , reutilization and recycling activities in these communities . local government initiatives , public education programs , and federal / provincial support for , and participation in , regional efforts have been highly effective in reducing solid waste through programs aimed at waste reduction , reuse and recycling of waste ( 3rs ) in off - reserve communities.41 the creation and introduction of first nations developed and centered educational programs that promote these waste management strategies could complement and enhance their successful and sustainable implementation in first nations communities . federal and provincial agencies must understand that first nations communities are restricted in terms of economic capital , constrained in terms of available labour , operational capabilities , and technical expertise . long - term stable funding should be made available to support the engineered construction of landfills and the human and physical infrastructure required for the technical operation , inspection , monitoring and overall management of these landfills . adequate infrastructure , education and training for the segregation of hazardous from household waste could also facilitate better waste disposal practices and lead to the cessation of burning waste in both backyard pits and community dump sites thus alleviating the risks to human and environmental health in these communities . the public works department ( pwd ) of a first nations community , or other named group , is typically responsible for community services , and establishes and maintains the community dump site . the pwd seems to be the logical body to take on the main requirement of centralized collection and disposal of solid waste in first nations communities . however funding allocated to this department for the operation and management of solid waste activities is often diverted to other competing priorities such as water supply , sewage disposal , housing , road repairs and construction . secured funding for the pwd that is targeted strictly for solid waste management may be a critical step to ensure appropriate solid waste management in these communities . this could be achieved , at least in part , by utilizing indian and northern affairs canada ( inac ) funding provisions through the capital assets management system , and partly by the action of individuals within the communities themselves . based on concerns for health , safety and the environment , community members could vote and agree to dedicate funding to the management and monitoring of waste and to meet requirements of a centralized sanitary landfill where there would be daily covering of wastes , secured and controlled access to the site and no open trash burning . these actions may lead to greater separation and segregation of wastes and recycling initiatives . for example communities may agree to arrange for special collections of major items ( old appliances , furniture and automobiles ) once or twice a year or even the development of a drop - off facility for hazardous wastes . first nation communities could partner and share the costs of collection , disposal equipment and labour for the operation and management of a regionally centralized waste site . some first nations communities have relatively small land bases and geologically , land may not be suited for waste site placement . while other communities may have a larger land base , could serve a larger population and offer a more geologically sound site for waste disposal . agreements between partnering communities could be arranged to share existing or new collection and compaction trucks , any administration , monitoring , and management costs and revenue from the regional collection of recyclable materials . to better inform waste management practices , communities could take part in a waste inventory to gain a better understanding of the types and volume of hazardous , household and recyclable wastes generated and the need for , and degree of , segregation , waste reduction and recycling regimes required for these communities . the process of local waste disposal could also be explored to inform waste practices and the initiation of a composing regime to promote waste reduction could be implemented .

the impact of current and historical waste disposal practices on the environment and human health of indigenous people in first nations communities has yet to be adequately addressed . solid waste disposal has been identified as a major environmental threat to first nations communities . a community - based participatory research project ( cbpr ) was initiated by the saskatoon tribal council health and family services incorporated to investigate concerns related to waste disposal in three saskatchewan first nations communities . utilizing a qualitative approach , we aimed to gain an understanding of past and present waste disposal practices and to identify any human and environmental health concerns related to these practices . one to one interviews and sharing circles were conducted with elders . elders were asked to share their perspectives on past and present waste disposal practices and to comment on the possible impacts these practices may have on the environment and community health . historically waste disposal practices were similar among communities . the homeowner generated small volumes of waste , was exclusively responsible for disposal and utilized a backyard pit . overtime waste disposal evolved to weekly pick - up of un - segregated garbage with waste disposal and open trash burning in a community dump site . dump site locations and open trash burning were identified as significant health issues related to waste disposal practices in these communities . this research raises issues of inequity in the management of waste in first nations communities . it highlights the need for long - term sustainable funding to support community - based waste disposal and management strategies and the development of first nations centered and delivered educational programs to encourage the adoption and implementation of waste reduction , reutilization and recycling activities in these communities .

Introduction Methods Findings and Discussion: Past and Present Waste Disposal in First Nations Past waste disposal practices Present waste disposal practices Human and environmental health concerns related to waste disposal practices Recommendations for future waste management practices Conclusions

although these laws and regulations are in place , enforced , and utilized as tools to control an extensive assortment of environmental risks in off - reserve communities , no such laws of environmental protection are currently in place for first nations communities.24 for over a century ( from 1876 to the early 1980 s ) , the indian act has been the main mechanism employed to govern the use of first nations lands).5 under this act , indian and northern affairs canada ( inac ) is the authority responsible for regulating first nations land use , environmental management and protection . in the early 1980 s , through the development of land management and administrative programs , inac provided some options to first nations to enable communities to assume more land management responsibilities . the regional lands administration program ( rlap ) and the delegated lands management programs ( dlmp ) , introduced in the 1980 s , but then replaced in 2005 by the reserve land and environment management program ( rlemp ) , enabled the transfer of various land use planning , environmental and natural resource management and administrative land transaction responsibilities from inac to first nations3,6 the only current alternative , under the indian act , is the first nations land management act ( fnlma ) , a federal law enacted in 1999 which provided signatory first nations law - making authority over reserve lands , resources and the environment.7 the fnlma sanctioned a framework agreement on first nations land management , negotiated and signed , in 1996 , between 14 first nations and the minister of indian affairs and northern development . in 2002 , the land management regime was opened to other interested first nations , however , policy decisions were made in 2008 to close the regime to new entrants due to the lack of sustainable long - term funding and thus the majority of first nations communities are governed by inac under the indian act.3,6 during negotiations of the framework agreement , solid waste was identified as one of 4 major environmental threats to first nations communities.3,8 in addition , discharge of household sewage and industrial and commercial wastewater into surface water , fuel storage tanks and environmental emergencies , such as chemical spills , were considered essential for environmental protection and recognized to pose significant risks on reserves . in 2007 , environment canada also recognized landfills , solid and hazardous wastes , as well as the air emissions from incineration and open burning of garbage as significant on - reserve risks requiring immediate attention.3 the minister of indian and northern affairs canada , under the indian reserve waste disposal regulations , is responsible for the operation of landfills and waste dumps in first nations communities.9 to date , inac neither promotes nor conducts significant surveillance of dumping sites and is not equipped to monitor compliance , conduct inspections or enforce waste disposal regulations.3,10 thus the majority of waste sites in first nations communities across canada remain unregulated.1,2 under current national and provincial legislation , the location of landfills on off - reserve communities requires a geological site selection process ; however in most first nations communities , derivation of landfill ( more appropriately referred to as community dump site ) location does not consider this process . community dump sites are known to be located in areas with silty sandy soils , near surface water features , rather than in more geologically acceptable areas composed of less permeable soils and distant from major water sources.1,4 additionally , abandoned dump sites are not monitored for gas emissions or appropriately sealed with a clay cap , to prevent vertical penetration of water into wastes.1,4,11 at present there are no central records documenting past as well as current waste disposal practices in saskatchewan first nations and the number and location of active and inactive dump sites are not known.1,11 inadequately managed solid waste disposal sites are common sources of pollution in first nations communities12 and the detrimental effects of ineffective waste management practices on human and environmental health is well documented.13 first nations communities are highly dependent on the health of the environment and the concerns of first nations people with respect to the adverse effects of inadequate dump site locations , garbage burning , and poor waste management practices have increased over the years.1,4,11,14 the historical poor management , monitoring and remediation of solid waste facilities across canada s first nations communities and the lack of current resolve over this issue has left many first nations people feeling the consequences of environmental pollution . some thirty years later , there are still severe restrictions on game fish consumption,1517 unemployment is at a high of 80% , social problems have arisen as a result of the loss of the communities self - sufficiency,15 community members are more reliant on market foods due to the growing lack of confidence in traditional foods due to the pollution of their river system and today , still suffer the effects of mercury poisoning.18 the disruption and decay of cultural practices and participation in traditional economy ( hunting , fishing trapping ) , as a result of perceived , or documented , cases of environmental contamination , has had a negative impact on the health and well - being of individuals , their families , and communities and has contributed to the current inequity in the health status of first nations in canada.19,20 in 1976 the united states environmental protection agency ( usepa ) stated : 90 percent of municipal and industrial wastes are disposed of on land in environmentally questionable ways . the results are potential public health problems , groundwater contamination by leachate , surface water pollution by runoff , air pollution from open burning , fires , and explosions at dumps , and risks to ecological systems.21 in fact , cases have been documented where leachate from municipal solid waste disposal sites have contaminated groundwater that supplied residential wells in sayvill , long island new york,22 and rockford illinois.23 hazardous substances associated with waste management have been identified as persistent environmental pollutants and known or suspected human carcinogens.13 waste incineration is known to produce a variety of pollutants from the combustion of chemical , industrial and household wastes . these can be grouped as particles and gases , metals and organic compounds.24 metals such as cadmium , mercury , arsenic , chromium , nickel ; organics that include , dioxins , polychlorinated biphenyls ( pcb ) , polyaromatic hydrocarbons ( pahs ) ; particulate matter and sulphur dioxide , are waste associated pollutants that have been classified as having the greatest potential impact on human health based on their environmental persistence , bioaccumulation , amount emitted and their inherent toxicity.13 the impact of current and historical waste disposal practices of the environment and human health of first nations communities across canada has yet to be adequately addressed or investigated . this issue was identified as critical since most first nations rely on local surface or groundwater for their drinking water supplies , and the potential impacts to health and environment associated with waste disposal practices is potentially significant through contamination of drinking water supplies in first nations communities . parameters found to exceed gcdwq25 included : chloride , sulphate , ph , arsenic , barium , iron , manganese , lead , total dissolved solids and total coliform bacteria.1 the results of the water analysis demonstrated a risk to groundwater quality in the areas of waste disposal . chemical analysis of soil and ash samples collected from one active community dump site indicated the presence of dioxins and furans at concentrations well above the canadian council of ministers of the environment ( ccme)26 soil quality guidelines for the protection of human and environmental health . a community - based participatory research project ( cbpr ) was initiated to describe waste disposal practices and to explore health and environmental concerns related to these practices in three saskatchewan first nations communities . this research project serves to present the views of first nations elders on past and present waste disposal practices in their communities . this article is a presentation and analysis of the knowledge presented on waste management practices during sharing circles and one to one interviews with participating elders from three saskatchewan first nations communities . the community - based participatory research project was initiated by the saskatoon tribal council health and family services to investigate concerns related to waste disposal and possible groundwater contamination in first nations communities in saskatchewan . the research reported here was part of the larger cbpr project that examined the hydrological , microbiological , and toxicological aspects of active and inactive waste sites in three saskatchewan first nations communities . community - based participatory research strives to be community situated , collaborative and action oriented.2729 this specific project was based on a respectful collaboration built between researchers and communities for the purpose of creating new knowledge and understanding of waste disposal that was of practical relevance to the involved communities . with the inclusion of a qualitative approach , we aimed to gain an understanding of the past and present waste disposal practices in three first nations communities and the potential human and environmental health concerns related to these practices of waste disposal . the muskoday first nation is located northeast of saskatoon , spans approximately 9687 hectares and has 1583 registered members.1 depending on community preference , information was gathered utilizing one of two approaches ; a one to one interview or a sharing circle.30,31 two one to one interviews were conducted with two female elders from one of the participating communities . interviews and sharing circles were held at the health centre s of the participating communities . they were invited to share their experiences of waste disposal as they remembered it when they were younger ( late 1950 s ) . elders were also asked to share their perspectives on present waste management practices and on the possible impacts of waste practices on community and environmental health . in general , one to one interviews took place with female participants and ranged in duration from seven to twenty five minutes . interviews and sharing circles were videotaped and recorded into dvd format and conducted in the summer of 2004 . in keeping with the principles of ownership , control , access , possession ( ocap ) and community - based participatory research,32 a consultative and approval process was undertaken with elders along with chief and council prior to the engagement of research and dissemination activities.28 thematic content analysis of transcribed interview and sharing circles was conducted to identify main themes arising from participants responses.33 video recordings were digitally transcribed verbatim . utilizing atlas.ti software , transcribed data were managed , organized , and coded by use of topic and descriptive categories , into emerging themes.34,35 major emergent themes were identified using an iterative process of comparison and evaluation across interviews and sharing circles . in the past options for waste disposal were limited and the homeowner was exclusively responsible for disposing waste . typically , households generated small volumes of garbage and a backyard pit or personal waste site was the most common past practice of waste disposal . waste disposal practices evolved from backyard pits and larger waste sites to weekly pick - up of un - segregated garbage with waste disposal into larger community dump sites . this present practice , a convention on many first nations communities across canada today , includes open - air dumping coupled with trash burning . weekly garbage truck pick - up with off - reserve disposal in an appropriately engineered landfill , governed by a local municipality , was currently practiced in the third community . elders expressed that community dumpsite locations as well as the practice of open trash burning were significant health issues related to waste disposal practices in their communities . overall , discussions with elders were centered on the description of past and present practices of waste disposal , the health and environmental issues related to these waste practices and the options for future waste management . primarily due to convenience and as a result of the limitations in waste disposal methodologies , historically , a small pit dug in the backyard was the most common past practice for disposing waste . an alternate site in the backyard the methods of past waste disposal were expressed by three elders as follows : they used to dig a hole . in summary , past waste disposal practices were similar among communities , and homeowners were primarily responsible for disposing waste . historically , households generated small volumes of garbage and a backyard pit was the most convenient , practical and common practice for waste disposal . although backyard pits were primarily utilized for household waste disposal in the past , it was established that in one community , a backyard pit was utilized for the preservation of wooden fence posts and still contained traces of chromium , copper and arsenic . segregation and recycling of waste materials was not currently practiced in these communities . with the exception of two concerns ; burning garbage and waste site location , the elders of these communities were generally satisfied with the current waste disposal practices in their communities . weekly garbage truck pick - up with off - reserve disposal , to a neighbouring city landfill , was the current practice in one of the three communities involved in the project . as a result the practice of waste disposal evolved from the backyard pit to the community dump site . however , the modification in disposal methods may have occurred in time with , and as a consequence of , population growth and the increased availability of over - packaged commercial commodities within these communities . on the whole , all elders raised concerns over the potential effects of past and present waste disposal practices on human and environmental health . health related discussions were centered on dump site locations and the current practice of open air trash burning at the community dump sites . methods of dump and burn are commonly utilized in first nations across saskatchewan , yet these methods came to a halt in non - reserve communities with the enforcement of the provincial new clean air regulations act in 1989.37 the community waste sites contain a variety of waste materials including ; plastics , wood , paper , cardboard , tires , and electronics . carbon monoxide , carbon dioxide , and nitrogen oxides represent the largest portion of pollutants emitted from open trash burning.26,38 however , other chemicals such as ; benzene , styrene , formaldehyde , polychlorinated dibenzodioxins ( pcdds ; also known as dioxins ) , polychlorinated biphenyls ( pcbs ) , polychlorinated dibenzofurans ( pcdfs ; also known as furans ) , and heavy metals ( lead , mercury and arsenic ) have been detected in smoke produced through the incineration of waste materials.3840 open air burning was conducted in two of the three communities . all elders were dissatisfied with the practice of open air trash burning in their communities . elders demonstrated concern for community members living in close proximity to the community dump and spoke of potential risks to their health , the health of their children and the air quality in their communities in relation to this practice of waste disposal . another elder expressed great concern over the impacts to the ambient air quality in their community as a result of burning tires at the community dump site . the burning of refuse helps to minimize the build - up of materials in the community dump sites ; however , as a number of respondents pointed out , the smoke is a nuisance , and they are concerned with the possible impacts to ambient air quality , exposures to airborne contaminants and the impacts to the health of their children and community members as a result of these airborne exposures . all participating elders noted that past and present dump site locations were an environmental issue . the elders from these communities provided several relevant solutions to remedy some of the issues with current waste disposal practices in their communities . an alternate site in the backyard the methods of past waste disposal were expressed by three elders as follows : they used to dig a hole . in summary , past waste disposal practices were similar among communities , and homeowners were primarily responsible for disposing waste . historically , households generated small volumes of garbage and a backyard pit was the most convenient , practical and common practice for waste disposal . although backyard pits were primarily utilized for household waste disposal in the past , it was established that in one community , a backyard pit was utilized for the preservation of wooden fence posts and still contained traces of chromium , copper and arsenic . segregation and recycling of waste materials was not currently practiced in these communities . with the exception of two concerns ; burning garbage and waste site location , the elders of these communities were generally satisfied with the current waste disposal practices in their communities . looking at it from past years and now , it s much cleaner weekly garbage truck pick - up with off - reserve disposal , to a neighbouring city landfill , was the current practice in one of the three communities involved in the project . elders noted that overtime , the volume of household waste increased and the responsibility of waste disposal on reserve transferred from the individual to the community . as a result the practice of waste disposal evolved from the backyard pit to the community dump site . however , the modification in disposal methods may have occurred in time with , and as a consequence of , population growth and the increased availability of over - packaged commercial commodities within these communities . on the whole , all elders raised concerns over the potential effects of past and present waste disposal practices on human and environmental health . health related discussions were centered on dump site locations and the current practice of open air trash burning at the community dump sites . methods of dump and burn are commonly utilized in first nations across saskatchewan , yet these methods came to a halt in non - reserve communities with the enforcement of the provincial new clean air regulations act in 1989.37 the community waste sites contain a variety of waste materials including ; plastics , wood , paper , cardboard , tires , and electronics . elders demonstrated concern for community members living in close proximity to the community dump and spoke of potential risks to their health , the health of their children and the air quality in their communities in relation to this practice of waste disposal . the burning of refuse helps to minimize the build - up of materials in the community dump sites ; however , as a number of respondents pointed out , the smoke is a nuisance , and they are concerned with the possible impacts to ambient air quality , exposures to airborne contaminants and the impacts to the health of their children and community members as a result of these airborne exposures . all participating elders noted that past and present dump site locations were an environmental issue . for example ; one of the current operational community dumpsites , was located in an area of silty sandy soils close to a lake.1 elders from all three communities recalled that in the past , a major staple of their diet was fish . the elders from these communities provided several relevant solutions to remedy some of the issues with current waste disposal practices in their communities . first nations communities across canada are not homogenous , have various waste disposal requirements and perhaps in some communities , due to their geographic locations may not have the opportunities to partner with municipalities . thus , community - based strategies for solid waste disposal and management may be the best option for first nations communities across canada . possible strategies of addressing immediate waste management issues could include the adoption of waste reduction , reutilization and recycling activities in these communities . local government initiatives , public education programs , and federal / provincial support for , and participation in , regional efforts have been highly effective in reducing solid waste through programs aimed at waste reduction , reuse and recycling of waste ( 3rs ) in off - reserve communities.41 the creation and introduction of first nations developed and centered educational programs that promote these waste management strategies could complement and enhance their successful and sustainable implementation in first nations communities . long - term stable funding should be made available to support the engineered construction of landfills and the human and physical infrastructure required for the technical operation , inspection , monitoring and overall management of these landfills . adequate infrastructure , education and training for the segregation of hazardous from household waste could also facilitate better waste disposal practices and lead to the cessation of burning waste in both backyard pits and community dump sites thus alleviating the risks to human and environmental health in these communities . the public works department ( pwd ) of a first nations community , or other named group , is typically responsible for community services , and establishes and maintains the community dump site . the pwd seems to be the logical body to take on the main requirement of centralized collection and disposal of solid waste in first nations communities . based on concerns for health , safety and the environment , community members could vote and agree to dedicate funding to the management and monitoring of waste and to meet requirements of a centralized sanitary landfill where there would be daily covering of wastes , secured and controlled access to the site and no open trash burning . to better inform waste management practices , communities could take part in a waste inventory to gain a better understanding of the types and volume of hazardous , household and recyclable wastes generated and the need for , and degree of , segregation , waste reduction and recycling regimes required for these communities . the process of local waste disposal could also be explored to inform waste practices and the initiation of a composing regime to promote waste reduction could be implemented .

tobacco is one of the major causes of death and disease in india , accounting for nearly 900,000 deaths every year . about 53% of all deaths in india are caused by ncds , for which tobacco use is one of the major risk factors . in india , 35% of all adults ( 275 million ) aged 15 years and above are users of tobacco , according to the global adult tobacco survey india , 200910 . india is the second largest consumer and third largest producer of tobacco and a plethora of tobacco products are available at very low prices . smoking among health care personnel , such as medical students , is an important public health issue . health professionals , while recognizing smoking as the leading preventable cause of death and disability , are not aware of their fundamental role in helping people quit smoking . in some countries , teaching about the effects of the use of tobacco and its related diseases is essential for undergraduate medical students , especially to counter the deadly effects of the same . physicians occupy a key position in this regard , as they are uniquely placed to lead smoking cessation programs in the community . in most developed countries where tobacco use has decreased , doctors often have set an example by being the first group to quit tobacco use . health professionals have an important role to play in the fight against tobacco . as individuals , health professionals can help educate the population ; as community members , they can support anti - smoking policies ; and at a societal level , they can influence national and global tobacco control efforts . to date , very few studies had been conducted in india to understand the magnitude of knowledge of smoking as a public health problem among medical students . therefore , it is important to understand the factors affecting tobacco use among medical students and to know whether medical students perceive tobacco use as a public health problem . hence , this study was undertaken to estimate the prevalence of tobacco use among medical students in chennai and to measure the extent of attitude toward , behavior around and knowledge of tobacco usage among medical students . this cross - sectional study was conducted among 4 randomly selected medical colleges , comprised of one government and three private medical colleges from a total of three government and nine private medical colleges in and around chennai , india . the study was conducted from september 2013 to august 2014 . with the prevalence of 22.4% , as per kumar et al . , at a 5% significance level and 20% allowable error , the sample size was determined using the formula n = 4pq / l2 , which came to 340 . considering a 15% non - response rate , the global health professional students survey ( ghpss ) , a standardized college based tool , was used to collect data among medical students . the questionnaire was pre - tested in a pilot study to assess the feasibility of adopting the questionnaire , and it was adopted without modification . the protocol was submitted to the institution ethics committee and approval was obtained . after obtaining the permission of heads of the institutions , was obtained from the students and the students were informed about the confidentiality of the data . data regarding demographics , knowledge of , attitude toward and behavior regarding tobacco were collected . data was entered into microsoft excel 2010 spreadsheet and analyzed using epi info version 3.4.3 . descriptive analyses were conducted ( means , percentages , proportions , and 95% confidence intervals ) for demographic data . this study was conducted among 479 third year medical college students at four medical colleges . the mean age of study participants was 20.46 years , with 200 ( 41.8% ) males and 279 ( 58.2% ) females . the proportion of students who ever tried cigarette smoking was found to be 10.9% ( males , 23.5% ; females , 1.8% ) . the prevalence of current cigarette smoking among students was found to be 4.8% ( males , 11.5% ; females , 0% ) . the proportion of cigarette smoking on college premises during the past year was found to be 1.9% . the proportion of students who ever used smokeless tobacco ( chewing tobacco , snuff ) was found to be 1.9% ( males 4.5% ; females , 0% ) . the prevalence of current smokeless tobacco use among students was found to be 1.0% ( males , 2.5% ; females , 0% ) . the proportion of students using smokeless tobacco on college premises during the past year was 0.6% . the prevalence of exposure to tobacco smoke at home among students was found to be 34.2% ( males , 49.5% ; females , 23.3% ) . the prevalence of exposure to tobacco smoke at public places was found to be 50.3% ( males , 58.5% ; females , 44.4% ; table 1 ) . a majority of the students ( 96.5% ) supported a tobacco sales ban to adolescents , as well as a ban of tobacco product advertisements ( 91.6% ) . most students agreed that smoking should be banned in restaurants ( 94.6% ) , in public places ( 93.7% ) and in discos / bars / pubs ( 73.9% ) . many students , 75.6% and 95.2% , indicated that health professionals should serve as a role model for patients and receive specific training on cessation techniques , respectively . the proportion of students who currently wanted to quit smoking cigarettes was 26.9% the proportion of students who ever tried to stop smoking cigarettes during the past year was 34.6% and the proportions of students who stopped smoking cigarettes and using smokeless tobacco were 13.5% and 44.4% , respectively . a majority of students , 59.6% , reported they received help or advice to stop smoking cigarettes . among students who completed the questionnaire , 70.6% reported that health professionals who smoke are less likely to advise patients to stop smoking . females were more likely than males to agree that health professionals should receive special training about smoking cessation techniques ; 76% of males agreed that advice would enhance the possibility of patients quitting smoking compared to 87.5% of females . the difference in attitudes between males and females was significant ( p = 0.007 ) . the proportion of students who said health professionals who smoke are less likely to advise patients to stop smoking was 70.6% ( table 2 ) . among the students who tried smoking / smokeless tobacco ( n = 52 ) , 29.8% and 34.6% wanted to quit smoking cigarettes and tried to stop smoking , respectively . the proportion of students who stopped smoking cigarettes and received help or advice to stop smoking cigarettes were 13.5% and 59.6% , respectively . the proportion of students who wanted to stop using smokeless tobacco was 44.4% ( table 3 ) . table 4 depicts that more than 90% of the students reported they were taught about the dangers of smoking and learned to record tobacco use history as a part of a patient s general medical history . only 23.6% of the students reported that they received formal training in smoking cessation techniques . a majority of students , 57.6% , indicated that they discussed the reasons why people smoke with their classmates . the current study was conducted to estimate the prevalence of tobacco use and to measure the extent of knowledge of , attitude toward and behavior around tobacco usage among third year medical students in chennai , india . the number of study participants was 479 students , with 200 males and 279 females . similar findings were reported by selokar et al . and lam et al . , with 10.3% and 11.8% of students having tried cigarette smoking , respectively . in contrast , alrsheedi et al . , sreeramareddy et al . and surani et al reported that 23% , 31.7% and 40% of students tried smoking , respectively . in the present study , among male medical students , in contrast , a study conducted by sinha et al . showed 7% and surani et al . showed 23.5% of females tried cigarette smoking . in the present study , less than 5% of students were current smokers . similar findings were reported by abdulghani et al . with 4.3% and ramakrishna et al . with 3.7% . in contrast , a higher proportion of current smokers was reported by other researchers in india . . found 14.2% , bartwal et al . reported 14.5% and kumar et al . reported none of the female students were current smokers , while 11.5% of male students indicated they were current smokers . similar finding were reported by ramakrishna et al . , with 12.4% of male and 0.8% of female medical students were current smokers . in contrast , saulle et al . reported 20.4% of male and 19.1% of female medical students were smokers , and jradi et al . reported 18% of male and 12% of female medical students were current smokers . all studies conducted among medical students reported higher rates of male current smokers relative to females . in the present study , in contrast , studies by alkawari et al . and surani et al . found that one - third of students smoked on college premises / property and 17.1% smoked in college buildings during the past year , respectively . in the present study , the prevalence of smokeless tobacco ever used was found to be 1.9% and the prevalence of current smokeless tobacco use was found to be 1.0% . in contrast , a study by joge et al . 2.5% of males and none of the females were current smokeless tobacco users , whereas surani et al . in our study , about 0.6% had used smokeless tobacco on college premises during the past year , whereas surani et al . found that 34.2% used smokeless tobacco on campus during the past year . in our study , approximately one - third of the students had exposure to smoke at home . nearly half of the male students and one - fourth of the female students had exposure to smoke at home . similar findings was reported by alkawari et al . , where 27.9% were exposed to smoke at home . reported that upward of fifty percent of the students had exposure to smoke at home . in the present study , approximately half of the participants had exposure to smoke at public places ( 58.5% among male students and 44.4% among female students ) . similar findings were reported by alkawari et al . and inandi et al . . in contrast , studies by sinha et al . and saade et al . reported that two - thirds of the students were exposed to smoke at public places . the study findings revealed that more medical students were exposed to environmental tobacco smoke than to active smoking among both males and females . this indicates the need for health education of families and the community about the health hazards of passive smoking . of the students in the study , 79.1% reported that their college has an official policy banning smoking in college buildings and clinics . a similar finding was reported by inandi et al . , with 88.2% of students reporting that their college had an official policy banning smoking on college premises . in contrast , studies by sinha et al . in 20052011 and saade et al . found that only half the students reported that their college had an official policy banning smoking in college premises . in the present study , two - thirds of the students said an official smoking ban in college buildings and clinics has been enforced . a slightly higher percentage of students reported a smoking ban was enforced in the studies done by inandi et al . with 87.2% and alkawari et al . with 88.1% . . found that a slightly lower number of students ( 53% ) reported that there was official smoking ban enforcement in their medical school . in the present study , more than 90% of the students favored banning the sale of tobacco products to adolescents , banning the advertisement of tobacco products , and banning smoking in restaurants and in public places . in the present study , nearly three - fourths of the students reported that smoking should be banned in discos / bars / pubs . mehrotra et al . reported that 70.5% and 66.9% , respectively , of students indicated smoking should be banned in discos / bars / pubs . in our study , even though more than 90% of the students supported bans of tobacco products in public places and restaurants , sales to adolescents and advertisements of tobacco products , only three - fourths of the students believed that tobacco products should be banned in discos , bars and pubs ; this finding was supported by other published studies . in the present study , approximately three - fourths of the students reported that health professionals should serve as a role model for their patients . similar findings were reported by sinha et al . , where 73.8% of medical students believed that health professionals served as role models for their patients and the public . reported that a slightly higher proportion ( 89% ) of medical students believe that health professionals serve as role models for their patients and the public . in the present study , 95.2% of the students reported that health professionals should get specific training in smoking cessation . in contrast , the study done by other investigators found that more than 50% of medical students had tried to quit smoking . in the present study , similarly , studies conducted by other investigators found that more than half of smokers ever received help or advice to stop smoking cigarettes . in our study , more than 70% of students reported health professionals who smoke are less likely to advise patients to stop smoking . adeel khan et al . found that a slightly higher proportion of students ( 77.5% ) reported that health professionals who smoke are less likely to advise patients to stop smoking . in the present study , more than 90% of students in contrast , adeel khan et al . reported that only 43.7% of students were taught about the dangers of smoking . in addition , in the present study , more than 90% of students were taught that it is important to record tobacco use as a part of a patient s history . similarly , adeel khan et al . found that 88.9% of students were taught the importance of recording tobacco usage history . in our study , in contrast , adeel khan et al . found that 34.2% of students received formal training in smoking cessation . in the present study , nearly three - fourths of the students are taught that it is important to provide educational materials on smoking to patients . in contrast , adeel khan et al . found that 58.5% of students were taught the importance of providing educational materials on smoking to patients . in the present study , more than three - fourths of students lam et al . found a slightly higher proportion ( 90.8% ) in their study . in contrast , jradi reported that only 18.4% have heard of nicotine replacement products . in the current study , more than half of the students are aware of the use of antidepressants in tobacco cessation programs . jradi found that a slightly higher proportion ( 63% ) of students were aware of the use of antidepressants in tobacco cessation programs . in the present study , approximately one - third of the medical students tried cigarette smoking . similar findings were reported by selokar et al . and lam et al . , with 10.3% and 11.8% of students having tried cigarette smoking , respectively . in contrast , alrsheedi et al . , sreeramareddy et al . and surani et al reported that 23% , 31.7% and 40% of students tried smoking , respectively . in the present study , among male medical students , in contrast , a study conducted by sinha et al . showed 7% and surani et al . showed 23.5% of females tried cigarette smoking . in the present study , less than 5% of students were current smokers . similar findings were reported by abdulghani et al . with 4.3% and ramakrishna et al . with 3.7% . in contrast , a higher proportion of current smokers was reported by other researchers in india . . found 14.2% , bartwal et al . reported 14.5% and kumar et al . reported 22.4% of medical students were current smokers none of the female students were current smokers , while 11.5% of male students indicated they were current smokers . similar finding were reported by ramakrishna et al . , with 12.4% of male and 0.8% of female medical students were current smokers . in contrast , saulle et al . reported 20.4% of male and 19.1% of female medical students were smokers , and jradi et al . reported all studies conducted among medical students reported higher rates of male current smokers relative to females . in the present study , in contrast , studies by alkawari et al . and surani et al . found that one - third of students smoked on college premises / property and 17.1% smoked in college buildings during the past year , respectively . in the present study , the prevalence of smokeless tobacco ever used was found to be 1.9% and the prevalence of current smokeless tobacco use was found to be 1.0% . in contrast , a study by joge et al . reported the prevalence of smokeless tobacco was 5.8% , selokar et al . reported 10% and surani et al . , 11.6% . in the present study , 2.5% of males and none of the females reported that 13.7% of males and 7.5% of females were current smokeless tobacco users . in our study , about 0.6% had used smokeless tobacco on college premises during the past year , whereas surani et al . found that 34.2% used smokeless tobacco on campus during the past year in our study , approximately one - third of the students had exposure to smoke at home . nearly half of the male students and one - fourth of the female students had exposure to smoke at home . similar findings was reported by alkawari et al . , where 27.9% were exposed to smoke at home . reported that upward of fifty percent of the students had exposure to smoke at home . in the present study , approximately half of the participants had exposure to smoke at public places ( 58.5% among male students and 44.4% among female students ) . similar findings were reported by alkawari et al . and inandi et al . . in contrast , studies by sinha et al . and saade et al . reported that two - thirds of the students were exposed to smoke at public places . the study findings revealed that more medical students were exposed to environmental tobacco smoke than to active smoking among both males and females . this indicates the need for health education of families and the community about the health hazards of passive smoking . of the students in the study , 79.1% reported that their college has an official policy banning smoking in college buildings and clinics . a similar finding was reported by inandi et al . , with 88.2% of students reporting that their college had an official policy banning smoking on college premises . in contrast , studies by sinha et al . in 20052011 and saade et al . found that only half the students reported that their college had an official policy banning smoking in college premises . in the present study , two - thirds of the students said an official smoking ban in college buildings and clinics has been enforced . a slightly higher percentage of students reported a smoking ban was enforced in the studies done by inandi et al . with 87.2% and alkawari et al . with 88.1% . . found that a slightly lower number of students ( 53% ) reported that there was official smoking ban enforcement in their medical school . in the present study , more than 90% of the students favored banning the sale of tobacco products to adolescents , banning the advertisement of tobacco products , and banning smoking in restaurants and in public places . in the present study , nearly three - fourths of the students reported that smoking should be banned in discos / bars / pubs . reported that 70.5% and 66.9% , respectively , of students indicated smoking should be banned in discos / bars / pubs . in our study , even though more than 90% of the students supported bans of tobacco products in public places and restaurants , sales to adolescents and advertisements of tobacco products , only three - fourths of the students believed that tobacco products should be banned in discos , bars and pubs ; this finding was supported by other published studies . in the present study , approximately three - fourths of the students reported that health professionals should serve as a role model for their patients . similar findings were reported by sinha et al . , where 73.8% of medical students believed that health professionals served as role models for their patients and the public . reported that a slightly higher proportion ( 89% ) of medical students believe that health professionals serve as role models for their patients and the public . in the present study , 95.2% of the students reported that health professionals should get specific training in smoking cessation . in the present study , one - third of students had ever tried to stop smoking . in contrast , the study done by other investigators found that more than 50% of medical students had tried to quit smoking . in the present study , similarly , studies conducted by other investigators found that more than half of smokers ever received help or advice to stop smoking cigarettes . in our study , more than 70% of students reported health professionals who smoke are less likely to advise patients to stop smoking . adeel khan et al . found that a slightly higher proportion of students ( 77.5% ) reported that health professionals who smoke are less likely to advise patients to stop smoking . in the present study , more than 90% of students reported they were taught about the dangers of smoking . in contrast , adeel khan et al . reported that only 43.7% of students were taught about the dangers of smoking . in addition , in the present study , more than 90% of students were taught that it is important to record tobacco use as a part of a patient s history . similarly , adeel khan et al . found that 88.9% of students were taught the importance of recording tobacco usage history . in our study , in contrast , adeel khan et al . found that 34.2% of students received formal training in smoking cessation . in the present study , nearly three - fourths of the students are taught that it is important to provide educational materials on smoking to patients . in contrast , adeel khan et al . found that 58.5% of students were taught the importance of providing educational materials on smoking to patients . in the present study , more than three - fourths of students lam et al . found a slightly higher proportion ( 90.8% ) in their study . in contrast , jradi reported that only 18.4% have heard of nicotine replacement products . in the current study , more than half of the students are aware of the use of antidepressants in tobacco cessation programs . jradi found that a slightly higher proportion ( 63% ) of students were aware of the use of antidepressants in tobacco cessation programs . in our study , less than 5% of medical students were current smokers , but upward of one - third to one - half were exposed to environmental tobacco smoke at home and in public places . this indicates that a health awareness campaign for the prevention and control of smoking at home and public places must be intensified . as indicated by the majority of the participants , medical students need to receive formal training in smoking cessation techniques and this training must be included in the medical curriculum so that students can instruct or counsel patients .

backgroundhealth professionals have an important role to play in the fight against tobacco . as individuals , health professionals can help educate the population ; as community members , they can support anti - smoking policies ; and , at a societal level , they can influence national and global tobacco control efforts . the objectives of the study was to estimate the prevalence of tobacco use among medical students in chennai and to measure the extent of attitude toward , behavior around and knowledge of tobacco use among medical students.methodsthis cross - sectional study was conducted among 400 medical students from 4 randomly selected medical colleges , comprised of 1 government and 3 private medical colleges in and around chennai , india . the global health professional students survey ( ghpss ) , a standardized college - based tool , was administered to students in the four medical colleges.resultsthe proportion of students who ever tried cigarette smoking was found to be 10.9% ( males , 23.5% and females , 1.8% ) . the prevalence of exposure to tobacco smoke at home was found to be 34.2% . a majority of students agreed that smoking should be banned . the proportion of students who wanted to quit smoking cigarettes and who ever tried to stop smoking were 29.8% and 34.6% respectively . only 23.6% of the students said they have received formal training in smoking cessation techniques.conclusionas indicated by the majority of the medical students queried , there is a need for formal training in smoking cessation techniques , and this training should be included in the medical curriculum , such that students can instruct or counsel their patients .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION 1. Prevalence of tobacco use among medical students 2. Exposure to environmental tobacco smoke 3. Attitude towards smoking and sale of tobacco products 4. Behavior/cessation of smoking and smokeless tobacco 5. Training/curriculum on smoking and tobacco use CONCLUSION

tobacco is one of the major causes of death and disease in india , accounting for nearly 900,000 deaths every year . about 53% of all deaths in india are caused by ncds , for which tobacco use is one of the major risk factors . in india , 35% of all adults ( 275 million ) aged 15 years and above are users of tobacco , according to the global adult tobacco survey india , 200910 . smoking among health care personnel , such as medical students , is an important public health issue . health professionals , while recognizing smoking as the leading preventable cause of death and disability , are not aware of their fundamental role in helping people quit smoking . in some countries , teaching about the effects of the use of tobacco and its related diseases is essential for undergraduate medical students , especially to counter the deadly effects of the same . physicians occupy a key position in this regard , as they are uniquely placed to lead smoking cessation programs in the community . in most developed countries where tobacco use has decreased , doctors often have set an example by being the first group to quit tobacco use . health professionals have an important role to play in the fight against tobacco . as individuals , health professionals can help educate the population ; as community members , they can support anti - smoking policies ; and at a societal level , they can influence national and global tobacco control efforts . to date , very few studies had been conducted in india to understand the magnitude of knowledge of smoking as a public health problem among medical students . therefore , it is important to understand the factors affecting tobacco use among medical students and to know whether medical students perceive tobacco use as a public health problem . hence , this study was undertaken to estimate the prevalence of tobacco use among medical students in chennai and to measure the extent of attitude toward , behavior around and knowledge of tobacco usage among medical students . this cross - sectional study was conducted among 4 randomly selected medical colleges , comprised of one government and three private medical colleges from a total of three government and nine private medical colleges in and around chennai , india . the study was conducted from september 2013 to august 2014 . with the prevalence of 22.4% , as per kumar et al . , at a 5% significance level and 20% allowable error , the sample size was determined using the formula n = 4pq / l2 , which came to 340 . considering a 15% non - response rate , the global health professional students survey ( ghpss ) , a standardized college based tool , was used to collect data among medical students . the questionnaire was pre - tested in a pilot study to assess the feasibility of adopting the questionnaire , and it was adopted without modification . after obtaining the permission of heads of the institutions , was obtained from the students and the students were informed about the confidentiality of the data . data regarding demographics , knowledge of , attitude toward and behavior regarding tobacco were collected . descriptive analyses were conducted ( means , percentages , proportions , and 95% confidence intervals ) for demographic data . this study was conducted among 479 third year medical college students at four medical colleges . the mean age of study participants was 20.46 years , with 200 ( 41.8% ) males and 279 ( 58.2% ) females . the proportion of students who ever tried cigarette smoking was found to be 10.9% ( males , 23.5% ; females , 1.8% ) . the prevalence of current cigarette smoking among students was found to be 4.8% ( males , 11.5% ; females , 0% ) . the proportion of cigarette smoking on college premises during the past year was found to be 1.9% . the proportion of students who ever used smokeless tobacco ( chewing tobacco , snuff ) was found to be 1.9% ( males 4.5% ; females , 0% ) . the prevalence of current smokeless tobacco use among students was found to be 1.0% ( males , 2.5% ; females , 0% ) . the proportion of students using smokeless tobacco on college premises during the past year was 0.6% . the prevalence of exposure to tobacco smoke at home among students was found to be 34.2% ( males , 49.5% ; females , 23.3% ) . the prevalence of exposure to tobacco smoke at public places was found to be 50.3% ( males , 58.5% ; females , 44.4% ; table 1 ) . a majority of the students ( 96.5% ) supported a tobacco sales ban to adolescents , as well as a ban of tobacco product advertisements ( 91.6% ) . most students agreed that smoking should be banned in restaurants ( 94.6% ) , in public places ( 93.7% ) and in discos / bars / pubs ( 73.9% ) . many students , 75.6% and 95.2% , indicated that health professionals should serve as a role model for patients and receive specific training on cessation techniques , respectively . the proportion of students who currently wanted to quit smoking cigarettes was 26.9% the proportion of students who ever tried to stop smoking cigarettes during the past year was 34.6% and the proportions of students who stopped smoking cigarettes and using smokeless tobacco were 13.5% and 44.4% , respectively . a majority of students , 59.6% , reported they received help or advice to stop smoking cigarettes . among students who completed the questionnaire , 70.6% reported that health professionals who smoke are less likely to advise patients to stop smoking . females were more likely than males to agree that health professionals should receive special training about smoking cessation techniques ; 76% of males agreed that advice would enhance the possibility of patients quitting smoking compared to 87.5% of females . the difference in attitudes between males and females was significant ( p = 0.007 ) . the proportion of students who said health professionals who smoke are less likely to advise patients to stop smoking was 70.6% ( table 2 ) . among the students who tried smoking / smokeless tobacco ( n = 52 ) , 29.8% and 34.6% wanted to quit smoking cigarettes and tried to stop smoking , respectively . the proportion of students who stopped smoking cigarettes and received help or advice to stop smoking cigarettes were 13.5% and 59.6% , respectively . the proportion of students who wanted to stop using smokeless tobacco was 44.4% ( table 3 ) . table 4 depicts that more than 90% of the students reported they were taught about the dangers of smoking and learned to record tobacco use history as a part of a patient s general medical history . only 23.6% of the students reported that they received formal training in smoking cessation techniques . a majority of students , 57.6% , indicated that they discussed the reasons why people smoke with their classmates . the current study was conducted to estimate the prevalence of tobacco use and to measure the extent of knowledge of , attitude toward and behavior around tobacco usage among third year medical students in chennai , india . , with 10.3% and 11.8% of students having tried cigarette smoking , respectively . and surani et al reported that 23% , 31.7% and 40% of students tried smoking , respectively . in the present study , among male medical students , in contrast , a study conducted by sinha et al . showed 23.5% of females tried cigarette smoking . in the present study , less than 5% of students were current smokers . in contrast , a higher proportion of current smokers was reported by other researchers in india . reported none of the female students were current smokers , while 11.5% of male students indicated they were current smokers . , with 12.4% of male and 0.8% of female medical students were current smokers . reported 20.4% of male and 19.1% of female medical students were smokers , and jradi et al . reported 18% of male and 12% of female medical students were current smokers . all studies conducted among medical students reported higher rates of male current smokers relative to females . in the present study , in contrast , studies by alkawari et al . found that one - third of students smoked on college premises / property and 17.1% smoked in college buildings during the past year , respectively . in the present study , the prevalence of smokeless tobacco ever used was found to be 1.9% and the prevalence of current smokeless tobacco use was found to be 1.0% . in contrast , a study by joge et al . 2.5% of males and none of the females were current smokeless tobacco users , whereas surani et al . found that 34.2% used smokeless tobacco on campus during the past year . in our study , approximately one - third of the students had exposure to smoke at home . nearly half of the male students and one - fourth of the female students had exposure to smoke at home . , where 27.9% were exposed to smoke at home . reported that upward of fifty percent of the students had exposure to smoke at home . in the present study , approximately half of the participants had exposure to smoke at public places ( 58.5% among male students and 44.4% among female students ) . reported that two - thirds of the students were exposed to smoke at public places . the study findings revealed that more medical students were exposed to environmental tobacco smoke than to active smoking among both males and females . this indicates the need for health education of families and the community about the health hazards of passive smoking . of the students in the study , 79.1% reported that their college has an official policy banning smoking in college buildings and clinics . , with 88.2% of students reporting that their college had an official policy banning smoking on college premises . found that only half the students reported that their college had an official policy banning smoking in college premises . in the present study , two - thirds of the students said an official smoking ban in college buildings and clinics has been enforced . a slightly higher percentage of students reported a smoking ban was enforced in the studies done by inandi et al . found that a slightly lower number of students ( 53% ) reported that there was official smoking ban enforcement in their medical school . in the present study , more than 90% of the students favored banning the sale of tobacco products to adolescents , banning the advertisement of tobacco products , and banning smoking in restaurants and in public places . in the present study , nearly three - fourths of the students reported that smoking should be banned in discos / bars / pubs . reported that 70.5% and 66.9% , respectively , of students indicated smoking should be banned in discos / bars / pubs . in our study , even though more than 90% of the students supported bans of tobacco products in public places and restaurants , sales to adolescents and advertisements of tobacco products , only three - fourths of the students believed that tobacco products should be banned in discos , bars and pubs ; this finding was supported by other published studies . in the present study , approximately three - fourths of the students reported that health professionals should serve as a role model for their patients . , where 73.8% of medical students believed that health professionals served as role models for their patients and the public . reported that a slightly higher proportion ( 89% ) of medical students believe that health professionals serve as role models for their patients and the public . in the present study , 95.2% of the students reported that health professionals should get specific training in smoking cessation . in contrast , the study done by other investigators found that more than 50% of medical students had tried to quit smoking . in the present study , similarly , studies conducted by other investigators found that more than half of smokers ever received help or advice to stop smoking cigarettes . in our study , more than 70% of students reported health professionals who smoke are less likely to advise patients to stop smoking . found that a slightly higher proportion of students ( 77.5% ) reported that health professionals who smoke are less likely to advise patients to stop smoking . in the present study , more than 90% of students in contrast , adeel khan et al . reported that only 43.7% of students were taught about the dangers of smoking . in addition , in the present study , more than 90% of students were taught that it is important to record tobacco use as a part of a patient s history . found that 88.9% of students were taught the importance of recording tobacco usage history . found that 34.2% of students received formal training in smoking cessation . in the present study , nearly three - fourths of the students are taught that it is important to provide educational materials on smoking to patients . found that 58.5% of students were taught the importance of providing educational materials on smoking to patients . in the present study , more than three - fourths of students lam et al . found a slightly higher proportion ( 90.8% ) in their study . in the current study , more than half of the students are aware of the use of antidepressants in tobacco cessation programs . jradi found that a slightly higher proportion ( 63% ) of students were aware of the use of antidepressants in tobacco cessation programs . in the present study , approximately one - third of the medical students tried cigarette smoking . , with 10.3% and 11.8% of students having tried cigarette smoking , respectively . in the present study , among male medical students , in contrast , a study conducted by sinha et al . showed 23.5% of females tried cigarette smoking . in the present study , less than 5% of students were current smokers . in contrast , a higher proportion of current smokers was reported by other researchers in india . reported 22.4% of medical students were current smokers none of the female students were current smokers , while 11.5% of male students indicated they were current smokers . , with 12.4% of male and 0.8% of female medical students were current smokers . reported 20.4% of male and 19.1% of female medical students were smokers , and jradi et al . reported all studies conducted among medical students reported higher rates of male current smokers relative to females . in the present study , in contrast , studies by alkawari et al . found that one - third of students smoked on college premises / property and 17.1% smoked in college buildings during the past year , respectively . in the present study , the prevalence of smokeless tobacco ever used was found to be 1.9% and the prevalence of current smokeless tobacco use was found to be 1.0% . in contrast , a study by joge et al . reported the prevalence of smokeless tobacco was 5.8% , selokar et al . in the present study , 2.5% of males and none of the females reported that 13.7% of males and 7.5% of females were current smokeless tobacco users . found that 34.2% used smokeless tobacco on campus during the past year in our study , approximately one - third of the students had exposure to smoke at home . nearly half of the male students and one - fourth of the female students had exposure to smoke at home . , where 27.9% were exposed to smoke at home . reported that upward of fifty percent of the students had exposure to smoke at home . in the present study , approximately half of the participants had exposure to smoke at public places ( 58.5% among male students and 44.4% among female students ) . reported that two - thirds of the students were exposed to smoke at public places . the study findings revealed that more medical students were exposed to environmental tobacco smoke than to active smoking among both males and females . this indicates the need for health education of families and the community about the health hazards of passive smoking . of the students in the study , 79.1% reported that their college has an official policy banning smoking in college buildings and clinics . , with 88.2% of students reporting that their college had an official policy banning smoking on college premises . found that only half the students reported that their college had an official policy banning smoking in college premises . in the present study , two - thirds of the students said an official smoking ban in college buildings and clinics has been enforced . a slightly higher percentage of students reported a smoking ban was enforced in the studies done by inandi et al . in the present study , more than 90% of the students favored banning the sale of tobacco products to adolescents , banning the advertisement of tobacco products , and banning smoking in restaurants and in public places . in the present study , nearly three - fourths of the students reported that smoking should be banned in discos / bars / pubs . reported that 70.5% and 66.9% , respectively , of students indicated smoking should be banned in discos / bars / pubs . in our study , even though more than 90% of the students supported bans of tobacco products in public places and restaurants , sales to adolescents and advertisements of tobacco products , only three - fourths of the students believed that tobacco products should be banned in discos , bars and pubs ; this finding was supported by other published studies . in the present study , approximately three - fourths of the students reported that health professionals should serve as a role model for their patients . , where 73.8% of medical students believed that health professionals served as role models for their patients and the public . reported that a slightly higher proportion ( 89% ) of medical students believe that health professionals serve as role models for their patients and the public . in the present study , 95.2% of the students reported that health professionals should get specific training in smoking cessation . in the present study , one - third of students had ever tried to stop smoking . in contrast , the study done by other investigators found that more than 50% of medical students had tried to quit smoking . in the present study , similarly , studies conducted by other investigators found that more than half of smokers ever received help or advice to stop smoking cigarettes . in our study , more than 70% of students reported health professionals who smoke are less likely to advise patients to stop smoking . found that a slightly higher proportion of students ( 77.5% ) reported that health professionals who smoke are less likely to advise patients to stop smoking . in the present study , more than 90% of students reported they were taught about the dangers of smoking . reported that only 43.7% of students were taught about the dangers of smoking . in addition , in the present study , more than 90% of students were taught that it is important to record tobacco use as a part of a patient s history . found that 88.9% of students were taught the importance of recording tobacco usage history . found that 34.2% of students received formal training in smoking cessation . in the present study , nearly three - fourths of the students are taught that it is important to provide educational materials on smoking to patients . found that 58.5% of students were taught the importance of providing educational materials on smoking to patients . in the present study , more than three - fourths of students lam et al . found a slightly higher proportion ( 90.8% ) in their study . in the current study , more than half of the students are aware of the use of antidepressants in tobacco cessation programs . jradi found that a slightly higher proportion ( 63% ) of students were aware of the use of antidepressants in tobacco cessation programs . in our study , less than 5% of medical students were current smokers , but upward of one - third to one - half were exposed to environmental tobacco smoke at home and in public places . as indicated by the majority of the participants , medical students need to receive formal training in smoking cessation techniques and this training must be included in the medical curriculum so that students can instruct or counsel patients .

brain cells release a variety of lipid mediators , which act in close proximity of their site of production to modulate synaptic plasticity and neural development . the reliability of this highly localized form of neural communication depends on the existence of deactivation mechanisms that ensure the rapid termination of lipid - mediated signaling , but few such mechanisms have been discovered so far . anandamide is an arachidonic acid derivative that regulates ion - channel activity and neurotransmitter release by engaging cb1 cannabinoid receptors on axon terminals . there is evidence that the intensity and duration of anandamide signaling are controlled by a two - step elimination process in which the substance is first internalized by neurons and astrocytes and then hydrolyzed by the intracellular membrane - bound amidases , faah-1 and faah-2 . removal of anandamide from the extracellular space exhibits several identifying features of a carrier - mediated facilitated diffusion process : ( i ) it is saturable and displays low micromolar affinity for anandamide ( apparent michaelis constant , km , 1.2 m in rat cortical neurons ) ; ( ii ) it preferentially recognizes anandamide over similar molecules , including the non - cannabinoid faah substrates oleoylethanolamide ( oea ) and palmitoylethanolamide ( pea ) ; ( iii ) it is inhibited in a competitive and stereoselective manner by substrate mimics ; and ( iv ) it does not require cellular energy . inhibitors of anandamide transport - which include the compounds am404 and omdm-1 - increase the levels of this endocannabinoid substance in vivo and produce a spectrum of cb1-mediated responses that only partially overlap with those elicited by faah blockade , presumably owing to the different kinetic properties and substrate preferences of the two deactivation mechanisms . these data indicate that carrier - mediated transport may play an important role in terminating the actions of anandamide and might represent a potential drug target . nevertheless , the molecular entity ( or entities ) involved in anandamide translocation is still unknown and the mechanistic bases of this process remain controversial . here , we identify a partly cytosolic variant of faah-1 , termed flat , which lacks amidase activity but binds anandamide with low micromolar affinity and confers anandamide transport to cells that are engineered to express it . am404 and other anandamide transport inhibitors suppress these effects . moreover , we disclose a small - molecule competitive inhibitor of the interaction of anandamide with flat , the compound arn272 , and show that this agent suppresses anandamide translocation in vitro and interrupts anandamide deactivation in vivo . we isolated total rna from brain and other rat tissues , and amplified products of the faah-1 gene using reverse - transcriptase polymerase chain reaction ( rt - pcr ) . one of the complementary dna products obtained was identical to faah-1 except that it lacked a 2 0 4 base - pair segment encoding for amino - acid residues 976 ( fig . 1a , supplementary fig . ribonuclease protection assays and southern blot analyses of rt - generated cdna confirmed the normal occurrence of flat mrna in rat brain and liver tissue ( supplementary fig . quantitative rt - pcr measurements showed that flat is unevenly transcribed in the rat brain , with highest levels in neocortex and hippocampus and lowest levels in brainstem and hypothalamus ( supplementary fig detectable levels of flat mrna were also found in rat primary astrocytes in cultures , rat neuroblastoma cells , and human astrocytoma cells ( supplementary fig . 1c ) , which were previously shown to express anandamide transport . an antibody raised against the c - terminus of faah-1 identified in brain cytosolic and membrane fractions obtained from wild - type mice , but not in those obtained from faah-1-deficient mice , a band with an apparent molecular weight of 56 kda , which is consistent with the calculated molecular weight of flat ( 56,008 da ) ( supplementary fig . this suggests that flat might be a product of the faah-1 gene generated by alternative splicing at non - canonical sites . the predicted structure of flat lacks most of faah-1 's 1 helix , which spans the lipid bilayer of intracellular membranes , and the entire 2 helix , which flanks the globular body of the protein exposed to the cytosol ( fig . when expressed in hek293 cells , flat displayed no detectable amidase activity toward anandamide or oea ( supplementary fig . computational studies identified two factors that might contribute to this loss of activity : ( i ) increased flexibility of regions proximal to the missing 1 and 2 helices - such as the ' 2-interacting loop ' ( fig . 1b ) - could facilitate access of water to the catalytic site buried inside the enzyme 's hydrophobic core ( supplementary fig . 3 ) ; and ( ii ) deletion of the 2 helix , which carries a positively charged surface , could lower the electrostatic potential in the region surrounding the catalytic triad component lys ( corresponding to lys in faah-1 ) ( supplementary fig . both factors are expected to impair amidase activity by interrupting the proton transfer from ser ( corresponding to catalytic ser in faah-1 ) to neutral lys . though critical for the amidase activity of faah-1 , lys does not influence the ability of this enzyme to cleave ester substrates . consistent with those data and the model proposed here , we found that recombinant flat effectively hydrolyzes the fatty acyl ester , 2-oleoyl - sn - glycerol ( supplementary fig . like faah-1 , recombinant purified flat forms homodimers in aqueous solution ( supplementary fig . 5 ) . to test whether flat also retains the ability to ligate anandamide , we expressed the protein fused with glutathione - s - transferase ( gst ) in e. coli and purified it by affinity chromatography . saturation binding studies showed that [ h]-anandamide associates with flat - gst ( dissociation constant , kd=2 m ) , but not with gst alone ( fig . the binding of [ h]-anandamide to flat is displaced by the anandamide transport inhibitors am404 and omdm-1 ( fig . 2b ) , with median inhibitory concentrations similar to those required for the inhibition of neuronal [ h]-anandamide internalization ( ic50 : am404 , 5.3 m ; omdm-1 , 4.8 m ) . 2b ) , likely because the productive interaction of this compound with the ser nucleophile of faah-1 requires a fully functional catalytic triad . collectively , the experiments described above indicate that flat lacks amidase activity , but binds anandamide with low micromolar affinity . we detected significant amounts of flat in cytosol fractions prepared from mouse brain ( supplementary fig . 2c ) , and found that the protein can be readily detached from hek293 cell membranes by incubation with sodium carbonate ( 0.1 m ) ( supplementary fig . 6 ) . these properties , along with the observation that am404 and omdm-1 antagonize the binding of [ h]-anandamide to flat ( fig . such a role was further implied by the reduced [ h]-anandamide accumulation observed in cultures of brain neurons obtained from faah-1 mice ( supplementary fig . we examined therefore whether heterologous expression of flat might increase anandamide transport in hek293 cells . we incubated control and flat - expressing cells for 5 min at 37c in a buffer containing [ h]-anandamide , and measured cell - associated radioactivity after removal of excess tracer . compared to controls , flat - expressing cells displayed a significantly higher level of [ h]-anandamide accumulation , which ( i ) was prevented by am404 ( ic50 = 4 m ) and other transport inhibitors ( omdm-1 , vdm11 , ucm707 ) , as well as by non - radioactive anandamide ( 100 m ) ( fig . 2d , e ) ; and ( ii ) was selective for anandamide compared to four structurally related lipids : the faah substrates [ h]-oea and [ h]-pea , the eicosanoid precursor [ h]-arachidonic acid , and the endocannabinoid fatty acyl ester [ h]-2-arachidonoyl - sn - glycerol ( 2-ag ) ( fig . [ h]-anandamide accumulation in flat - expressing hek293 cells may not be attributed to passive diffusion driven by faah - mediated hydrolysis , because the very low amidase activity present in native hek293 cells was not increased by flat expression ( supplementary fig . moreover , treatment with a maximally active concentration of the faah inhibitor urb597 ( 1 m ) or mutation of ser ( corresponding to the nucleophile ser in faah-1 ) did not affect [ h]-anandamide uptake by flat - expressing hek293 cells ( fig . urb597 did not affect [ h]-anandamide uptake at any of the concentration tested ( up to 10 m : 1035% of vehicle control ) . in addition to internalizing anandamide , the anandamide transport system may also facilitate the release of this lipid mediator from cells . to test whether flat contributes to this process , we over - expressed the protein in mouse neuro-2a cells and measured , by liquid chromatography / mass spectrometry , the levels of endogenously produced anandamide in the incubation medium . consistent with a role in anandamide release , flat over - expression was accompanied by a significant elevation in the extracellular levels of anandamide , but not 2-ag or oea ( supplementary fig . the results suggest that flat facilitates anandamide translocation through a mechanism that is selective for anandamide , independent of amidase activity , and prevented by known inhibitors of anandamide transport . to further investigate the functions of flat , and differentiate them from those of faah-1 , we searched for small drug - like molecules that might selectively interfere with flat 's ability to sequester anandamide . we subjected a virtual 4.3 million compound library to a ligand - screening campaign structured in multiple steps of progressively increasing stringency ( supplementary fig . the campaign returned a set of 46 structurally diverse compounds , which were tested for their interaction with flat in vitro . 3a ) , competitively antagonized [ h]-anandamide binding to purified flat ( ic50=1.8 m ; fig . 3a ) and inhibited [ h]-anandamide accumulation in both flat - expressing hek293 cells ( ic503 m ; fig . by contrast , arn272 exerted no significant effect on the residual [ h]-anandamide accumulation observed in cortical neurons of faah-1 mice ( supplementary fig . flat inhibition by arn272 appeared to be selective , because the compound had little or no inhibitory activity on several endocannabinoid - metabolizing enzymes , including n - acylphosphatidyl - ethanolamine - selective phospholipase d , diacylglycerol lipase- , monoacylglycerol lipase , and n - acylethanolamide - hydrolyzing acid amidase ( supplementary fig . moreover , arn272 produced only a weak and incomplete inhibition of rat brain faah activity ( fig . 3d ) , and was not significantly hydrolyzed after incubation with recombinant human faah-1 ( 5% hydrolysis after 24h at 37c ) . consistent with these observations , administration of arn272 ( 1 mg - kg , intraperitoneal , i.p . ) in mice increased plasma levels of anandamide ( fig . the inhibitory effects of arn272 on anandamide internalization in vitro and anandamide deactivation in vivo , along with the diminished anandamide accumulation observed in faah-1 mice , suggest that flat plays an important role in the membrane translocation of this endocannabinoid transmitter . anandamide transport inhibitors produce a variety of cb1-mediated responses , which include analgesia in animal models of nociceptive and inflammatory pain . we tested therefore whether arn272 might alleviate pain - related behaviors elicited in mice by intraplantar injection of the chemical irritant , formalin . systemic administration of arn272 ( 0.011 mg - kg , i.p . ) caused a dose - dependent reduction of formalin - induced pain behavior ( fig . substantial antinociceptive activity was observed on both the first phase of formalin pain , which involves acute activation of sensory c fibers , and the second phase of formalin pain , in which sensory fiber activity is accompanied by inflammation and central sensitization ( fig . similar effects were observed when arn272 ( 0.013 g per animal ) was injected into the cerebral ventricles ( supplementary fig . the cb1 antagonist am251 suppressed the antinociceptive actions of systemic arn272 , whereas the cb2 antagonist am630 and the transient receptor potential vanilloid-1 ( trpv-1 ) antagonist amg9810 were ineffective ( all drugs administered at 1 mg - kg , i.p ) ( fig . the cb1-mediated antinociceptive effects demonstrated by arn272 in the formalin test are similar to those previously reported for anandamide transport inhibitors such as am404 , omdm-1 and ucm707 . in another set of experiments , we evaluated the ability of arn272 to alleviate thermal hyperalgesia and paw edema elicited in mice by the pro - inflammatory polysaccharide , carrageenan . similarly to faah inhibitors , arn272 ( 0.013 mg - kg , i.p . ) exerted anti - hyperalgesic ( fig . these were suppressed by blockade of cb1 , but not cb2 or trpv-1 receptors ( fig . arn272 did not evoke any detectable nocifensive response when administered alone into the mouse paw ( supplementary table 1 ) , confirming its inability to interact productively with trpv-1 . finally , arn272 did not significantly alter the binding of the cannabinoid ligands [ h]-cp55940 or [ h]-anandamide to rat brain membranes ( supplementary fig . together , the findings indicate that arn272 elicits profound analgesic effects in mice , which result from inhibition of anandamide transport . we isolated total rna from brain and other rat tissues , and amplified products of the faah-1 gene using reverse - transcriptase polymerase chain reaction ( rt - pcr ) . one of the complementary dna products obtained was identical to faah-1 except that it lacked a 2 0 4 base - pair segment encoding for amino - acid residues 976 ( fig . 1a , supplementary fig . ribonuclease protection assays and southern blot analyses of rt - generated cdna confirmed the normal occurrence of flat mrna in rat brain and liver tissue ( supplementary fig . quantitative rt - pcr measurements showed that flat is unevenly transcribed in the rat brain , with highest levels in neocortex and hippocampus and lowest levels in brainstem and hypothalamus ( supplementary fig detectable levels of flat mrna were also found in rat primary astrocytes in cultures , rat neuroblastoma cells , and human astrocytoma cells ( supplementary fig . 1c ) , which were previously shown to express anandamide transport . an antibody raised against the c - terminus of faah-1 identified in brain cytosolic and membrane fractions obtained from wild - type mice , but not in those obtained from faah-1-deficient mice , a band with an apparent molecular weight of 56 kda , which is consistent with the calculated molecular weight of flat ( 56,008 da ) ( supplementary fig . this suggests that flat might be a product of the faah-1 gene generated by alternative splicing at non - canonical sites . the predicted structure of flat lacks most of faah-1 's 1 helix , which spans the lipid bilayer of intracellular membranes , and the entire 2 helix , which flanks the globular body of the protein exposed to the cytosol ( fig . when expressed in hek293 cells , flat displayed no detectable amidase activity toward anandamide or oea ( supplementary fig . computational studies identified two factors that might contribute to this loss of activity : ( i ) increased flexibility of regions proximal to the missing 1 and 2 helices - such as the ' 2-interacting loop ' ( fig . 1b ) - could facilitate access of water to the catalytic site buried inside the enzyme 's hydrophobic core ( supplementary fig . 3 ) ; and ( ii ) deletion of the 2 helix , which carries a positively charged surface , could lower the electrostatic potential in the region surrounding the catalytic triad component lys ( corresponding to lys in faah-1 ) ( supplementary fig . both factors are expected to impair amidase activity by interrupting the proton transfer from ser ( corresponding to catalytic ser in faah-1 ) to neutral lys . though critical for the amidase activity of faah-1 , lys does not influence the ability of this enzyme to cleave ester substrates . consistent with those data and the model proposed here , we found that recombinant flat effectively hydrolyzes the fatty acyl ester , 2-oleoyl - sn - glycerol ( supplementary fig . like faah-1 , recombinant purified flat forms homodimers in aqueous solution ( supplementary fig . 5 ) . to test whether flat also retains the ability to ligate anandamide , we expressed the protein fused with glutathione - s - transferase ( gst ) in e. coli and purified it by affinity chromatography . saturation binding studies showed that [ h]-anandamide associates with flat - gst ( dissociation constant , kd=2 m ) , but not with gst alone ( fig . the binding of [ h]-anandamide to flat is displaced by the anandamide transport inhibitors am404 and omdm-1 ( fig . 2b ) , with median inhibitory concentrations similar to those required for the inhibition of neuronal [ h]-anandamide internalization ( ic50 : am404 , 5.3 m ; omdm-1 , 4.8 m ) . 2b ) , likely because the productive interaction of this compound with the ser nucleophile of faah-1 requires a fully functional catalytic triad . collectively , the experiments described above indicate that flat lacks amidase activity , but binds anandamide with low micromolar affinity . we detected significant amounts of flat in cytosol fractions prepared from mouse brain ( supplementary fig . 2c ) , and found that the protein can be readily detached from hek293 cell membranes by incubation with sodium carbonate ( 0.1 m ) ( supplementary fig . 6 ) . these properties , along with the observation that am404 and omdm-1 antagonize the binding of [ h]-anandamide to flat ( fig . such a role was further implied by the reduced [ h]-anandamide accumulation observed in cultures of brain neurons obtained from faah-1 mice ( supplementary fig . we examined therefore whether heterologous expression of flat might increase anandamide transport in hek293 cells . we incubated control and flat - expressing cells for 5 min at 37c in a buffer containing [ h]-anandamide , and measured cell - associated radioactivity after removal of excess tracer . compared to controls , flat - expressing cells displayed a significantly higher level of [ h]-anandamide accumulation , which ( i ) was prevented by am404 ( ic50 = 4 m ) and other transport inhibitors ( omdm-1 , vdm11 , ucm707 ) , as well as by non - radioactive anandamide ( 100 m ) ( fig . 2d , e ) ; and ( ii ) was selective for anandamide compared to four structurally related lipids : the faah substrates [ h]-oea and [ h]-pea , the eicosanoid precursor [ h]-arachidonic acid , and the endocannabinoid fatty acyl ester [ h]-2-arachidonoyl - sn - glycerol ( 2-ag ) ( fig . [ h]-anandamide accumulation in flat - expressing hek293 cells may not be attributed to passive diffusion driven by faah - mediated hydrolysis , because the very low amidase activity present in native hek293 cells was not increased by flat expression ( supplementary fig . moreover , treatment with a maximally active concentration of the faah inhibitor urb597 ( 1 m ) or mutation of ser ( corresponding to the nucleophile ser in faah-1 ) did not affect [ h]-anandamide uptake by flat - expressing hek293 cells ( fig . urb597 did not affect [ h]-anandamide uptake at any of the concentration tested ( up to 10 m : 1035% of vehicle control ) . in addition to internalizing anandamide , the anandamide transport system may also facilitate the release of this lipid mediator from cells . to test whether flat contributes to this process , we over - expressed the protein in mouse neuro-2a cells and measured , by liquid chromatography / mass spectrometry , the levels of endogenously produced anandamide in the incubation medium . consistent with a role in anandamide release , flat over - expression was accompanied by a significant elevation in the extracellular levels of anandamide , but not 2-ag or oea ( supplementary fig . 7 ) . the results suggest that flat facilitates anandamide translocation through a mechanism that is selective for anandamide , independent of amidase activity , and prevented by known inhibitors of anandamide transport . to further investigate the functions of flat , and differentiate them from those of faah-1 , we searched for small drug - like molecules that might selectively interfere with flat 's ability to sequester anandamide . we subjected a virtual 4.3 million compound library to a ligand - screening campaign structured in multiple steps of progressively increasing stringency ( supplementary fig . the campaign returned a set of 46 structurally diverse compounds , which were tested for their interaction with flat in vitro . the substituted phthalazine arn272 ( fig . 3a ) , competitively antagonized [ h]-anandamide binding to purified flat ( ic50=1.8 m ; fig . 3a ) and inhibited [ h]-anandamide accumulation in both flat - expressing hek293 cells ( ic503 m ; fig . by contrast , arn272 exerted no significant effect on the residual [ h]-anandamide accumulation observed in cortical neurons of faah-1 mice ( supplementary fig . flat inhibition by arn272 appeared to be selective , because the compound had little or no inhibitory activity on several endocannabinoid - metabolizing enzymes , including n - acylphosphatidyl - ethanolamine - selective phospholipase d , diacylglycerol lipase- , monoacylglycerol lipase , and n - acylethanolamide - hydrolyzing acid amidase ( supplementary fig . 10a moreover , arn272 produced only a weak and incomplete inhibition of rat brain faah activity ( fig . 3d ) , and was not significantly hydrolyzed after incubation with recombinant human faah-1 ( 5% hydrolysis after 24h at 37c ) . consistent with these observations , administration of arn272 ( 1 mg - kg , intraperitoneal , i.p . ) in mice increased plasma levels of anandamide ( fig . the inhibitory effects of arn272 on anandamide internalization in vitro and anandamide deactivation in vivo , along with the diminished anandamide accumulation observed in faah-1 mice , suggest that flat plays an important role in the membrane translocation of this endocannabinoid transmitter . anandamide transport inhibitors produce a variety of cb1-mediated responses , which include analgesia in animal models of nociceptive and inflammatory pain . we tested therefore whether arn272 might alleviate pain - related behaviors elicited in mice by intraplantar injection of the chemical irritant , formalin . systemic administration of arn272 ( 0.011 mg - kg , i.p . ) caused a dose - dependent reduction of formalin - induced pain behavior ( fig . substantial antinociceptive activity was observed on both the first phase of formalin pain , which involves acute activation of sensory c fibers , and the second phase of formalin pain , in which sensory fiber activity is accompanied by inflammation and central sensitization ( fig . similar effects were observed when arn272 ( 0.013 g per animal ) was injected into the cerebral ventricles ( supplementary fig . the cb1 antagonist am251 suppressed the antinociceptive actions of systemic arn272 , whereas the cb2 antagonist am630 and the transient receptor potential vanilloid-1 ( trpv-1 ) antagonist amg9810 were ineffective ( all drugs administered at 1 mg - kg , i.p ) ( fig . the cb1-mediated antinociceptive effects demonstrated by arn272 in the formalin test are similar to those previously reported for anandamide transport inhibitors such as am404 , omdm-1 and ucm707 . in another set of experiments , we evaluated the ability of arn272 to alleviate thermal hyperalgesia and paw edema elicited in mice by the pro - inflammatory polysaccharide , carrageenan . similarly to faah inhibitors , arn272 ( 0.013 mg - kg , i.p . ) exerted anti - hyperalgesic ( fig . these were suppressed by blockade of cb1 , but not cb2 or trpv-1 receptors ( fig . arn272 did not evoke any detectable nocifensive response when administered alone into the mouse paw ( supplementary table 1 ) , confirming its inability to interact productively with trpv-1 . finally , arn272 did not significantly alter the binding of the cannabinoid ligands [ h]-cp55940 or [ h]-anandamide to rat brain membranes ( supplementary fig . together , the findings indicate that arn272 elicits profound analgesic effects in mice , which result from inhibition of anandamide transport . the functional properties of flat suggest that this protein is a key molecular component of the anandamide transport system in neural cells , and a potential target for therapeutic drugs . the findings presented here indicate that flat selectively binds to and internalizes anandamide , and that several known inhibitors of anandamide translocation - am404 , omdm-1 , u c m-707 and vdm-11 - interfere with these properties . moreover , our results show that arn272 , a small - molecule inhibitor of the interaction of anandamide with flat , suppresses anandamide accumulation by rat brain neurons in vitro and reproduces two key effects of transport blockade in vivo : elevation of plasma anandamide levels , and analgesia in models of nociceptive and inflammatory pain . consistent with these data and previous reports , deletion of the faah-1 gene substantially reduced anandamide transport in mouse cortical neurons , whereas acute pharmacological blockade of faah activity failed to do so . while implying an important role for flat in anandamide transport , our findings do not rule out the possibility that additional components of the endocannabinoid transport system remain to be discovered . in this context , it is important to point out that flat expression did not confer [ h]-2-ag or [ h]-oea transport to hek293 cells , and administration of the flat inhibitor arn272 did not increase plasma levels of 2-ag or oea in mice , which indicates that the translocation of these lipid mediators may be independent of flat . because of its ability to inhibit anandamide deactivation selectively , arn272 may be useful to differentiate the functional roles of anandamide from those of other lipid amides that are substrates for faah ( e.g. , oea and pea ) . multicellular organisms utilize protein carriers to coordinate the traffic of functionally important lipids , and target these biomolecules toward specific cells and subcellular compartments . two main types of lipid - carrier proteins are employed for this task : integral membrane transporters , such as cd36 and pgt , and lipid chaperones , such as ap2 and mal1 ( fatty acid - binding protein-4 and 5 , respectively ) . for example , membrane - bound cd36 in small - intestinal enterocytes captures dietary oleic acid and directs it toward the intracellular biosynthesis of oea , an important gut hormone . on the other hand , cytosolic ap2 in adipocytes encapsulates fatty acids derived from the circulation and partitions them toward appropriate cellular sites for storage or oxidative metabolism . our experiments suggest that , similarly to a lipid chaperone , flat might function by desorbing anandamide from the cell membrane and delivering it to intracellular organelles where faah-1 is located ( supplementary fig . it is also possible , though remains to be fully tested , that flat might contribute to anandamide release by facilitating the intracellular transfer of this lipophilic molecule from its as - yet - unknown site of biosynthesis to the cell membrane . despite its similarities with other lipid chaperones first , its substrate preference and sensitivity to pharmacological agents distinguish it from other carriers for lipophilic ligands , such as serum albumin and fatty acid - binding proteins , which are known to sequester anandamide . second , our studies suggest that the capacity of flat to ligate anandamide may be based on structural modifications that silence the amidase activity of faah-1 without compromising its anandamide - binding function . this mechanism provides an elegant example of phylogenetic parsimony and raises the possibility that other lipid transporters might have evolved following similar principles .

the endocannabinoid anandamide is removed from the synaptic space by a selective transport system , expressed in neurons and astrocytes , which remains molecularly uncharacterized . here we describe a partly cytosolic variant of the intracellular anandamide - degrading enzyme , fatty acid amide hydrolase-1 ( faah-1 ) , termed faah - like anandamide transporter ( flat ) , which lacks amidase activity but binds anandamide with low micromolar affinity and facilitates its translocation into cells . known anandamide transport inhibitors , such as am404 and omdm-1 , block these effects . additionally , we identify a competitive antagonist of the interaction of anandamide with flat , the phthalazine derivative arn272 , which prevents anandamide internalization in vitro , interrupts anandamide deactivation in vivo , and exerts profound analgesic effects in rodent models of nociceptive and inflammatory pain , which are mediated by cb1 cannabinoid receptors . the results identify flat as a critical molecular component of anandamide transport in neural cells and a potential target for therapeutic drugs .

INTRODUCTION RESULTS FLAT is an intracellular anandamide-binding protein A role for FLAT in anandamide transport Discovery of a competitive FLAT inhibitor ARN272 attenuates nociceptive and inflammatory pain DISCUSSION Supplementary Material

brain cells release a variety of lipid mediators , which act in close proximity of their site of production to modulate synaptic plasticity and neural development . anandamide is an arachidonic acid derivative that regulates ion - channel activity and neurotransmitter release by engaging cb1 cannabinoid receptors on axon terminals . there is evidence that the intensity and duration of anandamide signaling are controlled by a two - step elimination process in which the substance is first internalized by neurons and astrocytes and then hydrolyzed by the intracellular membrane - bound amidases , faah-1 and faah-2 . removal of anandamide from the extracellular space exhibits several identifying features of a carrier - mediated facilitated diffusion process : ( i ) it is saturable and displays low micromolar affinity for anandamide ( apparent michaelis constant , km , 1.2 m in rat cortical neurons ) ; ( ii ) it preferentially recognizes anandamide over similar molecules , including the non - cannabinoid faah substrates oleoylethanolamide ( oea ) and palmitoylethanolamide ( pea ) ; ( iii ) it is inhibited in a competitive and stereoselective manner by substrate mimics ; and ( iv ) it does not require cellular energy . inhibitors of anandamide transport - which include the compounds am404 and omdm-1 - increase the levels of this endocannabinoid substance in vivo and produce a spectrum of cb1-mediated responses that only partially overlap with those elicited by faah blockade , presumably owing to the different kinetic properties and substrate preferences of the two deactivation mechanisms . these data indicate that carrier - mediated transport may play an important role in terminating the actions of anandamide and might represent a potential drug target . here , we identify a partly cytosolic variant of faah-1 , termed flat , which lacks amidase activity but binds anandamide with low micromolar affinity and confers anandamide transport to cells that are engineered to express it . am404 and other anandamide transport inhibitors suppress these effects . moreover , we disclose a small - molecule competitive inhibitor of the interaction of anandamide with flat , the compound arn272 , and show that this agent suppresses anandamide translocation in vitro and interrupts anandamide deactivation in vivo . we isolated total rna from brain and other rat tissues , and amplified products of the faah-1 gene using reverse - transcriptase polymerase chain reaction ( rt - pcr ) . quantitative rt - pcr measurements showed that flat is unevenly transcribed in the rat brain , with highest levels in neocortex and hippocampus and lowest levels in brainstem and hypothalamus ( supplementary fig detectable levels of flat mrna were also found in rat primary astrocytes in cultures , rat neuroblastoma cells , and human astrocytoma cells ( supplementary fig . 1c ) , which were previously shown to express anandamide transport . an antibody raised against the c - terminus of faah-1 identified in brain cytosolic and membrane fractions obtained from wild - type mice , but not in those obtained from faah-1-deficient mice , a band with an apparent molecular weight of 56 kda , which is consistent with the calculated molecular weight of flat ( 56,008 da ) ( supplementary fig . this suggests that flat might be a product of the faah-1 gene generated by alternative splicing at non - canonical sites . the predicted structure of flat lacks most of faah-1 's 1 helix , which spans the lipid bilayer of intracellular membranes , and the entire 2 helix , which flanks the globular body of the protein exposed to the cytosol ( fig . when expressed in hek293 cells , flat displayed no detectable amidase activity toward anandamide or oea ( supplementary fig . computational studies identified two factors that might contribute to this loss of activity : ( i ) increased flexibility of regions proximal to the missing 1 and 2 helices - such as the ' 2-interacting loop ' ( fig . 3 ) ; and ( ii ) deletion of the 2 helix , which carries a positively charged surface , could lower the electrostatic potential in the region surrounding the catalytic triad component lys ( corresponding to lys in faah-1 ) ( supplementary fig . both factors are expected to impair amidase activity by interrupting the proton transfer from ser ( corresponding to catalytic ser in faah-1 ) to neutral lys . though critical for the amidase activity of faah-1 , lys does not influence the ability of this enzyme to cleave ester substrates . consistent with those data and the model proposed here , we found that recombinant flat effectively hydrolyzes the fatty acyl ester , 2-oleoyl - sn - glycerol ( supplementary fig . saturation binding studies showed that [ h]-anandamide associates with flat - gst ( dissociation constant , kd=2 m ) , but not with gst alone ( fig . the binding of [ h]-anandamide to flat is displaced by the anandamide transport inhibitors am404 and omdm-1 ( fig . 2b ) , with median inhibitory concentrations similar to those required for the inhibition of neuronal [ h]-anandamide internalization ( ic50 : am404 , 5.3 m ; omdm-1 , 4.8 m ) . 2b ) , likely because the productive interaction of this compound with the ser nucleophile of faah-1 requires a fully functional catalytic triad . collectively , the experiments described above indicate that flat lacks amidase activity , but binds anandamide with low micromolar affinity . 2c ) , and found that the protein can be readily detached from hek293 cell membranes by incubation with sodium carbonate ( 0.1 m ) ( supplementary fig . these properties , along with the observation that am404 and omdm-1 antagonize the binding of [ h]-anandamide to flat ( fig . we examined therefore whether heterologous expression of flat might increase anandamide transport in hek293 cells . we incubated control and flat - expressing cells for 5 min at 37c in a buffer containing [ h]-anandamide , and measured cell - associated radioactivity after removal of excess tracer . compared to controls , flat - expressing cells displayed a significantly higher level of [ h]-anandamide accumulation , which ( i ) was prevented by am404 ( ic50 = 4 m ) and other transport inhibitors ( omdm-1 , vdm11 , ucm707 ) , as well as by non - radioactive anandamide ( 100 m ) ( fig . 2d , e ) ; and ( ii ) was selective for anandamide compared to four structurally related lipids : the faah substrates [ h]-oea and [ h]-pea , the eicosanoid precursor [ h]-arachidonic acid , and the endocannabinoid fatty acyl ester [ h]-2-arachidonoyl - sn - glycerol ( 2-ag ) ( fig . [ h]-anandamide accumulation in flat - expressing hek293 cells may not be attributed to passive diffusion driven by faah - mediated hydrolysis , because the very low amidase activity present in native hek293 cells was not increased by flat expression ( supplementary fig . moreover , treatment with a maximally active concentration of the faah inhibitor urb597 ( 1 m ) or mutation of ser ( corresponding to the nucleophile ser in faah-1 ) did not affect [ h]-anandamide uptake by flat - expressing hek293 cells ( fig . in addition to internalizing anandamide , the anandamide transport system may also facilitate the release of this lipid mediator from cells . to test whether flat contributes to this process , we over - expressed the protein in mouse neuro-2a cells and measured , by liquid chromatography / mass spectrometry , the levels of endogenously produced anandamide in the incubation medium . consistent with a role in anandamide release , flat over - expression was accompanied by a significant elevation in the extracellular levels of anandamide , but not 2-ag or oea ( supplementary fig . the results suggest that flat facilitates anandamide translocation through a mechanism that is selective for anandamide , independent of amidase activity , and prevented by known inhibitors of anandamide transport . to further investigate the functions of flat , and differentiate them from those of faah-1 , we searched for small drug - like molecules that might selectively interfere with flat 's ability to sequester anandamide . the campaign returned a set of 46 structurally diverse compounds , which were tested for their interaction with flat in vitro . 3a ) , competitively antagonized [ h]-anandamide binding to purified flat ( ic50=1.8 m ; fig . 3d ) , and was not significantly hydrolyzed after incubation with recombinant human faah-1 ( 5% hydrolysis after 24h at 37c ) . the inhibitory effects of arn272 on anandamide internalization in vitro and anandamide deactivation in vivo , along with the diminished anandamide accumulation observed in faah-1 mice , suggest that flat plays an important role in the membrane translocation of this endocannabinoid transmitter . anandamide transport inhibitors produce a variety of cb1-mediated responses , which include analgesia in animal models of nociceptive and inflammatory pain . we tested therefore whether arn272 might alleviate pain - related behaviors elicited in mice by intraplantar injection of the chemical irritant , formalin . substantial antinociceptive activity was observed on both the first phase of formalin pain , which involves acute activation of sensory c fibers , and the second phase of formalin pain , in which sensory fiber activity is accompanied by inflammation and central sensitization ( fig . the cb1-mediated antinociceptive effects demonstrated by arn272 in the formalin test are similar to those previously reported for anandamide transport inhibitors such as am404 , omdm-1 and ucm707 . together , the findings indicate that arn272 elicits profound analgesic effects in mice , which result from inhibition of anandamide transport . we isolated total rna from brain and other rat tissues , and amplified products of the faah-1 gene using reverse - transcriptase polymerase chain reaction ( rt - pcr ) . quantitative rt - pcr measurements showed that flat is unevenly transcribed in the rat brain , with highest levels in neocortex and hippocampus and lowest levels in brainstem and hypothalamus ( supplementary fig detectable levels of flat mrna were also found in rat primary astrocytes in cultures , rat neuroblastoma cells , and human astrocytoma cells ( supplementary fig . 1c ) , which were previously shown to express anandamide transport . this suggests that flat might be a product of the faah-1 gene generated by alternative splicing at non - canonical sites . the predicted structure of flat lacks most of faah-1 's 1 helix , which spans the lipid bilayer of intracellular membranes , and the entire 2 helix , which flanks the globular body of the protein exposed to the cytosol ( fig . when expressed in hek293 cells , flat displayed no detectable amidase activity toward anandamide or oea ( supplementary fig . 3 ) ; and ( ii ) deletion of the 2 helix , which carries a positively charged surface , could lower the electrostatic potential in the region surrounding the catalytic triad component lys ( corresponding to lys in faah-1 ) ( supplementary fig . both factors are expected to impair amidase activity by interrupting the proton transfer from ser ( corresponding to catalytic ser in faah-1 ) to neutral lys . saturation binding studies showed that [ h]-anandamide associates with flat - gst ( dissociation constant , kd=2 m ) , but not with gst alone ( fig . the binding of [ h]-anandamide to flat is displaced by the anandamide transport inhibitors am404 and omdm-1 ( fig . 2b ) , with median inhibitory concentrations similar to those required for the inhibition of neuronal [ h]-anandamide internalization ( ic50 : am404 , 5.3 m ; omdm-1 , 4.8 m ) . 2b ) , likely because the productive interaction of this compound with the ser nucleophile of faah-1 requires a fully functional catalytic triad . collectively , the experiments described above indicate that flat lacks amidase activity , but binds anandamide with low micromolar affinity . 2c ) , and found that the protein can be readily detached from hek293 cell membranes by incubation with sodium carbonate ( 0.1 m ) ( supplementary fig . these properties , along with the observation that am404 and omdm-1 antagonize the binding of [ h]-anandamide to flat ( fig . we examined therefore whether heterologous expression of flat might increase anandamide transport in hek293 cells . compared to controls , flat - expressing cells displayed a significantly higher level of [ h]-anandamide accumulation , which ( i ) was prevented by am404 ( ic50 = 4 m ) and other transport inhibitors ( omdm-1 , vdm11 , ucm707 ) , as well as by non - radioactive anandamide ( 100 m ) ( fig . 2d , e ) ; and ( ii ) was selective for anandamide compared to four structurally related lipids : the faah substrates [ h]-oea and [ h]-pea , the eicosanoid precursor [ h]-arachidonic acid , and the endocannabinoid fatty acyl ester [ h]-2-arachidonoyl - sn - glycerol ( 2-ag ) ( fig . [ h]-anandamide accumulation in flat - expressing hek293 cells may not be attributed to passive diffusion driven by faah - mediated hydrolysis , because the very low amidase activity present in native hek293 cells was not increased by flat expression ( supplementary fig . moreover , treatment with a maximally active concentration of the faah inhibitor urb597 ( 1 m ) or mutation of ser ( corresponding to the nucleophile ser in faah-1 ) did not affect [ h]-anandamide uptake by flat - expressing hek293 cells ( fig . in addition to internalizing anandamide , the anandamide transport system may also facilitate the release of this lipid mediator from cells . to test whether flat contributes to this process , we over - expressed the protein in mouse neuro-2a cells and measured , by liquid chromatography / mass spectrometry , the levels of endogenously produced anandamide in the incubation medium . consistent with a role in anandamide release , flat over - expression was accompanied by a significant elevation in the extracellular levels of anandamide , but not 2-ag or oea ( supplementary fig . the results suggest that flat facilitates anandamide translocation through a mechanism that is selective for anandamide , independent of amidase activity , and prevented by known inhibitors of anandamide transport . to further investigate the functions of flat , and differentiate them from those of faah-1 , we searched for small drug - like molecules that might selectively interfere with flat 's ability to sequester anandamide . the campaign returned a set of 46 structurally diverse compounds , which were tested for their interaction with flat in vitro . 3d ) , and was not significantly hydrolyzed after incubation with recombinant human faah-1 ( 5% hydrolysis after 24h at 37c ) . the inhibitory effects of arn272 on anandamide internalization in vitro and anandamide deactivation in vivo , along with the diminished anandamide accumulation observed in faah-1 mice , suggest that flat plays an important role in the membrane translocation of this endocannabinoid transmitter . anandamide transport inhibitors produce a variety of cb1-mediated responses , which include analgesia in animal models of nociceptive and inflammatory pain . we tested therefore whether arn272 might alleviate pain - related behaviors elicited in mice by intraplantar injection of the chemical irritant , formalin . substantial antinociceptive activity was observed on both the first phase of formalin pain , which involves acute activation of sensory c fibers , and the second phase of formalin pain , in which sensory fiber activity is accompanied by inflammation and central sensitization ( fig . the cb1-mediated antinociceptive effects demonstrated by arn272 in the formalin test are similar to those previously reported for anandamide transport inhibitors such as am404 , omdm-1 and ucm707 . together , the findings indicate that arn272 elicits profound analgesic effects in mice , which result from inhibition of anandamide transport . the functional properties of flat suggest that this protein is a key molecular component of the anandamide transport system in neural cells , and a potential target for therapeutic drugs . the findings presented here indicate that flat selectively binds to and internalizes anandamide , and that several known inhibitors of anandamide translocation - am404 , omdm-1 , u c m-707 and vdm-11 - interfere with these properties . moreover , our results show that arn272 , a small - molecule inhibitor of the interaction of anandamide with flat , suppresses anandamide accumulation by rat brain neurons in vitro and reproduces two key effects of transport blockade in vivo : elevation of plasma anandamide levels , and analgesia in models of nociceptive and inflammatory pain . consistent with these data and previous reports , deletion of the faah-1 gene substantially reduced anandamide transport in mouse cortical neurons , whereas acute pharmacological blockade of faah activity failed to do so . while implying an important role for flat in anandamide transport , our findings do not rule out the possibility that additional components of the endocannabinoid transport system remain to be discovered . in this context , it is important to point out that flat expression did not confer [ h]-2-ag or [ h]-oea transport to hek293 cells , and administration of the flat inhibitor arn272 did not increase plasma levels of 2-ag or oea in mice , which indicates that the translocation of these lipid mediators may be independent of flat . because of its ability to inhibit anandamide deactivation selectively , arn272 may be useful to differentiate the functional roles of anandamide from those of other lipid amides that are substrates for faah ( e.g. multicellular organisms utilize protein carriers to coordinate the traffic of functionally important lipids , and target these biomolecules toward specific cells and subcellular compartments . two main types of lipid - carrier proteins are employed for this task : integral membrane transporters , such as cd36 and pgt , and lipid chaperones , such as ap2 and mal1 ( fatty acid - binding protein-4 and 5 , respectively ) . despite its similarities with other lipid chaperones first , its substrate preference and sensitivity to pharmacological agents distinguish it from other carriers for lipophilic ligands , such as serum albumin and fatty acid - binding proteins , which are known to sequester anandamide . second , our studies suggest that the capacity of flat to ligate anandamide may be based on structural modifications that silence the amidase activity of faah-1 without compromising its anandamide - binding function .

a biological species is usually defined in principle as a set of actually or potentially interbreeding organisms , but as interbreeding is very difficult to measure , species have in practice been identified by their phenotypic traits . until recently , progress in most microbe - related disciplines has been hampered by the enormous effort needed to identify less prominent traits . we are now in an age when we can identify species based on the genome ( genotype ) , although this does not change the principle that taxonomy is defined by phenotypes : according to the generally accepted rules of taxonomy , a strain belongs to a species if it falls within the range of phenotypes that define that species . this situation has been brought about by the success of the ribosomal rna approach to phylogenetics . well - conserved molecules , such as 16s rrna in particular , have been used to give a species a molecular identifier and to draw phylogenetic relationships . the 16s rrna - based approach has been widely accepted and has proved successful in phylogenetic tree - making and even in identifying species . in this context , the ribosomal database project has been established . there are other similar approaches , such as one based on the gyrase gene and multilocus sequence typing . nonetheless , it has been impossible in practice to analyze all the constituents of a microbial population , not only because of the huge size of such populations ( more than 10 cells per ml ) but also because of lack of suitable methodology . although there are methods other than gene and genome sequencing for analyzing genomes , such as restriction - fragment length polymorphism ( rflp ) , amplified fragment - length polymorphism ( aflp ) , octamer - based genome scanning ( obgs ) , random polymerase chain reaction ( pcr ) and others , most can not be used to identify species without a knowledge of phenotypic traits . in reality , there is no general methodology that enables us to identify species by genotype only , although many approaches use genotypic information ( dna sequences ) to complement phenotypic information . we have recently demonstrated the possibility of species identification by genotype using genome profiling , which is a temperature - gradient gel electrophoresis ( tgge ) analysis of random pcr products . in particular , the use of ' species - identification dots ' ( spiddos ) , which are feature points in genome profiles , is very useful for objective and reproducible data processing . we present here a universal method for provisional genotype - based species identification based on these technological advances and using the internet environment , which enables us to identify species in general . this paper also presents the important concepts of genome distance and genome sequence space , which are essential for species identification based on genotype . figure 1 shows one of the results obtained using the protocols described in the materials and methods . for the query species , the closest species as judged by spiddos and a list of genome profiles within the tolerance ( ) , together with the annotation attached to it , is given ( figure 1 ) . if there is a genome profile among the list annotated with species , then it means that the query species is identified with the confidence defined by the pattern similarity score ( pass ; see materials and methods ) . if the value of pass is very high ( that is , close to unity ) , then it is highly probable that it is indeed an exact match . in contrast , if the value is not sufficiently close to unity , then it may be only a related species ( not the exact species ) , belonging to the same genus or family or any of the higher taxonomical categories , depending on the value of pass . although we do not yet have enough data to determine the pass value at which it is safe to identify a species , we have a preliminary idea , based on experience , that 0.95 ( z score 4 ) may be a critical value . the important challenge of how to reconcile the difference between identification of species by phenotype , which conventional taxonomy has adopted , with that based on genotype , is discussed later . an important aspect of this system is that one does not need to be a specialist in the relevant biological field to obtain an initial identification of an unknown organism . all that is required is to register the genome profile of the unknown species on the database . therefore , an incomplete set of phenotypic data , which do not reach the criteria for species identification ( say , peculiar behaviors or unusual properties ) , can also be registered and later used without having to undertake further laborious phenotypic identification ( figure 2 ) . all the information regarding a given species ( in other words , all the entries within a certain pass value ) scientists can work cooperatively to identify species and collect their phenotypic traits ( figure 2 ) . in conventional approaches to identification , most of which have been phenotype - based , those data that failed to meet the required criteria for identification were left unconnected , and could not be used later because there was no convenient way of correlating them with a given species without knowing the species name ( figure 2 ) . thus , our approach of genotype - based species identification , utilizing genome profile and the internet , will be of great help to the field of taxonomy . in evaluating the effectiveness of this methodology , the nature of pass must first be considered , as it plays the most important part in the method . as pass is calculated on the basis of the coordinates of spiddos ( see equation 1 in materials and methods ) , the nature of spiddos must be thoroughly investigated . if two genomic dnas contain common sequence regions that can be amplified by random pcr using the same primer , the resultant dnas will usually generate similar spiddos ( by definition , the spiddos obtained by tgge represent the crucial points of a genome profile , points at which the temperature corresponds to the beginning of a prominent structural transition in dna ) . as shown schematically in figure 3 , two corresponding spiddos derived from two closely related species can be connected by a displacement vector , which consists of two independent elements of mobility ( ) and temperature ( ) . the differences in each element ( and ) can be related to the differences between two sequences as shown in figure 3 . the displacement in the ordinate is caused by the difference in length between the two dnas and is caused by deletion or insertion , whereas that in the abscissa is mainly caused by point mutation ( although insertion / deletion can also contribute ) . as the extent of these changes is roughly proportional to the evolutionary time since the species diverged , we can expect that the summation of the displacement of each spiddo is approximately proportional to the time since divergence , and thus to the genome - to - genome distance . by using a sufficient number of spiddos empirically , we know that 8 - 10 spiddos , which can be obtained from a single genome profile , can be significant . however , since the more spiddos the better the result , we tentatively made it a rule to adopt four genome profiles ( 32 - 40 spiddos ) - that is , four random - pcr products - as a current standard of initial species identification . therefore , pass has the theoretical and empirical basis to be used as a measure of similarity between genomes , although the extent of its effectiveness remains to be shown experimentally as data accumulates . we have introduced a measure of distance , d ' , obtained from pass as formulated in equation 2 ( see materials and methods ) , for the sake of convenience . we call d ' a genome sub - distance because it is based not on the whole but a part of the genome sequence . thus , we introduce ( true ) genome distance , d , as in equation 3 ( see materials and methods ) . genome distance must have a close relationship with genetic distance , as defined by nei and others , although there is a difference in the definition . the genetic distance based on sequences is basically the hamming distance ( the number of different letters at each corresponding position of two sequences of letters that are optimally aligned ) between two nucleotide ( or amino - acid ) sequences . in aligning sequences arbitrariness another constraint on genetic distance is that it is usually obtained from a limited number of genes , although that is also the case for genome distance . as genome distance is easier to obtain in practice using our method , it should be easier to obtain a lot of data on it compared with genetic distance . on the basis of d ( in practice d ' ) , we can construct phylogenetic trees and genome sequence space ( an imaginary spherical space in which all the genomes ( individuals ) can be uniquely located in a finite manner based on the distance between genomes , providing clusters of species ( k.n . although the applicability and effectiveness of genome distance for such purposes needs to be further investigated , it is obvious that an organism that has near - zero genome distance from a certain standard species , as an average over four or more genome sub - distances obtained from as many genome profiles , can be easily assigned to that same species with a high level of confidence . we are not claiming , however , to be able to give the correct taxonomical name to any species using this method . the greater the number of strains registered in the database , the more easily will a species be assigned . basically , no special efforts , except expanding the database and using sophisticated algorithms , are necessary to raise the proportion of correct assignments . therefore , this methodology has two potential great advantages for tentative species identification : first , expertise is not always necessary ; and second , database building can be carried out in a self - developing manner ( that is , by acquiring more and more accurate data on species ) with no waste of information . in evaluating the effectiveness of this methodology , the nature of pass must first be considered , as it plays the most important part in the method . as pass is calculated on the basis of the coordinates of spiddos ( see equation 1 in materials and methods ) , the nature of spiddos must be thoroughly investigated . if two genomic dnas contain common sequence regions that can be amplified by random pcr using the same primer , the resultant dnas will usually generate similar spiddos ( by definition , the spiddos obtained by tgge represent the crucial points of a genome profile , points at which the temperature corresponds to the beginning of a prominent structural transition in dna ) . as shown schematically in figure 3 , two corresponding spiddos derived from two closely related species can be connected by a displacement vector , which consists of two independent elements of mobility ( ) and temperature ( ) . the differences in each element ( and ) can be related to the differences between two sequences as shown in figure 3 . the displacement in the ordinate is caused by the difference in length between the two dnas and is caused by deletion or insertion , whereas that in the abscissa is mainly caused by point mutation ( although insertion / deletion can also contribute ) . as the extent of these changes is roughly proportional to the evolutionary time since the species diverged , we can expect that the summation of the displacement of each spiddo is approximately proportional to the time since divergence , and thus to the genome - to - genome distance . by using a sufficient number of spiddos empirically , we know that 8 - 10 spiddos , which can be obtained from a single genome profile , can be significant . however , since the more spiddos the better the result , we tentatively made it a rule to adopt four genome profiles ( 32 - 40 spiddos ) - that is , four random - pcr products - as a current standard of initial species identification . therefore , pass has the theoretical and empirical basis to be used as a measure of similarity between genomes , although the extent of its effectiveness remains to be shown experimentally as data accumulates . we have introduced a measure of distance , d ' , obtained from pass as formulated in equation 2 ( see materials and methods ) , for the sake of convenience . we call d ' a genome sub - distance because it is based not on the whole but a part of the genome sequence . thus , we introduce ( true ) genome distance , d , as in equation 3 ( see materials and methods ) . genome distance must have a close relationship with genetic distance , as defined by nei and others , although there is a difference in the definition . the genetic distance based on sequences is basically the hamming distance ( the number of different letters at each corresponding position of two sequences of letters that are optimally aligned ) between two nucleotide ( or amino - acid ) sequences . in aligning sequences arbitrariness another constraint on genetic distance is that it is usually obtained from a limited number of genes , although that is also the case for genome distance . as genome distance is easier to obtain in practice using our method , it should be easier to obtain a lot of data on it compared with genetic distance . on the basis of d ( in practice d ' ) , we can construct phylogenetic trees and genome sequence space ( an imaginary spherical space in which all the genomes ( individuals ) can be uniquely located in a finite manner based on the distance between genomes , providing clusters of species ( k.n . , unpublished observations ) ) . although the applicability and effectiveness of genome distance for such purposes needs to be further investigated , it is obvious that an organism that has near - zero genome distance from a certain standard species , as an average over four or more genome sub - distances obtained from as many genome profiles , can be easily assigned to that same species with a high level of confidence . we are not claiming , however , to be able to give the correct taxonomical name to any species using this method . the greater the number of strains registered in the database , the more easily will a species be assigned . basically , no special efforts , except expanding the database and using sophisticated algorithms , are necessary to raise the proportion of correct assignments . therefore , this methodology has two potential great advantages for tentative species identification : first , expertise is not always necessary ; and second , database building can be carried out in a self - developing manner ( that is , by acquiring more and more accurate data on species ) with no waste of information . the principle that a species can be identified on the basis of its similarity to a standard species remains unchanged in the shift from phenotype - based to genotype - based methodology . therefore , the essence of our methodology resides in finding a sufficiently closely related species by way of a measure of similarity - a pattern similarity score ( pass ) . note that this genotype - based methodology can not define species under the current taxonomy regime , in which phenotype is used as the defining characteristic of species . although genome profiling is the basic technology for our current purpose , provisional species identification based on genotype can be fulfilled only by using computer - aided database technology , which is most effectively constructed in the internet environment . as this methodology is based on use by a large number of scientists , the protocol must be designed to be reproducible and easy to carry out . the processes have been deliberately designed with this in view , and are presented on our website . genome profiling consists of two basic technologies : random pcr and tgge , which have been well established . however , if it is to be used for the purpose of general and universal applications , well - defined standardization is absolutely required to obtain significant results . the main topics included in the protocol are : preparation of genome dnas ; the set of primers used for random pcr and the internal reference dnas used for tgge ; experimental conditions for random pcr ; and the experimental conditions for tgge . the protocol also includes the related procedures ( extraction of spiddos , calculation of pass , and others ) . briefly , the alkaline extraction method was selected for simplicity as follows : 10 mg of cells or tissue are placed in an eppendorf tube and heated for 1 min at 100c . the cells are mixed with 10 g 0.5 m naoh and stirred for 1 min ( or 5 min or so for stiffer cells such as yeast ) using a microhomogenizer , if necessary , with added quartz sand . immediately , a 5 l aliquot of the lysate is mixed with 495 l 100 mm tris - hcl ( ph 8.0 ) . usually , a 3 l aliquot of the mixture thus obtained is used as a template for 100-l - scale pcr . in some cases , such as escherichia coli , which does not have a strong cell envelope , these cell - breakdown processes can even be omitted and the cells can be directly used in pcr . in other cases , such as fungi , thorough mechanical treatment ( grinding with quartz sand ) is needed . thus , minimal and common procedures are preferred as much as possible for simplicity and generality in so far as they are consistent with the purity and integrity of the dna samples . dna samples thus prepared were shown to be identical with those dnas prepared by the more elaborate conventional method of thomas as a pcr template . this seems quite natural , as pcr can be carried out successfully in the presence of contaminating proteins or polysaccharides , irrespective of the dna cleavages introduced , unless the regions of dna to be amplified are completely cleaved . nonspecific binding of proteins , which gives footprint effects , will change the yield but not the molecular ratio of random pcr products as long as the binding is totally stochastic . we also adopt a universal , convenient definition for genome dna - that it is composed of all dnas thus prepared , including dynamic elements such as satellite and organelle dnas , and is irrespective of haploid or diploid status of the cells . therefore , the dna samples for genome profiling can be prepared in a common , technically well - defined method for all organisms . technically important restrictions are introduced by selecting a standard set of primers for random pcr ( t.w . it is important to carry out random pcr with all kinds of organisms using the same primers so that all species can be compared on the same platform . we have initially selected four oligonucleotides ( pfm12 : dagaacgcgcctg ; pfm19 : dcagggcgcgtac ; d(tgc)3 ; d(t3g3)2 ) as a standard set of random pcr primers . the primers pfm12 and pfm19 were selected on the basis of the abundant experimental background on them , whereas d(tgc)3 and d(t3g3)2 were rather theoretically favored ( k.n . and a.s . , unpublished observations ) . ' oligonucleotide - stickiness analysis ' , which monitors oligonucleotide - binding sites along the template dna ( k.n . and a.s . , unpublished observations ) , was exploited to determine the universal primers and moderately sticky oligonucleotides were selected . more primers can be used to obtain more detailed information or to supplement insufficient information provided by the four primers about particular pairs of organisms . the information provided by such extra primers can explore in a more detailed manner the local landscape in genome sequence space . in contrast , the standard primers give us rough relationship between any pair of organisms . internal reference bands , which are provided by dnas of a known melting pattern , are used to calibrate each genome profile , giving highly reproducible results . random pcr is usually carried out under standard conditions : 10 ng template dna , 50 pmol primer dna , 250 m of each dntp , 50 mm tris - hcl ( ph 8.8 ) , 15 mm ( nh4)2so4 , 10 mm mgcl2 , 0.45% triton x-100 , 200 g / ml bovine serum albumin and 2 units of taq dna polymerase ( biotech international ) . pcr was carried out in 30 cycles of 30 sec at 94c , 2 min at 28c and 2 min at 47c , using a thermal cycler ptc-100tm ( mj research , ma ) . annealing temperature can be attenuated depending on the size of the template dna ( in general , the larger the template , the greater the number of dna fragments generated by random pcr ) . tgge analysis of random pcr products is carried out with co - migrating internal reference dnas . at least two feature points are extracted from the band pattern of the internal reference dna(s ) , and then used for calibration of genome profiles or species identification dots ( spiddos ) as described below . although the genome profile is a kind of reduction of information contained in the whole genome sequence , it is still too complicated to deal with as it is . thus , a second reduction is carried out by extracting feature points ( spiddos ) from the genome profiles . double - stranded dnas are known to melt in an intrinsically determined manner , depending on their sequence , when heated gradually . spiddos correspond to the structural transition points appearing in band patterns ( figure 4 ) . currently , there are four kinds of spiddos : initial melting point ( pini ) ; minimum mobility point ( pmin ) ; isomobility point ( piso ) ; and the end melting point ( pend ) . empirically , pini is the most reproducible . a set of spiddos ( around ten ) , assigned to a genome profile on a computer display , is processed to calculate the normalized mobility and temperature of each point . a measure of similarity of two genomes - the pass - is introduced as follows . of each spiddo ( 1 to n)is its position vector and is a function of temperature and mobility ( that is , || = p ( t , m ) ) . the superscripts 1 and 2 in parentheses in equation ( 1 ) represent genomes 1 and 2 , respectively . in general , 0 pass 1 . genome distance and genome sub - distance ( d ' ) are derived from pass as follows : d ' = ( 1 - pass)/pass ( 2 ) where d'(i ) is the ith genome sub - distance obtained with the ith primer used for random pcr . the overall process of obtaining an on - web genome profile is shown in figure 5 . there are two steps in this methodology : the local phase and the database phase . in the local phase , genome profiling is carried out for the organism of interest , following the standard protocol presented on our website and outlined in the previous sections . after obtaining a genome profile , the database is accessed and the database phase is begun as a client . the database site requires the client to input an image of the genome profile , to assign spiddos on the genome profile ( figure 4 ) , and to fill in relevant data on the online form . the site will search the database for species with the most similar pattern of spiddos by calculating the pass . although genome profiling is the basic technology for our current purpose , provisional species identification based on genotype can be fulfilled only by using computer - aided database technology , which is most effectively constructed in the internet environment . as this methodology is based on use by a large number of scientists , the protocol must be designed to be reproducible and easy to carry out . the processes have been deliberately designed with this in view , and are presented on our website . genome profiling consists of two basic technologies : random pcr and tgge , which have been well established . however , if it is to be used for the purpose of general and universal applications , well - defined standardization is absolutely required to obtain significant results . the main topics included in the protocol are : preparation of genome dnas ; the set of primers used for random pcr and the internal reference dnas used for tgge ; experimental conditions for random pcr ; and the experimental conditions for tgge . the protocol also includes the related procedures ( extraction of spiddos , calculation of pass , and others ) . briefly , the alkaline extraction method was selected for simplicity as follows : 10 mg of cells or tissue are placed in an eppendorf tube and heated for 1 min at 100c . the cells are mixed with 10 g 0.5 m naoh and stirred for 1 min ( or 5 min or so for stiffer cells such as yeast ) using a microhomogenizer , if necessary , with added quartz sand . immediately , a 5 l aliquot of the lysate is mixed with 495 l 100 mm tris - hcl ( ph 8.0 ) . usually , a 3 l aliquot of the mixture thus obtained is used as a template for 100-l - scale pcr . in some cases , such as escherichia coli , which does not have a strong cell envelope , these cell - breakdown processes can even be omitted and the cells can be directly used in pcr . in other cases , such as fungi , thorough mechanical treatment ( grinding with quartz sand ) is needed . thus , minimal and common procedures are preferred as much as possible for simplicity and generality in so far as they are consistent with the purity and integrity of the dna samples . dna samples thus prepared were shown to be identical with those dnas prepared by the more elaborate conventional method of thomas as a pcr template . this seems quite natural , as pcr can be carried out successfully in the presence of contaminating proteins or polysaccharides , irrespective of the dna cleavages introduced , unless the regions of dna to be amplified are completely cleaved . nonspecific binding of proteins , which gives footprint effects , will change the yield but not the molecular ratio of random pcr products as long as the binding is totally stochastic . we also adopt a universal , convenient definition for genome dna - that it is composed of all dnas thus prepared , including dynamic elements such as satellite and organelle dnas , and is irrespective of haploid or diploid status of the cells . therefore , the dna samples for genome profiling can be prepared in a common , technically well - defined method for all organisms . technically important restrictions are introduced by selecting a standard set of primers for random pcr ( t.w . it is important to carry out random pcr with all kinds of organisms using the same primers so that all species can be compared on the same platform . we have initially selected four oligonucleotides ( pfm12 : dagaacgcgcctg ; pfm19 : dcagggcgcgtac ; d(tgc)3 ; d(t3g3)2 ) as a standard set of random pcr primers . the primers pfm12 and pfm19 were selected on the basis of the abundant experimental background on them , whereas d(tgc)3 and d(t3g3)2 were rather theoretically favored ( k.n . and a.s . ' oligonucleotide - stickiness analysis ' , which monitors oligonucleotide - binding sites along the template dna ( k.n . and , unpublished observations ) , was exploited to determine the universal primers and moderately sticky oligonucleotides were selected . more primers can be used to obtain more detailed information or to supplement insufficient information provided by the four primers about particular pairs of organisms . the information provided by such extra primers can explore in a more detailed manner the local landscape in genome sequence space . in contrast , the standard primers give us rough relationship between any pair of organisms . internal reference bands , which are provided by dnas of a known melting pattern , are used to calibrate each genome profile , giving highly reproducible results . random pcr is usually carried out under standard conditions : 10 ng template dna , 50 pmol primer dna , 250 m of each dntp , 50 mm tris - hcl ( ph 8.8 ) , 15 mm ( nh4)2so4 , 10 mm mgcl2 , 0.45% triton x-100 , 200 g / ml bovine serum albumin and 2 units of taq dna polymerase ( biotech international ) . pcr was carried out in 30 cycles of 30 sec at 94c , 2 min at 28c and 2 min at 47c , using a thermal cycler ptc-100tm ( mj research , ma ) . annealing temperature can be attenuated depending on the size of the template dna ( in general , the larger the template , the greater the number of dna fragments generated by random pcr ) . tgge analysis of random pcr products is carried out with co - migrating internal reference dnas . at least two feature points are extracted from the band pattern of the internal reference dna(s ) , and then used for calibration of genome profiles or species identification dots ( spiddos ) as described below . briefly , the alkaline extraction method was selected for simplicity as follows : 10 mg of cells or tissue are placed in an eppendorf tube and heated for 1 min at 100c . the cells are mixed with 10 g 0.5 m naoh and stirred for 1 min ( or 5 min or so for stiffer cells such as yeast ) using a microhomogenizer , if necessary , with added quartz sand . immediately , a 5 l aliquot of the lysate is mixed with 495 l 100 mm tris - hcl ( ph 8.0 ) . usually , a 3 l aliquot of the mixture thus obtained is used as a template for 100-l - scale pcr . in some cases , such as escherichia coli , which does not have a strong cell envelope , these cell - breakdown processes can even be omitted and the cells can be directly used in pcr . in other cases , such as fungi , thorough mechanical treatment ( grinding with quartz sand ) is needed . thus , minimal and common procedures are preferred as much as possible for simplicity and generality in so far as they are consistent with the purity and integrity of the dna samples . dna samples thus prepared were shown to be identical with those dnas prepared by the more elaborate conventional method of thomas as a pcr template . this seems quite natural , as pcr can be carried out successfully in the presence of contaminating proteins or polysaccharides , irrespective of the dna cleavages introduced , unless the regions of dna to be amplified are completely cleaved . nonspecific binding of proteins , which gives footprint effects , will change the yield but not the molecular ratio of random pcr products as long as the binding is totally stochastic . we also adopt a universal , convenient definition for genome dna - that it is composed of all dnas thus prepared , including dynamic elements such as satellite and organelle dnas , and is irrespective of haploid or diploid status of the cells . therefore , the dna samples for genome profiling can be prepared in a common , technically well - defined method for all organisms . technically important restrictions are introduced by selecting a standard set of primers for random pcr ( t.w . it is important to carry out random pcr with all kinds of organisms using the same primers so that all species can be compared on the same platform . we have initially selected four oligonucleotides ( pfm12 : dagaacgcgcctg ; pfm19 : dcagggcgcgtac ; d(tgc)3 ; d(t3g3)2 ) as a standard set of random pcr primers . the primers pfm12 and pfm19 were selected on the basis of the abundant experimental background on them , whereas d(tgc)3 and d(t3g3)2 were rather theoretically favored ( k.n . and a.s . , unpublished observations ) . ' oligonucleotide - stickiness analysis ' , which monitors oligonucleotide - binding sites along the template dna ( k.n . and a.s . , unpublished observations ) , was exploited to determine the universal primers and moderately sticky oligonucleotides were selected . more primers can be used to obtain more detailed information or to supplement insufficient information provided by the four primers about particular pairs of organisms . the information provided by such extra primers can explore in a more detailed manner the local landscape in genome sequence space . in contrast , the standard primers give us rough relationship between any pair of organisms . internal reference bands , which are provided by dnas of a known melting pattern , are used to calibrate each genome profile , giving highly reproducible results . random pcr is usually carried out under standard conditions : 10 ng template dna , 50 pmol primer dna , 250 m of each dntp , 50 mm tris - hcl ( ph 8.8 ) , 15 mm ( nh4)2so4 , 10 mm mgcl2 , 0.45% triton x-100 , 200 g / ml bovine serum albumin and 2 units of taq dna polymerase ( biotech international ) . pcr was carried out in 30 cycles of 30 sec at 94c , 2 min at 28c and 2 min at 47c , using a thermal cycler ptc-100tm ( mj research , ma ) . annealing temperature can be attenuated depending on the size of the template dna ( in general , the larger the template , the greater the number of dna fragments generated by random pcr ) . tgge analysis of random pcr products is carried out with co - migrating internal reference dnas . at least two feature points are extracted from the band pattern of the internal reference dna(s ) , and then used for calibration of genome profiles or species identification dots ( spiddos ) as described below . although the genome profile is a kind of reduction of information contained in the whole genome sequence , it is still too complicated to deal with as it is . thus , a second reduction is carried out by extracting feature points ( spiddos ) from the genome profiles . double - stranded dnas are known to melt in an intrinsically determined manner , depending on their sequence , when heated gradually . spiddos correspond to the structural transition points appearing in band patterns ( figure 4 ) . currently , there are four kinds of spiddos : initial melting point ( pini ) ; minimum mobility point ( pmin ) ; isomobility point ( piso ) ; and the end melting point ( pend ) . empirically , pini is the most reproducible . a set of spiddos ( around ten ) , assigned to a genome profile on a computer display , is processed to calculate the normalized mobility and temperature of each point . a measure of similarity of two genomes - the pass - is introduced as follows . of each spiddo ( 1 to n)is its position vector and is a function of temperature and mobility ( that is , || = p ( t , m ) ) . the superscripts 1 and 2 in parentheses in equation ( 1 ) represent genomes 1 and 2 , respectively . pass will be unity for a complete match in two sets of spiddos . in general , genome distance and genome sub - distance ( d ' ) are derived from pass as follows : d ' = ( 1 - pass)/pass ( 2 ) where d'(i ) is the ith genome sub - distance obtained with the ith primer used for random pcr . the overall process of obtaining an on - web genome profile is shown in figure 5 . there are two steps in this methodology : the local phase and the database phase . in the local phase , genome profiling is carried out for the organism of interest , following the standard protocol presented on our website and outlined in the previous sections . after obtaining a genome profile , the database is accessed and the database phase is begun as a client . the database site requires the client to input an image of the genome profile , to assign spiddos on the genome profile ( figure 4 ) , and to fill in relevant data on the online form . the site will search the database for species with the most similar pattern of spiddos by calculating the pass . this study was supported in part by a grant - in - aid ( 09272203 ) from the ministry of education , science , sports and culture of japan . m.n . was supported by the japan society for promotion of science ( 13001147 ) . after uploading a genome - profile image and assigning spiddos and then subjecting it to a database search , a result will be displayed as shown , with the values of pass and genome distance to the closest species in the database . note that a pass value close to unity infers that the query species is close to ( or even the same as ) the one retrieved from the database . the information on the selected species ( right ) already registered in the database can be viewed by clicking the button . how to assign species in phenotype - based and genotype - based approaches . phenotype - based approaches ( indicated by p1 , p2 and p3 ) are heavily dependent on the traits ( phenotypic or behavioral , appearing as different shapes ) to identify species . in order to clarify such traits , sophisticated instruments and expert skills p2 represents a successful identification attempt , where all the required traits for identifying the species have been obtained , whereas p1 and p3 are not successful because of insufficient information . identity confirmed by genome profile in the genotype - based approaches makes it easy to compare and link unknown species ( g1-g3 ) to known ones ( g0 ) without the requirement for extensive knowledge of phenotypic traits . the displacement between two spiddos ( pi and pi ' ) from two genome profiles can be decomposed into two elements , and . , which results from the shift in melting temperature , must have been caused mainly by point mutation and sometimes by deletion / insertion . on the other hand , , which is a measure of length , must be a result of insertion / deletion events occurring in the dnas . ( a ) a genome profile before processing . the temperature gradient is set from left ( low ) to right ( high ) and the direction of migration is top to bottom ; ir , internal reference band used for normalization . ( b ) the spiddos of the genome profile are marked with red filled circles ; those of the ir are indicated with red open circles . all the spiddos except for the rightmost one are at the first transition of dna melting ( pini ) . although there are four kinds of spiddos ( dots ) , as described in materials and methods , pini is used for simplicity as these points are clearly visible . on - web genome profiling . the overall procedures to tentatively identify species by genotype only ( genome profiling ) are shown . genome profiles are prepared by tgge of random pcr products obtained from the genome dna of a particular organism at the client site ( the local phase ) . after accessing the database ( represented by the red cylinder ) , a client ( red circle ) has spiddos assigned to each genome profile , which are used to calculate the measure of similarity , pass , and will finally get an output of the nearest species registered in the database ( this phase of the process is called the database phase ) . genome sequence space , with the location of the genomes a and b , is shown in green above the database of genomes .

backgroundfor a long time one could not imagine being able to identify species on the basis of genotype only as there were no technological means to do so . but conventional phenotype - based identification requires much effort and a high level of skill , making it almost impossible to analyze a huge number of organisms , as , for example , in microbe - related biological disciplines . comparative analysis of 16s rrna has been changing the situation , however . we report here an approach that will allow rapid and accurate phylogenetic comparison of any unknown strain to all known type strains , enabling tentative assignments of strains to species . the approach is based on two main technologies : genome profiling and internet - based databases.resultsa complete procedure for provisional identification of species using only their genomes is presented , using random polymerase chain reaction , temperature - gradient gel electrophoresis , image processing to generate ' species - identification dots ' ( spiddos ) and data processing . a database website for this purpose was also constructed and operated successfully . the protocol was standardized to make the system reproducible and reliable . the overall methodology thus established has remarkable aspects in that it enables non - experts to obtain an initial species identification without a lot of effort and is self - developing ; that is , species can be determined more definitively as the database is used more and accumulates more genome profiles.conclusionswe have devised a methodology that enables provisional identification of species on the basis of their genotypes only . it is most useful for microbe - related disciplines as they face the most serious difficulties in species identification .

Background Results and discussion Key concepts of the on-web genome profiling Materials and methods General protocol for on-web genome profiling Preparation of genomic DNA Set of primers for random PCR Internal reference DNAs Conditions for random PCR Experimental conditions for TGGE Extraction of spiddos Calculation of PaSS and genome distance Computer-aided data acquisition Acknowledgements Figures and Tables

a biological species is usually defined in principle as a set of actually or potentially interbreeding organisms , but as interbreeding is very difficult to measure , species have in practice been identified by their phenotypic traits . until recently , progress in most microbe - related disciplines has been hampered by the enormous effort needed to identify less prominent traits . we are now in an age when we can identify species based on the genome ( genotype ) , although this does not change the principle that taxonomy is defined by phenotypes : according to the generally accepted rules of taxonomy , a strain belongs to a species if it falls within the range of phenotypes that define that species . this situation has been brought about by the success of the ribosomal rna approach to phylogenetics . well - conserved molecules , such as 16s rrna in particular , have been used to give a species a molecular identifier and to draw phylogenetic relationships . the 16s rrna - based approach has been widely accepted and has proved successful in phylogenetic tree - making and even in identifying species . there are other similar approaches , such as one based on the gyrase gene and multilocus sequence typing . nonetheless , it has been impossible in practice to analyze all the constituents of a microbial population , not only because of the huge size of such populations ( more than 10 cells per ml ) but also because of lack of suitable methodology . although there are methods other than gene and genome sequencing for analyzing genomes , such as restriction - fragment length polymorphism ( rflp ) , amplified fragment - length polymorphism ( aflp ) , octamer - based genome scanning ( obgs ) , random polymerase chain reaction ( pcr ) and others , most can not be used to identify species without a knowledge of phenotypic traits . in reality , there is no general methodology that enables us to identify species by genotype only , although many approaches use genotypic information ( dna sequences ) to complement phenotypic information . we have recently demonstrated the possibility of species identification by genotype using genome profiling , which is a temperature - gradient gel electrophoresis ( tgge ) analysis of random pcr products . in particular , the use of ' species - identification dots ' ( spiddos ) , which are feature points in genome profiles , is very useful for objective and reproducible data processing . we present here a universal method for provisional genotype - based species identification based on these technological advances and using the internet environment , which enables us to identify species in general . this paper also presents the important concepts of genome distance and genome sequence space , which are essential for species identification based on genotype . for the query species , the closest species as judged by spiddos and a list of genome profiles within the tolerance ( ) , together with the annotation attached to it , is given ( figure 1 ) . if the value of pass is very high ( that is , close to unity ) , then it is highly probable that it is indeed an exact match . although we do not yet have enough data to determine the pass value at which it is safe to identify a species , we have a preliminary idea , based on experience , that 0.95 ( z score 4 ) may be a critical value . the important challenge of how to reconcile the difference between identification of species by phenotype , which conventional taxonomy has adopted , with that based on genotype , is discussed later . an important aspect of this system is that one does not need to be a specialist in the relevant biological field to obtain an initial identification of an unknown organism . all that is required is to register the genome profile of the unknown species on the database . all the information regarding a given species ( in other words , all the entries within a certain pass value ) scientists can work cooperatively to identify species and collect their phenotypic traits ( figure 2 ) . in conventional approaches to identification , most of which have been phenotype - based , those data that failed to meet the required criteria for identification were left unconnected , and could not be used later because there was no convenient way of correlating them with a given species without knowing the species name ( figure 2 ) . thus , our approach of genotype - based species identification , utilizing genome profile and the internet , will be of great help to the field of taxonomy . in evaluating the effectiveness of this methodology , the nature of pass must first be considered , as it plays the most important part in the method . as pass is calculated on the basis of the coordinates of spiddos ( see equation 1 in materials and methods ) , the nature of spiddos must be thoroughly investigated . as shown schematically in figure 3 , two corresponding spiddos derived from two closely related species can be connected by a displacement vector , which consists of two independent elements of mobility ( ) and temperature ( ) . by using a sufficient number of spiddos empirically , we know that 8 - 10 spiddos , which can be obtained from a single genome profile , can be significant . however , since the more spiddos the better the result , we tentatively made it a rule to adopt four genome profiles ( 32 - 40 spiddos ) - that is , four random - pcr products - as a current standard of initial species identification . we call d ' a genome sub - distance because it is based not on the whole but a part of the genome sequence . the genetic distance based on sequences is basically the hamming distance ( the number of different letters at each corresponding position of two sequences of letters that are optimally aligned ) between two nucleotide ( or amino - acid ) sequences . in aligning sequences arbitrariness another constraint on genetic distance is that it is usually obtained from a limited number of genes , although that is also the case for genome distance . as genome distance is easier to obtain in practice using our method , it should be easier to obtain a lot of data on it compared with genetic distance . on the basis of d ( in practice d ' ) , we can construct phylogenetic trees and genome sequence space ( an imaginary spherical space in which all the genomes ( individuals ) can be uniquely located in a finite manner based on the distance between genomes , providing clusters of species ( k.n . although the applicability and effectiveness of genome distance for such purposes needs to be further investigated , it is obvious that an organism that has near - zero genome distance from a certain standard species , as an average over four or more genome sub - distances obtained from as many genome profiles , can be easily assigned to that same species with a high level of confidence . we are not claiming , however , to be able to give the correct taxonomical name to any species using this method . the greater the number of strains registered in the database , the more easily will a species be assigned . therefore , this methodology has two potential great advantages for tentative species identification : first , expertise is not always necessary ; and second , database building can be carried out in a self - developing manner ( that is , by acquiring more and more accurate data on species ) with no waste of information . in evaluating the effectiveness of this methodology , the nature of pass must first be considered , as it plays the most important part in the method . as pass is calculated on the basis of the coordinates of spiddos ( see equation 1 in materials and methods ) , the nature of spiddos must be thoroughly investigated . if two genomic dnas contain common sequence regions that can be amplified by random pcr using the same primer , the resultant dnas will usually generate similar spiddos ( by definition , the spiddos obtained by tgge represent the crucial points of a genome profile , points at which the temperature corresponds to the beginning of a prominent structural transition in dna ) . as shown schematically in figure 3 , two corresponding spiddos derived from two closely related species can be connected by a displacement vector , which consists of two independent elements of mobility ( ) and temperature ( ) . as the extent of these changes is roughly proportional to the evolutionary time since the species diverged , we can expect that the summation of the displacement of each spiddo is approximately proportional to the time since divergence , and thus to the genome - to - genome distance . by using a sufficient number of spiddos empirically , we know that 8 - 10 spiddos , which can be obtained from a single genome profile , can be significant . however , since the more spiddos the better the result , we tentatively made it a rule to adopt four genome profiles ( 32 - 40 spiddos ) - that is , four random - pcr products - as a current standard of initial species identification . we have introduced a measure of distance , d ' , obtained from pass as formulated in equation 2 ( see materials and methods ) , for the sake of convenience . we call d ' a genome sub - distance because it is based not on the whole but a part of the genome sequence . the genetic distance based on sequences is basically the hamming distance ( the number of different letters at each corresponding position of two sequences of letters that are optimally aligned ) between two nucleotide ( or amino - acid ) sequences . in aligning sequences arbitrariness another constraint on genetic distance is that it is usually obtained from a limited number of genes , although that is also the case for genome distance . as genome distance is easier to obtain in practice using our method , it should be easier to obtain a lot of data on it compared with genetic distance . on the basis of d ( in practice d ' ) , we can construct phylogenetic trees and genome sequence space ( an imaginary spherical space in which all the genomes ( individuals ) can be uniquely located in a finite manner based on the distance between genomes , providing clusters of species ( k.n . although the applicability and effectiveness of genome distance for such purposes needs to be further investigated , it is obvious that an organism that has near - zero genome distance from a certain standard species , as an average over four or more genome sub - distances obtained from as many genome profiles , can be easily assigned to that same species with a high level of confidence . we are not claiming , however , to be able to give the correct taxonomical name to any species using this method . the greater the number of strains registered in the database , the more easily will a species be assigned . therefore , this methodology has two potential great advantages for tentative species identification : first , expertise is not always necessary ; and second , database building can be carried out in a self - developing manner ( that is , by acquiring more and more accurate data on species ) with no waste of information . the principle that a species can be identified on the basis of its similarity to a standard species remains unchanged in the shift from phenotype - based to genotype - based methodology . note that this genotype - based methodology can not define species under the current taxonomy regime , in which phenotype is used as the defining characteristic of species . although genome profiling is the basic technology for our current purpose , provisional species identification based on genotype can be fulfilled only by using computer - aided database technology , which is most effectively constructed in the internet environment . as this methodology is based on use by a large number of scientists , the protocol must be designed to be reproducible and easy to carry out . genome profiling consists of two basic technologies : random pcr and tgge , which have been well established . however , if it is to be used for the purpose of general and universal applications , well - defined standardization is absolutely required to obtain significant results . this seems quite natural , as pcr can be carried out successfully in the presence of contaminating proteins or polysaccharides , irrespective of the dna cleavages introduced , unless the regions of dna to be amplified are completely cleaved . we also adopt a universal , convenient definition for genome dna - that it is composed of all dnas thus prepared , including dynamic elements such as satellite and organelle dnas , and is irrespective of haploid or diploid status of the cells . therefore , the dna samples for genome profiling can be prepared in a common , technically well - defined method for all organisms . it is important to carry out random pcr with all kinds of organisms using the same primers so that all species can be compared on the same platform . the primers pfm12 and pfm19 were selected on the basis of the abundant experimental background on them , whereas d(tgc)3 and d(t3g3)2 were rather theoretically favored ( k.n . and a.s . more primers can be used to obtain more detailed information or to supplement insufficient information provided by the four primers about particular pairs of organisms . annealing temperature can be attenuated depending on the size of the template dna ( in general , the larger the template , the greater the number of dna fragments generated by random pcr ) . at least two feature points are extracted from the band pattern of the internal reference dna(s ) , and then used for calibration of genome profiles or species identification dots ( spiddos ) as described below . thus , a second reduction is carried out by extracting feature points ( spiddos ) from the genome profiles . empirically , pini is the most reproducible . of each spiddo ( 1 to n)is its position vector and is a function of temperature and mobility ( that is , || = p ( t , m ) ) . after obtaining a genome profile , the database is accessed and the database phase is begun as a client . the database site requires the client to input an image of the genome profile , to assign spiddos on the genome profile ( figure 4 ) , and to fill in relevant data on the online form . the site will search the database for species with the most similar pattern of spiddos by calculating the pass . although genome profiling is the basic technology for our current purpose , provisional species identification based on genotype can be fulfilled only by using computer - aided database technology , which is most effectively constructed in the internet environment . as this methodology is based on use by a large number of scientists , the protocol must be designed to be reproducible and easy to carry out . genome profiling consists of two basic technologies : random pcr and tgge , which have been well established . however , if it is to be used for the purpose of general and universal applications , well - defined standardization is absolutely required to obtain significant results . thus , minimal and common procedures are preferred as much as possible for simplicity and generality in so far as they are consistent with the purity and integrity of the dna samples . this seems quite natural , as pcr can be carried out successfully in the presence of contaminating proteins or polysaccharides , irrespective of the dna cleavages introduced , unless the regions of dna to be amplified are completely cleaved . we also adopt a universal , convenient definition for genome dna - that it is composed of all dnas thus prepared , including dynamic elements such as satellite and organelle dnas , and is irrespective of haploid or diploid status of the cells . therefore , the dna samples for genome profiling can be prepared in a common , technically well - defined method for all organisms . it is important to carry out random pcr with all kinds of organisms using the same primers so that all species can be compared on the same platform . the primers pfm12 and pfm19 were selected on the basis of the abundant experimental background on them , whereas d(tgc)3 and d(t3g3)2 were rather theoretically favored ( k.n . more primers can be used to obtain more detailed information or to supplement insufficient information provided by the four primers about particular pairs of organisms . pcr was carried out in 30 cycles of 30 sec at 94c , 2 min at 28c and 2 min at 47c , using a thermal cycler ptc-100tm ( mj research , ma ) . annealing temperature can be attenuated depending on the size of the template dna ( in general , the larger the template , the greater the number of dna fragments generated by random pcr ) . at least two feature points are extracted from the band pattern of the internal reference dna(s ) , and then used for calibration of genome profiles or species identification dots ( spiddos ) as described below . usually , a 3 l aliquot of the mixture thus obtained is used as a template for 100-l - scale pcr . this seems quite natural , as pcr can be carried out successfully in the presence of contaminating proteins or polysaccharides , irrespective of the dna cleavages introduced , unless the regions of dna to be amplified are completely cleaved . we also adopt a universal , convenient definition for genome dna - that it is composed of all dnas thus prepared , including dynamic elements such as satellite and organelle dnas , and is irrespective of haploid or diploid status of the cells . therefore , the dna samples for genome profiling can be prepared in a common , technically well - defined method for all organisms . it is important to carry out random pcr with all kinds of organisms using the same primers so that all species can be compared on the same platform . the primers pfm12 and pfm19 were selected on the basis of the abundant experimental background on them , whereas d(tgc)3 and d(t3g3)2 were rather theoretically favored ( k.n . more primers can be used to obtain more detailed information or to supplement insufficient information provided by the four primers about particular pairs of organisms . annealing temperature can be attenuated depending on the size of the template dna ( in general , the larger the template , the greater the number of dna fragments generated by random pcr ) . tgge analysis of random pcr products is carried out with co - migrating internal reference dnas . at least two feature points are extracted from the band pattern of the internal reference dna(s ) , and then used for calibration of genome profiles or species identification dots ( spiddos ) as described below . thus , a second reduction is carried out by extracting feature points ( spiddos ) from the genome profiles . of each spiddo ( 1 to n)is its position vector and is a function of temperature and mobility ( that is , || = p ( t , m ) ) . after obtaining a genome profile , the database is accessed and the database phase is begun as a client . the database site requires the client to input an image of the genome profile , to assign spiddos on the genome profile ( figure 4 ) , and to fill in relevant data on the online form . the site will search the database for species with the most similar pattern of spiddos by calculating the pass . after uploading a genome - profile image and assigning spiddos and then subjecting it to a database search , a result will be displayed as shown , with the values of pass and genome distance to the closest species in the database . the information on the selected species ( right ) already registered in the database can be viewed by clicking the button . how to assign species in phenotype - based and genotype - based approaches . phenotype - based approaches ( indicated by p1 , p2 and p3 ) are heavily dependent on the traits ( phenotypic or behavioral , appearing as different shapes ) to identify species . the displacement between two spiddos ( pi and pi ' ) from two genome profiles can be decomposed into two elements , and . although there are four kinds of spiddos ( dots ) , as described in materials and methods , pini is used for simplicity as these points are clearly visible . on - web genome profiling . the overall procedures to tentatively identify species by genotype only ( genome profiling ) are shown . genome sequence space , with the location of the genomes a and b , is shown in green above the database of genomes .

many new patients , generally young ( in their 30 's ) and after a recent pregnancy , come to the plastic surgeon to restore their breasts to previous firm appearances and restoration without any scar . this seems logical for patients coming with moderate ptosis and wishing to have breast augmentation as well or at least their appearance before maternity . nevertheless , periareolar augmentation mastopexy is one of the leading contributors for malpractice claims in the united states . high patient expectations combined with less elaborate procedures may not always achieve desired goals . despite the large number of published articles and different surgical techniques described for periareolar augmentation mastopexy the outcomes are still not optimal . the common complications are [ figure 1 ] : see common complications after standard periareolar mastopexy ( flattened shape with low projection , early formation of waterfall , widening of periareolar scars ) tendency of the implant to cranially migrate due to insufficient space in the lower quadrant and the vertical traction by the pectoralis major muscle.immediate flattened appearance due to inadequate cutaneous envelope.early formation of waterfall appearance due to glandular drooping during the 1 year post - surgerywidening of periareolar scars . tendency of the implant to cranially migrate due to insufficient space in the lower quadrant and the vertical traction by the pectoralis major muscle . waterfall appearance due to glandular drooping during the 1 year post - surgery widening of periareolar scars . indeed , our patients presented a moderate incidence of these complications . from the need to improve on this much - requested technique , we studied these complications in our patients , taking into account the surgical indications and the outcomes . we established a hypothesis , and with all the findings noted , we developed a new therapeutic approach . usually , to justify the indication for a periareolar mastopexy , the patient has to fulfil two basic criteria : the nipple must lie below the level of the inframammary fold ( otherwise , merely an implant will solve the problem).the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the nipple must lie below the level of the inframammary fold ( otherwise , merely an implant will solve the problem ) . the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the appearance of the ideal breast has an inferior pole ( distance from the nipple to inframammary fold ) of 57 cm and the distance from the sternal notch to the nipple is 1921 cm with the nipple lying above the level of the inframammary fold . when there is an involution with a resulting ptosis , the nipple lies below the level of the inframammary fold , as shown in the diagram [ figure 2 ] . involution with a resulting ptosis , where the nipple - areola complex goes beyond the submammary crease . if the distance from nipple to submammary crease ( y , y ' ) is normal ( 68 cm ) , the sternal to nipple distance ( x , x ' ) will be higher than those 2324 cm established to perform a periareolar mastopexy if the inferior pole is normally constituted ( distance from nipple to inframammary fold : 57 cm minimum [ y , y ' ] ) , the distance from the sternal notch to the nipple ( x , x ' ) will obligatorily be higher than those 2324 cm established as a basic criteria to perform a periareolar mastopexy . for a breast to meet the two requirements established above to undergo a periareolar mastopexy ( nipple located below the level of the inframammary fold and distance from sternal notch to nipple < 24 cm ) , it should necessarily have an abnormally short inferior pole , meaning that it would belong to the group of the so - called anomalies of the breast base , widely known as tuberous breasts . from this criteria , the medical records and photographs of our patients having undergone a periareolar augmentation mastopexy were reviewed . it was observed that they already had an abnormally short inferior pole when the pre - operative measures were taken . checking the pre - operative measurements of these patients ( total amount 137 patients ) , 100% had an abnormally short inferior pole . the average measurement obtained from these patients was 4.5 cm 0.2 standard deviation ( sd ) from areola to mammary crease , and none of them exceeded the minimum for a normal breast ( 5 cm ) . to fix the indication to perform a periareolar augmentation mastopexy for ptosis correction , the breast has to be tuberous at any level and to have an abnormally short inferior pole ( < 5 cm ) . the above findings may explain the main complications from periareolar augmentation mastopexy with the standard surgical technique ( cranial migration , flattened shape , early formation of waterfall or cascade , widening of periareolar scars ) . consequently , we decided to tackle the problem differently and began to perform periareolar augmentation mastopexy dealing with the cases as tuberous breasts . our modified therapeutic plan was as following : extra - glandular approach , practically subcutaneous , based on freeing all the skin from the glandular complex until the new submammary crease was reached , with minimal invasion of the skin in the belly area to avoid differences between gland and implant and the formation of a double bubble [ figure 3].freeing of lateral and medial flaps of the gland allowing an adequate rise of the breast per se.gland dissection from the muscle to perform a dual plane type ii to allow an adequate gland rise , to let the gland drape freely over the implant.dissection of sub - muscular pocket.placement of an anatomical implant with maximum projection to create the most horizontal support plane to prevent , maximum projection is achieved to prevent a truncated cone shape [ figures 46].skin closure of nipple - areola complex with unabsorbable suture ( as it resists mechanical stresses better ) , performing round block technique by freeing all the dermis from the skin to better drape the nipple - areola complex . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . the unpleasant tendency to upper migration is thus avoided [ figures 5 and 6 ] . extra - glandular approach , practically subcutaneous , based on freeing all the skin from the glandular complex until the new submammary crease was reached , with minimal invasion of the skin in the belly area to avoid differences between gland and implant and the formation of a freeing of lateral and medial flaps of the gland allowing an adequate rise of the breast per se . gland dissection from the muscle to perform a dual plane type ii to allow an adequate gland rise , to let the gland drape freely over the implant . placement of an anatomical implant with maximum projection to create the most horizontal support plane to prevent , maximum projection is achieved to prevent a truncated cone shape [ figures 46 ] . skin closure of nipple - areola complex with unabsorbable suture ( as it resists mechanical stresses better ) , performing round block technique by freeing all the dermis from the skin to better drape the nipple - areola complex . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . the unpleasant tendency to upper migration is thus avoided [ figures 5 and 6 ] . extra - glandular approach , freeing all the skin from the glandular complex , reaching the new submammary crease , treating to invade the skin of upper abdomen as little as possible compare the different placements of the anatomical implants [ figures 4 and 6 ] . figure 4 shows subglandular placement , which would facilitate breast ptosis as the breast matures , being the prosthesis in the lower part of the breast in a is showed another incorrect placement of the anatomical implants : the subpectoral implantation at the same level as the gland , it would facilitate cranial migration of the prostheses with drop of the gland and waterfall effect , which can be seen in b we recommend performing a dual plane type ii and the placement of an anatomical implant with maximum projection to create the most horizontal support plane for the gland , and to prevent truncated cone shape at the same time , high profile implants make possible a support plane that permits the satisfactory evolution of the breast over time and adequate projection . following this new surgical technique we started a prospective observational study wherein , we included 56 patients who accomplished the former criteria for periareolar mastopexy but considered as tuberous breasts . usually , to justify the indication for a periareolar mastopexy , the patient has to fulfil two basic criteria : the nipple must lie below the level of the inframammary fold ( otherwise , merely an implant will solve the problem).the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the nipple must lie below the level of the inframammary fold ( otherwise , merely an implant will solve the problem ) . the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the appearance of the ideal breast has an inferior pole ( distance from the nipple to inframammary fold ) of 57 cm and the distance from the sternal notch to the nipple is 1921 cm with the nipple lying above the level of the inframammary fold . when there is an involution with a resulting ptosis , the nipple lies below the level of the inframammary fold , as shown in the diagram [ figure 2 ] . involution with a resulting ptosis , where the nipple - areola complex goes beyond the submammary crease . if the distance from nipple to submammary crease ( y , y ' ) is normal ( 68 cm ) , the sternal to nipple distance ( x , x ' ) will be higher than those 2324 cm established to perform a periareolar mastopexy if the inferior pole is normally constituted ( distance from nipple to inframammary fold : 57 cm minimum [ y , y ' ] ) , the distance from the sternal notch to the nipple ( x , x ' ) will obligatorily be higher than those 2324 cm established as a basic criteria to perform a periareolar mastopexy . for a breast to meet the two requirements established above to undergo a periareolar mastopexy ( nipple located below the level of the inframammary fold and distance from sternal notch to nipple < 24 cm ) , it should necessarily have an abnormally short inferior pole , meaning that it would belong to the group of the so - called anomalies of the breast base , widely known as tuberous breasts . from this criteria , the medical records and photographs of our patients having undergone a periareolar augmentation mastopexy were reviewed . it was observed that they already had an abnormally short inferior pole when the pre - operative measures were taken . checking the pre - operative measurements of these patients ( total amount 137 patients ) , 100% had an abnormally short inferior pole . the average measurement obtained from these patients was 4.5 cm 0.2 standard deviation ( sd ) from areola to mammary crease , and none of them exceeded the minimum for a normal breast ( 5 cm ) . to fix the indication to perform a periareolar augmentation mastopexy for ptosis correction , the breast has to be tuberous at any level and to have an abnormally short inferior pole ( < 5 cm ) . the above findings may explain the main complications from periareolar augmentation mastopexy with the standard surgical technique ( cranial migration , flattened shape , early formation of waterfall or cascade , widening of periareolar scars ) . consequently , we decided to tackle the problem differently and began to perform periareolar augmentation mastopexy dealing with the cases as tuberous breasts . our modified therapeutic plan was as following : extra - glandular approach , practically subcutaneous , based on freeing all the skin from the glandular complex until the new submammary crease was reached , with minimal invasion of the skin in the belly area to avoid differences between gland and implant and the formation of a double bubble [ figure 3].freeing of lateral and medial flaps of the gland allowing an adequate rise of the breast per se.gland dissection from the muscle to perform a dual plane type ii to allow an adequate gland rise , to let the gland drape freely over the implant.dissection of sub - muscular pocket.placement of an anatomical implant with maximum projection to create the most horizontal support plane to prevent , maximum projection is achieved to prevent a truncated cone shape [ figures 46].skin closure of nipple - areola complex with unabsorbable suture ( as it resists mechanical stresses better ) , performing round block technique by freeing all the dermis from the skin to better drape the nipple - areola complex . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . the unpleasant tendency to upper migration is thus avoided [ figures 5 and 6 ] . extra - glandular approach , practically subcutaneous , based on freeing all the skin from the glandular complex until the new submammary crease was reached , with minimal invasion of the skin in the belly area to avoid differences between gland and implant and the formation of a freeing of lateral and medial flaps of the gland allowing an adequate rise of the breast per se . gland dissection from the muscle to perform a dual plane type ii to allow an adequate gland rise , to let the gland drape freely over the implant . placement of an anatomical implant with maximum projection to create the most horizontal support plane to prevent , maximum projection is achieved to prevent a truncated cone shape [ figures 46 ] . skin closure of nipple - areola complex with unabsorbable suture ( as it resists mechanical stresses better ) , performing round block technique by freeing all the dermis from the skin to better drape the nipple - areola complex . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . the unpleasant tendency to upper migration is thus avoided [ figures 5 and 6 ] . extra - glandular approach , freeing all the skin from the glandular complex , reaching the new submammary crease , treating to invade the skin of upper abdomen as little as possible compare the different placements of the anatomical implants [ figures 4 and 6 ] . figure 4 shows subglandular placement , which would facilitate breast ptosis as the breast matures , being the prosthesis in the lower part of the breast in a is showed another incorrect placement of the anatomical implants : the subpectoral implantation at the same level as the gland , it would facilitate cranial migration of the prostheses with drop of the gland and waterfall effect , which can be seen in b we recommend performing a dual plane type ii and the placement of an anatomical implant with maximum projection to create the most horizontal support plane for the gland , and to prevent truncated cone shape at the same time , high profile implants make possible a support plane that permits the satisfactory evolution of the breast over time and adequate projection . following this new surgical technique we started a prospective observational study wherein , we included 56 patients who accomplished the former criteria for periareolar mastopexy but considered as tuberous breasts . usually , to justify the indication for a periareolar mastopexy , the patient has to fulfil two basic criteria : the nipple must lie below the level of the inframammary fold ( otherwise , merely an implant will solve the problem).the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the nipple must lie below the level of the inframammary fold ( otherwise , merely an implant will solve the problem ) . the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the appearance of the ideal breast has an inferior pole ( distance from the nipple to inframammary fold ) of 57 cm and the distance from the sternal notch to the nipple is 1921 cm with the nipple lying above the level of the inframammary fold . when there is an involution with a resulting ptosis , the nipple lies below the level of the inframammary fold , as shown in the diagram [ figure 2 ] . involution with a resulting ptosis , where the nipple - areola complex goes beyond the submammary crease . if the distance from nipple to submammary crease ( y , y ' ) is normal ( 68 cm ) , the sternal to nipple distance ( x , x ' ) will be higher than those 2324 cm established to perform a periareolar mastopexy if the inferior pole is normally constituted ( distance from nipple to inframammary fold : 57 cm minimum [ y , y ' ] ) , the distance from the sternal notch to the nipple ( x , x ' ) will obligatorily be higher than those 2324 cm established as a basic criteria to perform a periareolar mastopexy . for a breast to meet the two requirements established above to undergo a periareolar mastopexy ( nipple located below the level of the inframammary fold and distance from sternal notch to nipple < 24 cm ) , it should necessarily have an abnormally short inferior pole , meaning that it would belong to the group of the so - called anomalies of the breast base , widely known as tuberous breasts . from this criteria , the medical records and photographs of our patients having undergone a periareolar augmentation mastopexy were reviewed . it was observed that they already had an abnormally short inferior pole when the pre - operative measures were taken . checking the pre - operative measurements of these patients ( total amount 137 patients ) , 100% had an abnormally short inferior pole . the average measurement obtained from these patients was 4.5 cm 0.2 standard deviation ( sd ) from areola to mammary crease , and none of them exceeded the minimum for a normal breast ( 5 cm ) . to fix the indication to perform a periareolar augmentation mastopexy for ptosis correction , the breast has to be tuberous at any level and to have an abnormally short inferior pole ( < 5 cm ) . the above findings may explain the main complications from periareolar augmentation mastopexy with the standard surgical technique ( cranial migration , flattened shape , early formation of waterfall or cascade , widening of periareolar scars ) . consequently , we decided to tackle the problem differently and began to perform periareolar augmentation mastopexy dealing with the cases as tuberous breasts . extra - glandular approach , practically subcutaneous , based on freeing all the skin from the glandular complex until the new submammary crease was reached , with minimal invasion of the skin in the belly area to avoid differences between gland and implant and the formation of a double bubble [ figure 3].freeing of lateral and medial flaps of the gland allowing an adequate rise of the breast per se.gland dissection from the muscle to perform a dual plane type ii to allow an adequate gland rise , to let the gland drape freely over the implant.dissection of sub - muscular pocket.placement of an anatomical implant with maximum projection to create the most horizontal support plane to prevent waterfall effect . with this anatomical implant , maximum projection is achieved to prevent a truncated cone shape [ figures 46].skin closure of nipple - areola complex with unabsorbable suture ( as it resists mechanical stresses better ) , performing round block technique by freeing all the dermis from the skin to better drape the nipple - areola complex . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . the unpleasant tendency to upper migration is thus avoided [ figures 5 and 6 ] . extra - glandular approach , practically subcutaneous , based on freeing all the skin from the glandular complex until the new submammary crease was reached , with minimal invasion of the skin in the belly area to avoid differences between gland and implant and the formation of a freeing of lateral and medial flaps of the gland allowing an adequate rise of the breast per se . gland dissection from the muscle to perform a dual plane type ii to allow an adequate gland rise , to let the gland drape freely over the implant . placement of an anatomical implant with maximum projection to create the most horizontal support plane to prevent skin closure of nipple - areola complex with unabsorbable suture ( as it resists mechanical stresses better ) , performing round block technique by freeing all the dermis from the skin to better drape the nipple - areola complex . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . the unpleasant tendency to upper migration is thus avoided [ figures 5 and 6 ] . extra - glandular approach , freeing all the skin from the glandular complex , reaching the new submammary crease , treating to invade the skin of upper abdomen as little as possible compare the different placements of the anatomical implants [ figures 4 and 6 ] . figure 4 shows subglandular placement , which would facilitate breast ptosis as the breast matures , being the prosthesis in the lower part of the breast in a is showed another incorrect placement of the anatomical implants : the subpectoral implantation at the same level as the gland , it would facilitate cranial migration of the prostheses with drop of the gland and waterfall effect , which can be seen in b we recommend performing a dual plane type ii and the placement of an anatomical implant with maximum projection to create the most horizontal support plane for the gland , and to prevent truncated cone shape at the same time , high profile implants make possible a support plane that permits the satisfactory evolution of the breast over time and adequate projection . following this new surgical technique we started a prospective observational study wherein , we included 56 patients who accomplished the former criteria for periareolar mastopexy but considered as tuberous breasts . fifty - six periareolar augmentation mastopexies have been performed during the 3 years with this technique , from 2009 to 2012 [ figures 79 ] , with a minimum follow - up of 1 year . the patients ' age ranged from 25 to 45 years ( on average 33.32 5.89 sd ) . all the prostheses were high profiled ; the volume ranged from 170 cc to 420 cc ( average 288.39 79.91 sd ) , and the distance from the nipple to inframammary fold ranged from 4.00 to 5.00 ( on average 4.626 0.228 sd cm ) [ table 1 ] . there was 1 case of wound infection ( 1.7% ) which settled with antibiotics , removal of sutures and immediate closure after refreshing the skin margins . there was partial periareolar necrosis as well ( 1.7% ) in one heavy smoker , which needed debridement , advancing and delayed closure , without any major aesthetic sequelae [ table 2 ] . pre- and post - operative photos of periareolar mastopexy procedure pre- and post - operative photos of periareolar mastopexy procedure pre- and post - operative photos of periareolar mastopexy procedure age , prosthesis volumen and distance nipple to inframammary fold in patients with the procedure of periareolar mastopexy considering breasts as tuberous acute and chronic complications recorded during 1 year follow up , in the group of 56 patients with periareolar mastopexy considering breasts as tuberous breasts regarding patients ' satisfaction , 40 of them ( 71.43% ) described the results as very positive ; 8 patients ( 14.29% ) stated that the results were moderately satisfying ; 6 patients ( 10.71% ) were equivocal and 2 of them ( 3.57% ) did not accept the results ( one of them because of waterfall effect after weight loss , the other patient because of widening of periareolar scars ) [ table 3 ] . both cases required surgical correction to convert a periareolar augmentation mastopexy into augmentation mastopexy with vertical scar. patients satisfaction survey results tuberous breasts present skin and breast tissue deficiencies , especially in the lower quadrants . type i affects the inferomedial quadrant and type ii affects both lower quadrants . the article published by deluca - pytell in galveston showed that the prevalence of the pathology affected more than 50% of women attending a plastic surgery clinic for a breast augmentation or mastopexy . this frequency shows that this is a vast group , usually not diagnosed , camouflaged by the characteristics of the population requiring this surgery . most of them are young women who have recently delivered , present a moderate ptosis and an involution of the gland , after breastfeeding . moreover , they present most of the stigmata of tuberous breasts such as a certain level of areolar dysmorphia . when the breasts are measured , and the focus is on the inferior pole , all of them present an abnormally short lower segment as well . when a patient comes to the surgeon for a regular tuberous breast treatment , a well - defined surgical plan ( pucket ) is devised for her . why not follow the same procedure in those patients who demand periareolar augmentation mastopexy and present the described features ? throughout the 3 years that our study has been undertaken , we have modified the therapeutic approach given the following empirical observation : if a periareolar mastopexy can be performed , then it must be a tuberous breast. from this , a new surgical technique has been developed obtaining an improvement in our surgical results , and we have achieved a different view on this pathology , which has not been reported in literature yet . this clinical study was approved by the ethics committee of instituto de investigacin sanitaria del hospital universitario y politcnico la fe and has been performed in accordance with the ethical standards set forth in the 1964 declaration of helsinki and its later amendments . this clinical study was approved by the ethics committee of instituto de investigacin sanitaria del hospital universitario y politcnico la fe and has been performed in accordance with the ethical standards set forth in the 1964 declaration of helsinki and its later amendments .

background : periareolar augmentation mastopexy is one of the most demanded operations at plastic surgery clinics . nevertheless , it is one of the leads of malpractice claims in united states caused by the high patient expectations and the standard surgical techniques which may result in common complications . the aim of this report is to present a new surgical approach to solve these complications.methods:after establishing a working hypothesis , we performed a revision study of our patients and we came to the following conclusion : in order to perform a periareolar mastopexy for ptosis correction , breast has to be tuberous at any level and to have abnormally short inferior pole . these findings may explain the main complications from periareolar augmentation mastopexy with the standard surgical techniques . consequently , we started a prospective observational study including 56 patients following a new surgical technique which deals the cases as tuberous breasts.results:during three years , fifty - six periareolar mastopexies were performed with this new surgical approach with one year follow - up . no major complications were observed and 40 of the patients ( 71% ) described the results as very positive.conclusion:if a periareolar mastopexy can be performed , then it must be a tuberous breast . according to this , a new surgical technique for periareolar augmentation mastopexy has been developed obtaining an improvement in our surgical results and achieving a totally different view on this pathology , which has not been reported in literature yet .

INTRODUCTION MATERIALS AND METHODS Working hypothesis Patient selection for periareolar mastopexy Evolution of mammary ptosis Conclusion drawn from these two concepts Revision of our patients' database Final reasoning Impact on therapeutic level New therapeutic plan RESULTS DISCUSSION CONCLUSION Statement of ethical standards Financial support and sponsorship Conflicts of interest

nevertheless , periareolar augmentation mastopexy is one of the leading contributors for malpractice claims in the united states . despite the large number of published articles and different surgical techniques described for periareolar augmentation mastopexy the outcomes are still not optimal . the common complications are [ figure 1 ] : see common complications after standard periareolar mastopexy ( flattened shape with low projection , early formation of waterfall , widening of periareolar scars ) tendency of the implant to cranially migrate due to insufficient space in the lower quadrant and the vertical traction by the pectoralis major muscle.immediate flattened appearance due to inadequate cutaneous envelope.early formation of waterfall appearance due to glandular drooping during the 1 year post - surgerywidening of periareolar scars . tendency of the implant to cranially migrate due to insufficient space in the lower quadrant and the vertical traction by the pectoralis major muscle . from the need to improve on this much - requested technique , we studied these complications in our patients , taking into account the surgical indications and the outcomes . we established a hypothesis , and with all the findings noted , we developed a new therapeutic approach . usually , to justify the indication for a periareolar mastopexy , the patient has to fulfil two basic criteria : the nipple must lie below the level of the inframammary fold ( otherwise , merely an implant will solve the problem).the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the appearance of the ideal breast has an inferior pole ( distance from the nipple to inframammary fold ) of 57 cm and the distance from the sternal notch to the nipple is 1921 cm with the nipple lying above the level of the inframammary fold . if the distance from nipple to submammary crease ( y , y ' ) is normal ( 68 cm ) , the sternal to nipple distance ( x , x ' ) will be higher than those 2324 cm established to perform a periareolar mastopexy if the inferior pole is normally constituted ( distance from nipple to inframammary fold : 57 cm minimum [ y , y ' ] ) , the distance from the sternal notch to the nipple ( x , x ' ) will obligatorily be higher than those 2324 cm established as a basic criteria to perform a periareolar mastopexy . for a breast to meet the two requirements established above to undergo a periareolar mastopexy ( nipple located below the level of the inframammary fold and distance from sternal notch to nipple < 24 cm ) , it should necessarily have an abnormally short inferior pole , meaning that it would belong to the group of the so - called anomalies of the breast base , widely known as tuberous breasts . from this criteria , the medical records and photographs of our patients having undergone a periareolar augmentation mastopexy were reviewed . it was observed that they already had an abnormally short inferior pole when the pre - operative measures were taken . checking the pre - operative measurements of these patients ( total amount 137 patients ) , 100% had an abnormally short inferior pole . to fix the indication to perform a periareolar augmentation mastopexy for ptosis correction , the breast has to be tuberous at any level and to have an abnormally short inferior pole ( < 5 cm ) . the above findings may explain the main complications from periareolar augmentation mastopexy with the standard surgical technique ( cranial migration , flattened shape , early formation of waterfall or cascade , widening of periareolar scars ) . consequently , we decided to tackle the problem differently and began to perform periareolar augmentation mastopexy dealing with the cases as tuberous breasts . our modified therapeutic plan was as following : extra - glandular approach , practically subcutaneous , based on freeing all the skin from the glandular complex until the new submammary crease was reached , with minimal invasion of the skin in the belly area to avoid differences between gland and implant and the formation of a double bubble [ figure 3].freeing of lateral and medial flaps of the gland allowing an adequate rise of the breast per se.gland dissection from the muscle to perform a dual plane type ii to allow an adequate gland rise , to let the gland drape freely over the implant.dissection of sub - muscular pocket.placement of an anatomical implant with maximum projection to create the most horizontal support plane to prevent , maximum projection is achieved to prevent a truncated cone shape [ figures 46].skin closure of nipple - areola complex with unabsorbable suture ( as it resists mechanical stresses better ) , performing round block technique by freeing all the dermis from the skin to better drape the nipple - areola complex . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . figure 4 shows subglandular placement , which would facilitate breast ptosis as the breast matures , being the prosthesis in the lower part of the breast in a is showed another incorrect placement of the anatomical implants : the subpectoral implantation at the same level as the gland , it would facilitate cranial migration of the prostheses with drop of the gland and waterfall effect , which can be seen in b we recommend performing a dual plane type ii and the placement of an anatomical implant with maximum projection to create the most horizontal support plane for the gland , and to prevent truncated cone shape at the same time , high profile implants make possible a support plane that permits the satisfactory evolution of the breast over time and adequate projection . following this new surgical technique we started a prospective observational study wherein , we included 56 patients who accomplished the former criteria for periareolar mastopexy but considered as tuberous breasts . usually , to justify the indication for a periareolar mastopexy , the patient has to fulfil two basic criteria : the nipple must lie below the level of the inframammary fold ( otherwise , merely an implant will solve the problem).the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the appearance of the ideal breast has an inferior pole ( distance from the nipple to inframammary fold ) of 57 cm and the distance from the sternal notch to the nipple is 1921 cm with the nipple lying above the level of the inframammary fold . if the distance from nipple to submammary crease ( y , y ' ) is normal ( 68 cm ) , the sternal to nipple distance ( x , x ' ) will be higher than those 2324 cm established to perform a periareolar mastopexy if the inferior pole is normally constituted ( distance from nipple to inframammary fold : 57 cm minimum [ y , y ' ] ) , the distance from the sternal notch to the nipple ( x , x ' ) will obligatorily be higher than those 2324 cm established as a basic criteria to perform a periareolar mastopexy . for a breast to meet the two requirements established above to undergo a periareolar mastopexy ( nipple located below the level of the inframammary fold and distance from sternal notch to nipple < 24 cm ) , it should necessarily have an abnormally short inferior pole , meaning that it would belong to the group of the so - called anomalies of the breast base , widely known as tuberous breasts . from this criteria , the medical records and photographs of our patients having undergone a periareolar augmentation mastopexy were reviewed . it was observed that they already had an abnormally short inferior pole when the pre - operative measures were taken . checking the pre - operative measurements of these patients ( total amount 137 patients ) , 100% had an abnormally short inferior pole . to fix the indication to perform a periareolar augmentation mastopexy for ptosis correction , the breast has to be tuberous at any level and to have an abnormally short inferior pole ( < 5 cm ) . the above findings may explain the main complications from periareolar augmentation mastopexy with the standard surgical technique ( cranial migration , flattened shape , early formation of waterfall or cascade , widening of periareolar scars ) . consequently , we decided to tackle the problem differently and began to perform periareolar augmentation mastopexy dealing with the cases as tuberous breasts . our modified therapeutic plan was as following : extra - glandular approach , practically subcutaneous , based on freeing all the skin from the glandular complex until the new submammary crease was reached , with minimal invasion of the skin in the belly area to avoid differences between gland and implant and the formation of a double bubble [ figure 3].freeing of lateral and medial flaps of the gland allowing an adequate rise of the breast per se.gland dissection from the muscle to perform a dual plane type ii to allow an adequate gland rise , to let the gland drape freely over the implant.dissection of sub - muscular pocket.placement of an anatomical implant with maximum projection to create the most horizontal support plane to prevent , maximum projection is achieved to prevent a truncated cone shape [ figures 46].skin closure of nipple - areola complex with unabsorbable suture ( as it resists mechanical stresses better ) , performing round block technique by freeing all the dermis from the skin to better drape the nipple - areola complex . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . figure 4 shows subglandular placement , which would facilitate breast ptosis as the breast matures , being the prosthesis in the lower part of the breast in a is showed another incorrect placement of the anatomical implants : the subpectoral implantation at the same level as the gland , it would facilitate cranial migration of the prostheses with drop of the gland and waterfall effect , which can be seen in b we recommend performing a dual plane type ii and the placement of an anatomical implant with maximum projection to create the most horizontal support plane for the gland , and to prevent truncated cone shape at the same time , high profile implants make possible a support plane that permits the satisfactory evolution of the breast over time and adequate projection . following this new surgical technique we started a prospective observational study wherein , we included 56 patients who accomplished the former criteria for periareolar mastopexy but considered as tuberous breasts . usually , to justify the indication for a periareolar mastopexy , the patient has to fulfil two basic criteria : the nipple must lie below the level of the inframammary fold ( otherwise , merely an implant will solve the problem).the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the distance from the sternal notch to the nipple has to be lesser than or about 2324 cm ( if the distance is more than 2324 cm a mastopexy , with either a vertical scar or with an inverted - t technique is indicated ) . the appearance of the ideal breast has an inferior pole ( distance from the nipple to inframammary fold ) of 57 cm and the distance from the sternal notch to the nipple is 1921 cm with the nipple lying above the level of the inframammary fold . if the distance from nipple to submammary crease ( y , y ' ) is normal ( 68 cm ) , the sternal to nipple distance ( x , x ' ) will be higher than those 2324 cm established to perform a periareolar mastopexy if the inferior pole is normally constituted ( distance from nipple to inframammary fold : 57 cm minimum [ y , y ' ] ) , the distance from the sternal notch to the nipple ( x , x ' ) will obligatorily be higher than those 2324 cm established as a basic criteria to perform a periareolar mastopexy . for a breast to meet the two requirements established above to undergo a periareolar mastopexy ( nipple located below the level of the inframammary fold and distance from sternal notch to nipple < 24 cm ) , it should necessarily have an abnormally short inferior pole , meaning that it would belong to the group of the so - called anomalies of the breast base , widely known as tuberous breasts . from this criteria , the medical records and photographs of our patients having undergone a periareolar augmentation mastopexy were reviewed . it was observed that they already had an abnormally short inferior pole when the pre - operative measures were taken . checking the pre - operative measurements of these patients ( total amount 137 patients ) , 100% had an abnormally short inferior pole . to fix the indication to perform a periareolar augmentation mastopexy for ptosis correction , the breast has to be tuberous at any level and to have an abnormally short inferior pole ( < 5 cm ) . the above findings may explain the main complications from periareolar augmentation mastopexy with the standard surgical technique ( cranial migration , flattened shape , early formation of waterfall or cascade , widening of periareolar scars ) . consequently , we decided to tackle the problem differently and began to perform periareolar augmentation mastopexy dealing with the cases as tuberous breasts . extra - glandular approach , practically subcutaneous , based on freeing all the skin from the glandular complex until the new submammary crease was reached , with minimal invasion of the skin in the belly area to avoid differences between gland and implant and the formation of a double bubble [ figure 3].freeing of lateral and medial flaps of the gland allowing an adequate rise of the breast per se.gland dissection from the muscle to perform a dual plane type ii to allow an adequate gland rise , to let the gland drape freely over the implant.dissection of sub - muscular pocket.placement of an anatomical implant with maximum projection to create the most horizontal support plane to prevent waterfall effect . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . these new modifications result in a breast with wider space in the lower quadrants , which allow better adaptation of the implant and a pleasing shape to the breast . figure 4 shows subglandular placement , which would facilitate breast ptosis as the breast matures , being the prosthesis in the lower part of the breast in a is showed another incorrect placement of the anatomical implants : the subpectoral implantation at the same level as the gland , it would facilitate cranial migration of the prostheses with drop of the gland and waterfall effect , which can be seen in b we recommend performing a dual plane type ii and the placement of an anatomical implant with maximum projection to create the most horizontal support plane for the gland , and to prevent truncated cone shape at the same time , high profile implants make possible a support plane that permits the satisfactory evolution of the breast over time and adequate projection . following this new surgical technique we started a prospective observational study wherein , we included 56 patients who accomplished the former criteria for periareolar mastopexy but considered as tuberous breasts . fifty - six periareolar augmentation mastopexies have been performed during the 3 years with this technique , from 2009 to 2012 [ figures 79 ] , with a minimum follow - up of 1 year . pre- and post - operative photos of periareolar mastopexy procedure pre- and post - operative photos of periareolar mastopexy procedure pre- and post - operative photos of periareolar mastopexy procedure age , prosthesis volumen and distance nipple to inframammary fold in patients with the procedure of periareolar mastopexy considering breasts as tuberous acute and chronic complications recorded during 1 year follow up , in the group of 56 patients with periareolar mastopexy considering breasts as tuberous breasts regarding patients ' satisfaction , 40 of them ( 71.43% ) described the results as very positive ; 8 patients ( 14.29% ) stated that the results were moderately satisfying ; 6 patients ( 10.71% ) were equivocal and 2 of them ( 3.57% ) did not accept the results ( one of them because of waterfall effect after weight loss , the other patient because of widening of periareolar scars ) [ table 3 ] . both cases required surgical correction to convert a periareolar augmentation mastopexy into augmentation mastopexy with vertical scar. the article published by deluca - pytell in galveston showed that the prevalence of the pathology affected more than 50% of women attending a plastic surgery clinic for a breast augmentation or mastopexy . when the breasts are measured , and the focus is on the inferior pole , all of them present an abnormally short lower segment as well . when a patient comes to the surgeon for a regular tuberous breast treatment , a well - defined surgical plan ( pucket ) is devised for her . why not follow the same procedure in those patients who demand periareolar augmentation mastopexy and present the described features ? throughout the 3 years that our study has been undertaken , we have modified the therapeutic approach given the following empirical observation : if a periareolar mastopexy can be performed , then it must be a tuberous breast. from this , a new surgical technique has been developed obtaining an improvement in our surgical results , and we have achieved a different view on this pathology , which has not been reported in literature yet . this clinical study was approved by the ethics committee of instituto de investigacin sanitaria del hospital universitario y politcnico la fe and has been performed in accordance with the ethical standards set forth in the 1964 declaration of helsinki and its later amendments . this clinical study was approved by the ethics committee of instituto de investigacin sanitaria del hospital universitario y politcnico la fe and has been performed in accordance with the ethical standards set forth in the 1964 declaration of helsinki and its later amendments .

the expansive incorporation of laser technology into an array of medical , commercial , and military applications has introduced a multitude of laser based technology developments . the medical laser industry is projected to surge over the next decade and promises to deliver an array of commercial and clinical devices for the diagnostic evaluation and treatment of disease . the continually expanding use of laser technology requires careful monitoring of the laser safety standards with a full understanding of the mechanisms of damage and adequacy of the data supporting the standard . this is especially highlighted in the ophthalmic community with regard to a study by morgan et al . . using an adaptive optics ( ao ) confocal scanning laser ophthalmoscope ( ao cslo ) , they observed permanent change to the retinal pigment epithelium ( rpe ) from laser exposure an order of magnitude lower than previous experimental data which served as a base for current laser safety standards . their results underscore the need for the use of high - resolution imaging as an additional endpoint for evaluating retinal laser exposures for the establishment of laser safety standards . the ansi standard sets the maximum permissible exposure ( mpe ) as the level of laser radiation to which an unprotected person may be exposed without adverse biological changes in the eye or skin . the standard is based upon in vitro and in vivo [ 711 ] laser bioeffects experiments designed to determine the limits of tissue to laser damage as well as to describe the photobiological processes involved . experiment exposure parameters are based on the mechanisms governing light induced tissue damage : power density , total exposure duration , and wavelength of irradiating energy . with respect to the eye , the wavelength dependent optical properties of the anterior segment and vitreous also play an important role . retinal damage from laser or noncoherent light sources can occur via three principal light damage mechanisms : photothermal , photochemical , and photomechanical damage . retinal laser damage studies have traditionally used a direct - view funduscope ( ophthalmoscope ) to observe changes to retinal tissue following laser exposure . these studies have been limited by the resolution limits of the funduscope to resolve lesion formation in addition to lack of cross - sectional views necessitating histology and subsequent euthanasia of the subject . more recently , commercially available high - resolution retinal devices such as spectral domain optical coherence tomography ( sd - oct ) have been incorporated into studies of laser induced damage to the retina for laser safety recommendations [ 14 , 15 ] and to monitor the healing response of retinal laser treatments for clinical applications [ 1618 ] . since the transition of adaptive optics ( ao ) technology from astronomy to ophthalmology almost 17 years ago , ao has made significant progress in the performance of ophthalmic instruments . to date , there have been no studies utilizing ao sd - oct to evaluate laser injury to the retina nor the healing response . the work presented here is intended to facilitate laser lesion detection and identification . in this pilot study , the progression of photomechanical and photothermal / photochemical laser exposure to the retina of the nonhuman primate ( nhp ) was documented using a combined ao confocal scanning laser ophthalmoscope ( ao cslo ) and an ao sd - oct . real - time imaging of lesion development and characterization over time will enable better understanding of regimes ( thermal , photochemical , and photodisruptive ) of laser damage in the primate retina . the ao sd - oct and ao cslo provide researchers with high - resolution images of retinal layers to investigate laser damage in vivo and an opportunity to observe laser damage over multiple time points without the need for histopathology as a single end point for observation . sixteen eyes of eight male cynomolgus ( macaca fascicularis ) monkeys between the ages of 6 and 12 years ( 4.47.0 kg ) were used for this experiment only . all subjects were used for in vivo imaging and histological study of laser exposure to the retina . prior to retinal imaging , subjects were restrained utilizing 26 mg / kg intramuscular ( i m ) injection of telazol . once restrained , two drops each of proparacaine hcl 0.5% , phenylephrine hcl 2.5% , and tropicamide 1% were administered to both eyes . once initial sedation was achieved with telazol , lactated ringer 's solution ( 10 ml / kg per hour flow rate ) and propofol were introduced with placement of intravenous ( iv ) catheters in the saphenous veins of the lower limbs . an initial dose of propofol ( 512 mg / kg ) was followed by a continuous maintenance dose of 0.21.0 mg / kg / min as required to maintain the desired plane of anesthesia . prior to prone placement of the subject on a 5-axis goniometric translation stage , the subject was intubated , and a peribulbar injection of 4% lidocaine was administered in the first eye to be imaged to reduce extraocular muscular movement . the subject 's temperature , oxygen saturation , and pulse were continuously monitored throughout the experimental protocol . body temperature was maintained by the use of a temperature management unit ( bair hugger , arizant healthcare inc . , prior to each imaging session , axial length , used to scale images , was measured ( palmscan ap200 , mico medical devices ) . primate 's eyelids were held open with a wire lid speculum for each imaging session and a hard permeable contact lens was used to prevent corneal drying . all experimental procedures were approved by the brooks city base institutional animal care and use committee and conformed to all united states department of agriculture ( usda ) , national institutes of health ( nih ) guidelines , and the arvo statement for the use of animals in ophthalmic and vision research . monkeys were euthanized under deep anesthesia and transcardially perfused with a fixative solution ( ph 7.4 ) consisting of 10% formalin and 2% paraformaldehyde in 0.1 m sodium phosphate buffer . each animal was sedated with a dose of telazol ( 26 mg / kg ) and then brought to a surgical plane of anesthesia with the use of propofol . once this plane was achieved , euthanasia was accomplished with an overdose of pentobarbital sodium ( 10 mg / kg ) . after each perfusion was complete , eyes were enucleated and immediately were fixed in 10% neutral buffered formalin . a 360 incision was made at the ora serrata and the anterior eye structures were removed from the posterior eye cup . a notch suture was placed in the nasal sclera before enucleation to maintain proper orientation . after fixation , the caudal retina including the optic nerve , fovea , and injury sites was prepared as a flat - mount in paraffin blocks . five to seven m serial sections of the retina were placed on positively charged glass slides and stained with hematoxylin and eosin stain on an automated stainer . images were taken at 400x with an olympus dp72 camera ( center valley , pa ) attached to an olympus bx51 microscope . three types of laser exposure parameters were delivered to the retina to examine photomechanical and photothermal / photochemical light interactions . a beam splitter was used to reflect part of the beam to a reference energy meter to determine the ratio of the output beam energy to the energy delivered to the subject . the laser exposure parameters and subjects used for each experiment are summarized in tables 1 and 2 , respectively . retinal exposure spot sizes were approximated using an abcd propagation method under the assumption of a collimated 3 mm ( 1/e ) diameter at the corneal plane . retinal irradiance was calculated based on the assumption of 100% ocular transmission at all exposure wavelengths . experiment 1 ( pulsed photothermal regime : microsecond pulsed exposure)five eyes from three primates were exposed to single and multiple pulse 555 nm ( candela lfdl-8 , candela laser corporation , wayland , ma ) 2 s exposures to the macular region with an estimated retinal spot size of diameter of either 120 m ( n = 3 ) or 40 m ( n = 2 ) . five eyes from three primates were exposed to single and multiple pulse 555 nm ( candela lfdl-8 , candela laser corporation , wayland , ma ) 2 s exposures to the macular region with an estimated retinal spot size of diameter of either 120 m ( n = 3 ) or 40 m ( n = 2 ) . retinal exposure energies ranged from 72 to 4,536 mj / cm . experiment 2 ( photomechanical regime : picosecond pulsed exposure)a nd : yag regenerative amplifier ( spectra physics gcr-3ra , newport corp . , irvine , ca ) was used to produce 532 nm , 40 ps exposures . the beam was directed through a half - wave plate and polarizing beam splitter cube combination to control the energy delivered to the subject . retina test sites received laser exposures for energies ranging from 6.2 to 1,843 mj / cm ( 24 m retinal spot size ) . a nd : yag regenerative amplifier ( spectra physics gcr-3ra , newport corp . , irvine , ca ) was used to produce 532 nm , 40 ps exposures . the beam was directed through a half - wave plate and polarizing beam splitter cube combination to control the energy delivered to the subject . retina test sites received laser exposures for energies ranging from 6.2 to 1,843 mj / cm ( 24 m retinal spot size ) . experiment 3 ( photochemical / photothermal regime)seven eyes from four primates were exposed to collimated 413 nm ( 105 m retinal spot size ) krypton ion laser ( coherent sabre , coherent inc . , santa clara , ca ) for 20 seconds in the macular region to investigate photochemical / photothermal interactions . seven eyes from four primates were exposed to collimated 413 nm ( 105 m retinal spot size ) krypton ion laser ( coherent sabre , coherent inc . , santa clara , ca ) for 20 seconds in the macular region to investigate photochemical / photothermal interactions . retinal irradiance exposures ranged from 40.4 to 7,000 j / cm . for most of the experiments , the insult beam was passed through a safety shutter prior to coalignment with the combined ao retinal imager to enable real - time viewing of laser exposure ( figure 1 ) . , andover , ma ) with a wide field line scanning laser ophthalmoscope ( lslo ) and an ao cslo ( iris ao , berkley , ca ) were combined to simultaneously collect complementary in vivo images of the primate retina in high - resolution ( figure 2 ) . the ao sd - oct and cslo do not have common optics other than the combining beam splitters ; therefore , each image plane is collected independently of the other but is recorded simultaneously . the ao sd - oct and lslo have been previously described ; therefore we describe only the ao cslo and the integrated setup . briefly , the ao sd - oct portion of the integrated setup provides high - speed ( 15 frames / sec ) cross - sectional scans of the retina with a theoretical axial and transverse resolution of 4 m and ~10 m , respectively . the lslo provides a 30 field of view ( fov ) of the retina , which was used to facilitate the alignment of the imager beams on the retina . the ao cslo system consists of a series of focal telescopes that relay the 7 mm pupil to the optical conjugate planes at the galvanometric scanner , the resonant scanner , the dm ( 42 actuators mems ; iris ao , berkley , ca ) , and the wfs ( adaptive optics associates inc . , cambridge , ma ) . the pupil is demagnified to 3.3 mm at the plane of the dm and wfs . the resonant scanner frequency is 16 khz while the acquisition rate of 30 hz is set by the galvanometric scanner , enabling 512 512 images of the retina set to either a 1 or 2 fov . the ao cslo beam ( 632 nm ) is input into the combined system through a beam splitter and is reflected off the dm , scanners , and spherical mirrors into the eye . the reflected light follows the same reverse path through the system and onto the wfs and the photomultiplier tube ( hamamatsu h7422 - 40 , hamamatsu corp . , japan ) . the subject 's pupil was aligned to the instrument using the goniometric stage and pupil camera . the retinal features and location of the ao sd - oct and cslo probe beam were identified from the lslo image . trial lenses were placed at a pupil conjugate in front of the dm of both ao systems to remove lower - order defocus and astigmatism . oct defocus was removed with the focus adjustment ( 5 diopters ) by observing oct signal intensity and ws spots . both ao systems were used to simultaneously capture image sequences of the retinal area during laser exposure . experiments that did not image laser irradiations are denoted by an asterisk in table 2 . oct b - scans and cslo images were collected with and without adaptive optics correction . a line and/or raster scan configuration was used to record oct b - scans prior to and following laser exposure . line scans were collected up to a 5.5 scan length on the retina and consisted of 1024 a - scans . up to 400 b - scan frames were recorded to ensure capture of the longer 413 nm 20-second exposures . raster scans are multiple line scans swept through the x and y direction to create volumetric retinal maps and were sized as either 2 5.5 or 2 2 of retinal area with a b - scan density of 256 or 512 . the cslo frames were recorded in 2 or 1 fov and collected for duration of 10 or 20 seconds . prior to laser exposure , images of the macular region were collected using a 2 fov for the cslo and a raster scan configuration for oct . in addition , color fundus photography was performed to provide direct comparison with ao images at each experimental time point . three examiners evaluated all eyes using color fundus photos for each time point following laser exposure . visible lesions at a given exposure site were reported to be present only if two or more examiners identified a lesion in color fundus photos . lesion identification in oct c - scans and cslo retinal montages of the lesion field was performed by a single person in images with a signal - to - noise ratio greater than 10 db . volumetric oct images of the macular region were imported into avizo 6.3 ( visualization sciences group , burlington , ma ) for measurement of lesion width and area . lesions were characterized as regions of hyper- or hyporeflectivity with or without changes in retinal structure in comparison to undisturbed regions . lesion widths were manually measured as the furthest extent of damage in either the rpe or photoreceptor layer in b - scan images . seed grayscale pixel value within the hyper- or hyporeflective areas associated with injury . a manually adjusted threshold range based on the grayscale the number of visible lesions was determined after 1 hour and 24 hours after exposure , and a probit analysis was performed to determine the dose which corresponds to a 50% probability for damage ( ed50 ) . retinal sites within a single subject receiving multiple exposures of the same laser irradiance were grouped and averaged . paired t - tests were used to compare damage size in the rpe and photoreceptor layers . lesion widths measured from ao cslo scans of picosecond exposures were collected from multiple scans of the retinal area . no lesions were detected below the ansi mpe limit of 500 j / cm for the microsecond laser exposures . lesions detected in available oct and ao cslo images were always observed in color fundus photos . all subjects were reevaluated at the 24-hour observation point with the exception of subject s6 . most lesions appeared pale gray to white and transitioned to the former with increasing energy . exposures which did not result in visible lesions in color fundus photos within the first hour did not develop into lesions at any later time point . the energy required to create damage for the larger of the two retinal spot sizes ( 120 m ) required four to seven times greater energy than smaller spot size exposures ( 40 m ) . the range of retinal irradiances producing immediate reflectance changes or visible disruption in either ao sd - oct or cslo images was greater for the 40 m retinal spot size ( 1234,563 mj / cm ) than for the 120 m retinal spot size ( 721,362.4 mj / cm ) . the combined ed50 all of microsecond exposures was 495 mj / cm and 386 mj / cm for the 1-hour and 24-hour observation points ( n = 80 ) , respectively . pooling only the larger retinal spot size exposures ( n = 60 ) , the ed50 value remained unchanged between the 1- and 24-hour observation points ( 369 mj / cm ) . real - time in vivo imaging of higher energy microsecond exposures revealed disruptive events possibly associated with microcavitation . the ao cslo acquisition rate of 33 ms / frame is too long to record microbubble formation but can monitor the spatial rearrangement of the retina following exposure . the measured ed50 for immediate retinal rearrangement and/or disruption observed using the ao cslo in one subject was 826 mj / cm ( slope of 8.2 ) . this subject received 50 exposures ( 120 m spot size ) in both eyes ranging from 72 to 1,318 mj / cm . visible retinal rearrangement observed in ao cslo images did not coincide with the appearance of a minimal visible lesion ( mvl ) in color fundus photos for 10 instances ( false negatives ) . there were no instances in which a disruptive event was observed in an ao cslo image and did not result in the formation of a mvl in fundus photos ( false positive ) . immediately following the higher energy 120 m spot size exposures , there are slight disturbances to the rpe with increased reflectivity of the outer nuclear layer ( onl ) directly above the exposure site . figure 3 shows cross - sectional images of the formation of a lesion ( 870 mj / cm ) in the retina directly before , immediately after , and minutes following laser exposure . within approximately 10 minutes ( figure 3(c ) ) , the region surrounding the rpe up to the connecting cilia of the photoreceptors appears to swell and there is an increase in reflectivity extending from the rpe into the outer nuclear layer ( onl ) . lesions induced using lower energy exposures ( < 619.2 mj / cm ) appeared as a mild increase in reflectance of the rpe and onl in oct cross - sections and appeared within the hour . the 40 m retinal spot size exposures occupy a smaller damage footprint compared to the 120 m spot size exposures ( data not shown ) . disruptive events were not observed for smaller spot size exposures in oct b - scans or slo images . oct b - scans of multiple pulse - exposures ( 1,317 mj / cm ) in subject 2 are shown in figures 4(a ) and 4(b ) at the 1- and 72-hour follow - up , respectively . figure 4(c ) is an en face view of the retina photoreceptor layer of subject 3 after receiving a multiple - pulse exposure ( 874 j / cm ) . the energy range of 532 nm 40 ps exposures was 6.21,843 mj / cm . retinal damage immediately visible in ao cslo images occurred for energies of 139 mj / cm and greater . the damage appearance ranged from barely visible retinal disruption sometimes accompanied with reflectance changes to a distinguished explosive event . as exposure energy lesion appearance in color fundus photos ranged from pale gray to white increasing in reflectivity and size as energy was increased for each exposure . none of the 66221 mj / cm exposures in the os of subject s8 became visible using any imaging modality at any time point . however , exposures ranging from 66817 mj / cm in the od of s8 observed using ao cslo produced disruptive events or no changes . ao cslo movies of moderate energy exposures 221442 mj / cm show immediate nonuniform increases in reflectance with or without the appearance of a donut - like hole in the lesion center . one hour later , exposures greater than 199 mj / cm were visible using both ao imaging modalities . within a 24-hour period , most exposures became visible in color fundus photos . high energy exposures ( > 442 mj / cm ) produced few inner retinal hemorrhages and were more readily identifiable at the 1-hour observation points in oct and ao cslo images . oct b - scans collected within an hour of higher energy exposures reveal that the most damage was confined to the outer retina and extended further into the onl compared to moderate energy exposures . explosive photomechanical disruptions were restricted to exposures of 813 mj / cm or greater . lesion area increased in proportion to power , as observed from oct measurements ( figure 5 ) . the entire damage zone surrounding the lesion center is more distinguishable in cslo images at the 1-hour observation time point in comparison to oct en face and cross - sectional images . observations of the lesion area using cslo allowed viewing of a definite hyperreflective region sized approximately 60120 m in width with a hyporeflective center . oct cross - sections reveal a punctate increase in hyperreflectance extending from the rpe to the onl ( figures 6 and 7 ) . lesion widths measured from oct en face images of the photoreceptor inner segments ranged from 20 to 40 m . the lesion area measured from oct en face images at the 24-hour follow - up suggests that the lesions increase in size from 29 to 50% . the retinal sites exposed to 413 nm light had a pale yellow appearance in color fundus photos , which was maintained through all observation time points . for all subjects , retinal damage observed using sd - oct associated with laser exposure was not always positively identified as mvls in color fundus photos . of the total 173 lesions , there was a 46% improvement in lesion detection using oct en face images ( n = 93 ) versus color fundus photos ( n = 43 ) at the 1-hour time point . in figure 8 , of the 25 retinal sites receiving 413 nm exposures ranging from 165 to 1,183 j / cm , 24% ( 7 out of 25 ) qualified as mvls in color fundus photo taken 1 hour later even though hyperreflective spots coincident with exposure locations can be seen in the oct en face view ( figure 8(a ) ) . oct c - scans of the photoreceptor inner segments reveal that 88% ( 22 out of 25 ) of the exposed regions ten days later , 56% of the lesions were identified as mvl in color fundus photos ( figure 8(c ) ) compared to 80% identified using oct raster scans . oct b - scans and cslo images collected during 413 nm , 20-second exposures did not reveal any immediate changes to the retina during laser irradiation . exposures greater than 162 j / cm produced hyperreflective changes in retinal oct b - scans collected within 1 hour following exposure . retinal layers affected were the photoreceptor inner segments , rpe , and inner retina . figure 9 shows the retinal damage response of three retinal locations receiving a retinal exposure of 474 j / cm . figure 10 is a 1-hour oct retinal cross - section of a 1,696 j / cm exposure with complementary en face views . the damage extends full thickness but does not have a uniform appearance as it extends from the retinal ganglion cell ( rgc ) layer into the rpe . figure 11 shows histological sections from a retinal flat mount of the same lesion nine days following exposure . lesions which did not become visible in color fundus photos until the 72-hour observation point had retinal irradiance values ranging from 225 to 1,089 j / cm . oct and cslo scans captured during exposure and 1 hour after exposure did not present any changes in retinal structure or reflectance . raster oct scans collected 24 hours later revealed 1015 m round hyperreflective structures within the onl closest to the fovea with appearance of threading extending from round body like structures ( figure 12 ) . minimal or no damage was visible to the rpe for exposure energies less than 1,696 j / cm in oct c - scans for the first hour after exposure ; therefore , lesion width measurements were collected only at the photoreceptor layer for the first hour where retinal changes were visible . over the course of 24 hours , the damage zone of the photoreceptor layer significantly increased in size ( p = 0.02 ) for one subject ( figure 13 ) . damage at the 1-hour time point for the remaining subjects was not clearly demarcated or subjects did not have a 24-hour follow - on imaging session . the differences in lesion size between the photoreceptor layer and rpe at 24 hours for a single subject were significantly different ( p = 0.02 ) ( figure 13 ) . the ansi mpe calculations based on the experimental parameters provide a limit for both thermal and photochemical exposures . for this set of experiments , the limits for thermal and photochemical damage are 57.2 and 2.44 j / cm , respectively . exposures at the 1-hour time point which are greater than the lower limiting photochemical mpe were identified as inducing retinal reflectance changes in oct and fundus images for 62% and 42% of the exposures , respectively . there was an improvement in lesion identification using fundus photos 48 and 72 hours after exposure when compared to the number of lesions identified using oct . in particular , for subject s7 , none of the 15 lesions above the thermal limit was identified using fundus images at the 1- and 24-hour observation time points but was observed 72 hours later ( 73% ) . lesions were identified ( 27% ) at the 1-hour observation time point from oct en face images . the introduction of high - resolution retinal imaging into light damage studies has enabled greater detection of the effects of laser light exposure compared to traditional methods employed in past studies of laser damage thresholds . progress in understanding the mechanism for laser interaction with retinal tissue is driven both by advances in ophthalmic laser therapy and by the need to establish mechanistic underpinning for laser eye safety standards . for the first time , to our knowledge , we have been able to image the immediate effects of photomechanical disruption to the retina . the data presented within this study are a pilot study using high - resolution imaging to study laser - tissue interactions in vivo . short pulse laser exposure can damage the retina via photomechanical or photothermal mechanisms depending on the pulse duration and total energy delivered . real - time in vivo imaging of higher energy microsecond exposures revealed disruptive events likely associated with explosive vaporization of water and subsequent cavitation around melanin particles in the rpe cells . the transition from thermal denaturation into thermomechanical effects as the primary cause of damage to the rpe after laser exposure is approximately 50 s . demonstrated in experiments utilizing ex vivo porcine rpe samples that microbubble formation is the dominant damage mechanism to the rpe for 5 s pulse durations . photomechanical events possibly associated with microcavitation disruption may have been observed at the greater end of exposure energies for experiments 1 and 2 but in all likelihood were mostly thermal in nature . previous studies have used reflectometry [ 22 , 23 ] , stroboscopic imaging , and acoustic measurements [ 25 , 26 ] to monitor microbubble formation . the ao slo acquisition rate of 33 ms / frame does allow recording of actual microbubble formation ( ~20 s ) on the order of microseconds but can monitor the spatial rearrangement of the retina following exposure . although our experiments did not use any complementary independent methods , such as reflectometry , other than oct cross - sections to confirm the presence of microbubbles , the 2 s pulse duration used in our studies was likely associated with thermal damage . the ed50 threshold for detecting visible disruption from 2 s in ao cslo images was 827 mj / cm . the threshold for microbubble formation in ex vivo porcine rpe reported by schuele et al . beam profile distributions and size ( 40 m versus approximated 120 m retinal spot size ) could account for the difference in threshold . the false negatives for damage resulting from the measurement of the spatial disruption of the retinal layers as an indicator of bubble formation indicate that thermal denaturation may still be the critical damage mechanism for the laser parameters tested . however , the presence of retinal layer disruption should be a clear indicator of a mechanical process and thus this compels the belief that 2 s is close to the exposure time associated with the transition of damage from photothermal to photomechanical mechanisms . this observation is consistent with theoretical model results of laser induced damage processes in the retina . while false negatives of retinal layer disruption as an indicator of bubble formation suggest thermal denaturation as the predominate damage mechanism at threshold , other possibilities for the lack of physical evidence of microbubble formation include obscuration by imaging artifact and low signal - to - noise ratio in slo imaging . furthermore , slight misalignment of the insult and oct beam may have resulted in b - scans recorded to be slightly adjacent to the site of highest irradiance during exposure . none of the exposure levels was below the mpes set by ansi for respective laser parameters . the retinal effects of microsecond and picosecond laser pulses are dependent on the energy per pulse and laser spot size at the retina . lesion size and area of 3-picosecond exposures were logarithmically related to the amount of energy delivered to the retina . for the microsecond exposures , there was retinal spot size dependence when comparing energies required for creating mvls as well as the extent of injury . the radiant exposure needed to create damage is greater for the larger spot but results in a more exaggerated disruptive event in comparison to the smaller retinal beam size . one reason may be due to a larger volume of fluid being heated surrounding the increased number of melanosomes within the irradiated area . as a result , the formation of larger bubbles due to coalescence of multiple smaller bubbles could cause an exaggerated explosive disruption . using a laser with an annulus and top hat beam distribution profile , kennedy et al . 2004 show that the ed50 threshold for 3 us 590 nm pulses varies with retinal spot size . our results followed a similar trend in which more energy is required to produce damage with a larger retinal spot size and point to thermal mechanisms at play . previous picosecond laser damage studies [ 14 , 29 ] have compared the extent of damage and damage thresholds to rhesus monkey retinas based on spot size differences . the energy range of picosecond exposures at the cornea for this set of experiments 6.21,843 mj / cm ( 0.03 to 8.34 j at the cornea ) was less than the exposure energies to rhesus monkeys in the study by roach et al . the differences in beam size and two different primate species do not allow direct comparison of damage characteristics ; therefore , we will only compare damage trends . color fundus photos from subject s8 were out of focus due to corneal aberrations and therefore did not allow inclusion for probit analysis . the energy spread of picosecond exposures of subjects s7 and s8 resulted in all positive mvls . as a result , the energy distribution of applied exposures and number of eyes available for use in probit analysis ( n = 3 ) were not sufficient . results of aforementioned picosecond studies are similar in regard to the localization of damage in the outer retina when laser induced breakdown ( lib ) does not play a role . in addition , as the energy of the exposure increases , the extent of the damage also increases to include the inner retinal layers . the collapsed appearance of the photoreceptor layer following moderate to high power exposures suggests explosive mechanisms . low signal - to - noise ratio , frame rate , poor light transmission , eye movement , and misalignment of the imaging laser with insult laser may account for missed and unobserved photomechanical phenomena in vivo . the retinal radiant exposures used in this set of experiments were 40.47,000 mj / cm . the dominant light induced damage for this set of experiments is primarily driven by photochemical mechanisms . it is possible that the damage mechanism transitioned from a photochemical to thermal effect or was partly thermal and photochemical in nature . probit analysis of 413 nm data failed to converge to a solution for either 1- or 24-hour observations and may point to small differences in energy required to transition between damage mechanisms . not all 413 nm damage observable using high - resolution imaging modalities was noted as an mvl in color fundus photos . for example , in more than one subject and multiple eyes receiving 413 nm 20-second exposures , damage was visualized at the one - hour time point in oct b - scans as small nonuniform regions of speckled hyperreflectivity at the rpe and photoreceptor layers , but damage was not identified in color fundus photos . in addition , lesions from one subject were not visible in color fundus photos until the 72-hour time point despite evidence of light induced changes in oct scans at earlier time points . the time course of photochemical lesion development to become funduscopically visible is on the order of 2 - 3 days after exposure [ 31 , 32 ] . it is possible based on our findings that observable retinal changes associated with photochemical damage could occur on a much shorter time scale ( hours ) as compared to days but are not observed using traditional fundus photography . in another study investigating possible photochemical / photothermal damage , a reduction in rpe fluorescence occurred immediately following 900 s 568 nm laser exposures with an average power greater than 47 w . it is not known whether the exposures were funduscopically visible 13 days following injury as color fundus images were not collected until 6 days following injury . the majority of retinal exposures used to study 413 nm damage in this study are greater than 47 w and within ansi standards for the mpe at the lower limit photochemical light exposure . the 413 nm 20 exposures closest to the fovea with energies greater than 700 j / cm resulted in full retinal thickness reflectance changes associated with damage and edematous reactions . the macular pigment concentration is greatest at the foveal pit center and decreases exponentially out to approximately seven degrees eccentricity from the pit center . most of the macular pigment is localized within the henle fibers , ipl and opl . the macular pigment protects the retina from blue light damage as it serves as a blue light filter and antioxidant . previous studies [ 36 , 37 ] have demonstrated the difference in lesion localization with respect to the fovea and examining injury from argon and krypton laser wavelengths . marshall and bird examined 50300 mw argon laser exposures from 1 up to 10 eccentricity from the foveal center . argon lesions delivered outside of a 2 circle of eccentricity from the foveal pit were localized to the rpe while lesions within the 2 circle extended from the rpe into the inner retina and appeared full - thickness . the localization of damage described here closely resembles the histological analysis of argon laser damage to the fovea and its periphery . the ability to follow lesion progression over several time points after exposure ( up to 9 days ) revealed the full - thickness damage we observed at 1 hour and 24 hours after exposure is no longer visible in oct at a 9-day follow - up point . histology of those same lesions also did not show any observable damage within the inner retinal layers . the damage response following exposure for the 413 nm 20-second and 532 nm 40-picosecond exposures was observed using ao sd - oct . the thread - like structures develop within the hour following exposure and are localized to the lesion site at the external limiting membrane ( elm ) and reach into the opl and are generally seen beyond the foveal region but is limited to lesions closest to the fovea . lesions closer to the fovea display higher density threading than those further away . the filamentous structures were not observed at the 1- or 24-hour observation point for the 570 nm 2 s exposures but did appear following picosecond exposures but not as densely . at the 24-hour observation time point , the threading increases in reflectivity and size . a possible explanation includes damage to the henle fiber layer due to transsynaptic degeneration and direct damage from laser exposure . in particular , light ( 488 nm ) injury is associated with enlargement of mller cell processes , gliosis , and upregulation of gfap . gfap upregulation in mller glial cells following retinal injury and disease is the most sensitive nonspecific response to cellular dysfunction and can be used as a retinal stress indicator . such changes could cause an increase in the refractive index of the mller cell cytoplasm , with increased visibility of the structure by high - definition oct . the threading spans from the external limiting membrane ( elm ) and to the opl . the mller cells extend from the elm inward to the internal limiting membrane ( ilm ) and can extend their foot processes instead to rest upon intraretinal blood vessels where they form part of the blood - retinal barrier [ 40 , 41 ] . the cone axons of the henle fiber layer are bound and encased by the outer processes of mller cells and both run in parallel as they leave the central foveal area . the number of mller cells to cone terminals in the perifoveal opl is equal , and the mller cells per / mm decrease with increasing distance from the fovea . this would partially help to explain the reduced appearance of the threaded structures away from the fovea . another possible explanation for the threading process is the accumulation of serum or blood leakage from laser - damaged intraretinal vessels ; such vascular damage with a 488 nm laser has been illustrated [ 36 , 37 ] . the serum or blood would tend to infiltrate the retina along the path of least resistance , in spaces formerly occupied by intact mller cells . although this idea could explain the time frame of our observations , the very sharply defined quality of these reflective thread - like structures does not appear similar to blood vessel hemorrhage observed in oct b - scans after pulsed laser exposure . this is a pilot study to examine the feasibility of using ao sd - oct and ao cslo to examine regimes of laser damage in real - time in addition to the damage response . there is an increased detection of blue light damage at the 1-hour time mark using high - resolution imaging prior to lesion appearance in fundus photos at 24 hours and later . the results highlight the need for continued use of high - resolution imaging to resolve retinal light damage in support of laser safety guidelines . with regard to pulsed thermal laser exposure , high - resolution imaging systems with high frame rate may produce more accurate representation of the pulsed laser tissue phenomena . future studies should include histology to explain and interpret the filamentous threaded structures emanating from the damaged region . the ao sd - oct and cslo provide researchers with high - resolution images of retinal layers to resolve laser damage in vivo . this approach also provides an opportunity to observe laser damage over multiple time points reducing the need for histopathology as a single end point for observation . ao systems are developed and refined for clinical operation ; they will continue to improve the ability of the ophthalmologist to visualize diseased or damaged tissue and manage therapies .

purpose . to investigate fundamental mechanisms of regimes of laser induced damage to the retina and the morphological changes associated with the damage response . methods . varying grades of photothermal , photochemical , and photomechanical retinal laser damage were produced in eyes of eight cynomolgus monkeys . an adaptive optics confocal scanning laser ophthalmoscope and spectral domain optical coherence tomographer were combined to simultaneously collect complementary in vivo images of retinal laser damage during and following exposure . baseline color fundus photography was performed to complement high - resolution imaging . monkeys were perfused with 10% buffered formalin and eyes were enucleated for histological analysis . results . laser energies for visible retinal damage in this study were consistent with previously reported damage thresholds . lesions were identified in oct images that were not visible in direct ophthalmoscopic examination or fundus photos . unique diagnostic characteristics , specific to each damage regime , were identified and associated with shape and localization of lesions to specific retinal layers . previously undocumented retinal healing response to blue continuous wave laser exposure was recorded through a novel experimental methodology . conclusion . this study revealed increased sensitivity of lesion detection and improved specificity to the laser of origin utilizing high - resolution imaging when compared to traditional ophthalmic imaging techniques in the retina .

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

the expansive incorporation of laser technology into an array of medical , commercial , and military applications has introduced a multitude of laser based technology developments . the continually expanding use of laser technology requires careful monitoring of the laser safety standards with a full understanding of the mechanisms of damage and adequacy of the data supporting the standard . using an adaptive optics ( ao ) confocal scanning laser ophthalmoscope ( ao cslo ) , they observed permanent change to the retinal pigment epithelium ( rpe ) from laser exposure an order of magnitude lower than previous experimental data which served as a base for current laser safety standards . their results underscore the need for the use of high - resolution imaging as an additional endpoint for evaluating retinal laser exposures for the establishment of laser safety standards . the ansi standard sets the maximum permissible exposure ( mpe ) as the level of laser radiation to which an unprotected person may be exposed without adverse biological changes in the eye or skin . the standard is based upon in vitro and in vivo [ 711 ] laser bioeffects experiments designed to determine the limits of tissue to laser damage as well as to describe the photobiological processes involved . with respect to the eye , the wavelength dependent optical properties of the anterior segment and vitreous also play an important role . retinal damage from laser or noncoherent light sources can occur via three principal light damage mechanisms : photothermal , photochemical , and photomechanical damage . retinal laser damage studies have traditionally used a direct - view funduscope ( ophthalmoscope ) to observe changes to retinal tissue following laser exposure . more recently , commercially available high - resolution retinal devices such as spectral domain optical coherence tomography ( sd - oct ) have been incorporated into studies of laser induced damage to the retina for laser safety recommendations [ 14 , 15 ] and to monitor the healing response of retinal laser treatments for clinical applications [ 1618 ] . since the transition of adaptive optics ( ao ) technology from astronomy to ophthalmology almost 17 years ago , ao has made significant progress in the performance of ophthalmic instruments . to date , there have been no studies utilizing ao sd - oct to evaluate laser injury to the retina nor the healing response . the work presented here is intended to facilitate laser lesion detection and identification . in this pilot study , the progression of photomechanical and photothermal / photochemical laser exposure to the retina of the nonhuman primate ( nhp ) was documented using a combined ao confocal scanning laser ophthalmoscope ( ao cslo ) and an ao sd - oct . real - time imaging of lesion development and characterization over time will enable better understanding of regimes ( thermal , photochemical , and photodisruptive ) of laser damage in the primate retina . the ao sd - oct and ao cslo provide researchers with high - resolution images of retinal layers to investigate laser damage in vivo and an opportunity to observe laser damage over multiple time points without the need for histopathology as a single end point for observation . sixteen eyes of eight male cynomolgus ( macaca fascicularis ) monkeys between the ages of 6 and 12 years ( 4.47.0 kg ) were used for this experiment only . all subjects were used for in vivo imaging and histological study of laser exposure to the retina . once restrained , two drops each of proparacaine hcl 0.5% , phenylephrine hcl 2.5% , and tropicamide 1% were administered to both eyes . prior to prone placement of the subject on a 5-axis goniometric translation stage , the subject was intubated , and a peribulbar injection of 4% lidocaine was administered in the first eye to be imaged to reduce extraocular muscular movement . the subject 's temperature , oxygen saturation , and pulse were continuously monitored throughout the experimental protocol . all experimental procedures were approved by the brooks city base institutional animal care and use committee and conformed to all united states department of agriculture ( usda ) , national institutes of health ( nih ) guidelines , and the arvo statement for the use of animals in ophthalmic and vision research . monkeys were euthanized under deep anesthesia and transcardially perfused with a fixative solution ( ph 7.4 ) consisting of 10% formalin and 2% paraformaldehyde in 0.1 m sodium phosphate buffer . after each perfusion was complete , eyes were enucleated and immediately were fixed in 10% neutral buffered formalin . three types of laser exposure parameters were delivered to the retina to examine photomechanical and photothermal / photochemical light interactions . the laser exposure parameters and subjects used for each experiment are summarized in tables 1 and 2 , respectively . the beam was directed through a half - wave plate and polarizing beam splitter cube combination to control the energy delivered to the subject . the beam was directed through a half - wave plate and polarizing beam splitter cube combination to control the energy delivered to the subject . , santa clara , ca ) for 20 seconds in the macular region to investigate photochemical / photothermal interactions . , santa clara , ca ) for 20 seconds in the macular region to investigate photochemical / photothermal interactions . for most of the experiments , the insult beam was passed through a safety shutter prior to coalignment with the combined ao retinal imager to enable real - time viewing of laser exposure ( figure 1 ) . , andover , ma ) with a wide field line scanning laser ophthalmoscope ( lslo ) and an ao cslo ( iris ao , berkley , ca ) were combined to simultaneously collect complementary in vivo images of the primate retina in high - resolution ( figure 2 ) . briefly , the ao sd - oct portion of the integrated setup provides high - speed ( 15 frames / sec ) cross - sectional scans of the retina with a theoretical axial and transverse resolution of 4 m and ~10 m , respectively . the ao cslo system consists of a series of focal telescopes that relay the 7 mm pupil to the optical conjugate planes at the galvanometric scanner , the resonant scanner , the dm ( 42 actuators mems ; iris ao , berkley , ca ) , and the wfs ( adaptive optics associates inc . the resonant scanner frequency is 16 khz while the acquisition rate of 30 hz is set by the galvanometric scanner , enabling 512 512 images of the retina set to either a 1 or 2 fov . the ao cslo beam ( 632 nm ) is input into the combined system through a beam splitter and is reflected off the dm , scanners , and spherical mirrors into the eye . both ao systems were used to simultaneously capture image sequences of the retinal area during laser exposure . a line and/or raster scan configuration was used to record oct b - scans prior to and following laser exposure . line scans were collected up to a 5.5 scan length on the retina and consisted of 1024 a - scans . prior to laser exposure , images of the macular region were collected using a 2 fov for the cslo and a raster scan configuration for oct . in addition , color fundus photography was performed to provide direct comparison with ao images at each experimental time point . three examiners evaluated all eyes using color fundus photos for each time point following laser exposure . visible lesions at a given exposure site were reported to be present only if two or more examiners identified a lesion in color fundus photos . lesion identification in oct c - scans and cslo retinal montages of the lesion field was performed by a single person in images with a signal - to - noise ratio greater than 10 db . volumetric oct images of the macular region were imported into avizo 6.3 ( visualization sciences group , burlington , ma ) for measurement of lesion width and area . seed grayscale pixel value within the hyper- or hyporeflective areas associated with injury . a manually adjusted threshold range based on the grayscale the number of visible lesions was determined after 1 hour and 24 hours after exposure , and a probit analysis was performed to determine the dose which corresponds to a 50% probability for damage ( ed50 ) . lesions detected in available oct and ao cslo images were always observed in color fundus photos . exposures which did not result in visible lesions in color fundus photos within the first hour did not develop into lesions at any later time point . real - time in vivo imaging of higher energy microsecond exposures revealed disruptive events possibly associated with microcavitation . the ao cslo acquisition rate of 33 ms / frame is too long to record microbubble formation but can monitor the spatial rearrangement of the retina following exposure . visible retinal rearrangement observed in ao cslo images did not coincide with the appearance of a minimal visible lesion ( mvl ) in color fundus photos for 10 instances ( false negatives ) . there were no instances in which a disruptive event was observed in an ao cslo image and did not result in the formation of a mvl in fundus photos ( false positive ) . figure 3 shows cross - sectional images of the formation of a lesion ( 870 mj / cm ) in the retina directly before , immediately after , and minutes following laser exposure . the 40 m retinal spot size exposures occupy a smaller damage footprint compared to the 120 m spot size exposures ( data not shown ) . disruptive events were not observed for smaller spot size exposures in oct b - scans or slo images . retinal damage immediately visible in ao cslo images occurred for energies of 139 mj / cm and greater . the damage appearance ranged from barely visible retinal disruption sometimes accompanied with reflectance changes to a distinguished explosive event . as exposure energy lesion appearance in color fundus photos ranged from pale gray to white increasing in reflectivity and size as energy was increased for each exposure . within a 24-hour period , most exposures became visible in color fundus photos . high energy exposures ( > 442 mj / cm ) produced few inner retinal hemorrhages and were more readily identifiable at the 1-hour observation points in oct and ao cslo images . oct b - scans collected within an hour of higher energy exposures reveal that the most damage was confined to the outer retina and extended further into the onl compared to moderate energy exposures . the retinal sites exposed to 413 nm light had a pale yellow appearance in color fundus photos , which was maintained through all observation time points . for all subjects , retinal damage observed using sd - oct associated with laser exposure was not always positively identified as mvls in color fundus photos . of the total 173 lesions , there was a 46% improvement in lesion detection using oct en face images ( n = 93 ) versus color fundus photos ( n = 43 ) at the 1-hour time point . in figure 8 , of the 25 retinal sites receiving 413 nm exposures ranging from 165 to 1,183 j / cm , 24% ( 7 out of 25 ) qualified as mvls in color fundus photo taken 1 hour later even though hyperreflective spots coincident with exposure locations can be seen in the oct en face view ( figure 8(a ) ) . oct c - scans of the photoreceptor inner segments reveal that 88% ( 22 out of 25 ) of the exposed regions ten days later , 56% of the lesions were identified as mvl in color fundus photos ( figure 8(c ) ) compared to 80% identified using oct raster scans . oct b - scans and cslo images collected during 413 nm , 20-second exposures did not reveal any immediate changes to the retina during laser irradiation . retinal layers affected were the photoreceptor inner segments , rpe , and inner retina . figure 9 shows the retinal damage response of three retinal locations receiving a retinal exposure of 474 j / cm . lesions which did not become visible in color fundus photos until the 72-hour observation point had retinal irradiance values ranging from 225 to 1,089 j / cm . minimal or no damage was visible to the rpe for exposure energies less than 1,696 j / cm in oct c - scans for the first hour after exposure ; therefore , lesion width measurements were collected only at the photoreceptor layer for the first hour where retinal changes were visible . exposures at the 1-hour time point which are greater than the lower limiting photochemical mpe were identified as inducing retinal reflectance changes in oct and fundus images for 62% and 42% of the exposures , respectively . there was an improvement in lesion identification using fundus photos 48 and 72 hours after exposure when compared to the number of lesions identified using oct . lesions were identified ( 27% ) at the 1-hour observation time point from oct en face images . the introduction of high - resolution retinal imaging into light damage studies has enabled greater detection of the effects of laser light exposure compared to traditional methods employed in past studies of laser damage thresholds . for the first time , to our knowledge , we have been able to image the immediate effects of photomechanical disruption to the retina . the data presented within this study are a pilot study using high - resolution imaging to study laser - tissue interactions in vivo . short pulse laser exposure can damage the retina via photomechanical or photothermal mechanisms depending on the pulse duration and total energy delivered . real - time in vivo imaging of higher energy microsecond exposures revealed disruptive events likely associated with explosive vaporization of water and subsequent cavitation around melanin particles in the rpe cells . the transition from thermal denaturation into thermomechanical effects as the primary cause of damage to the rpe after laser exposure is approximately 50 s . photomechanical events possibly associated with microcavitation disruption may have been observed at the greater end of exposure energies for experiments 1 and 2 but in all likelihood were mostly thermal in nature . the ao slo acquisition rate of 33 ms / frame does allow recording of actual microbubble formation ( ~20 s ) on the order of microseconds but can monitor the spatial rearrangement of the retina following exposure . the false negatives for damage resulting from the measurement of the spatial disruption of the retinal layers as an indicator of bubble formation indicate that thermal denaturation may still be the critical damage mechanism for the laser parameters tested . however , the presence of retinal layer disruption should be a clear indicator of a mechanical process and thus this compels the belief that 2 s is close to the exposure time associated with the transition of damage from photothermal to photomechanical mechanisms . this observation is consistent with theoretical model results of laser induced damage processes in the retina . lesion size and area of 3-picosecond exposures were logarithmically related to the amount of energy delivered to the retina . previous picosecond laser damage studies [ 14 , 29 ] have compared the extent of damage and damage thresholds to rhesus monkey retinas based on spot size differences . color fundus photos from subject s8 were out of focus due to corneal aberrations and therefore did not allow inclusion for probit analysis . results of aforementioned picosecond studies are similar in regard to the localization of damage in the outer retina when laser induced breakdown ( lib ) does not play a role . in addition , as the energy of the exposure increases , the extent of the damage also increases to include the inner retinal layers . low signal - to - noise ratio , frame rate , poor light transmission , eye movement , and misalignment of the imaging laser with insult laser may account for missed and unobserved photomechanical phenomena in vivo . not all 413 nm damage observable using high - resolution imaging modalities was noted as an mvl in color fundus photos . for example , in more than one subject and multiple eyes receiving 413 nm 20-second exposures , damage was visualized at the one - hour time point in oct b - scans as small nonuniform regions of speckled hyperreflectivity at the rpe and photoreceptor layers , but damage was not identified in color fundus photos . in addition , lesions from one subject were not visible in color fundus photos until the 72-hour time point despite evidence of light induced changes in oct scans at earlier time points . it is possible based on our findings that observable retinal changes associated with photochemical damage could occur on a much shorter time scale ( hours ) as compared to days but are not observed using traditional fundus photography . it is not known whether the exposures were funduscopically visible 13 days following injury as color fundus images were not collected until 6 days following injury . the majority of retinal exposures used to study 413 nm damage in this study are greater than 47 w and within ansi standards for the mpe at the lower limit photochemical light exposure . the 413 nm 20 exposures closest to the fovea with energies greater than 700 j / cm resulted in full retinal thickness reflectance changes associated with damage and edematous reactions . argon lesions delivered outside of a 2 circle of eccentricity from the foveal pit were localized to the rpe while lesions within the 2 circle extended from the rpe into the inner retina and appeared full - thickness . the localization of damage described here closely resembles the histological analysis of argon laser damage to the fovea and its periphery . the ability to follow lesion progression over several time points after exposure ( up to 9 days ) revealed the full - thickness damage we observed at 1 hour and 24 hours after exposure is no longer visible in oct at a 9-day follow - up point . the damage response following exposure for the 413 nm 20-second and 532 nm 40-picosecond exposures was observed using ao sd - oct . the thread - like structures develop within the hour following exposure and are localized to the lesion site at the external limiting membrane ( elm ) and reach into the opl and are generally seen beyond the foveal region but is limited to lesions closest to the fovea . a possible explanation includes damage to the henle fiber layer due to transsynaptic degeneration and direct damage from laser exposure . in particular , light ( 488 nm ) injury is associated with enlargement of mller cell processes , gliosis , and upregulation of gfap . such changes could cause an increase in the refractive index of the mller cell cytoplasm , with increased visibility of the structure by high - definition oct . the number of mller cells to cone terminals in the perifoveal opl is equal , and the mller cells per / mm decrease with increasing distance from the fovea . although this idea could explain the time frame of our observations , the very sharply defined quality of these reflective thread - like structures does not appear similar to blood vessel hemorrhage observed in oct b - scans after pulsed laser exposure . this is a pilot study to examine the feasibility of using ao sd - oct and ao cslo to examine regimes of laser damage in real - time in addition to the damage response . there is an increased detection of blue light damage at the 1-hour time mark using high - resolution imaging prior to lesion appearance in fundus photos at 24 hours and later . the results highlight the need for continued use of high - resolution imaging to resolve retinal light damage in support of laser safety guidelines . with regard to pulsed thermal laser exposure , high - resolution imaging systems with high frame rate may produce more accurate representation of the pulsed laser tissue phenomena . the ao sd - oct and cslo provide researchers with high - resolution images of retinal layers to resolve laser damage in vivo .

in the last decade , the use of 3d image processing has been increased especially for medical applications ; this leads to increase the qualified radiologists ' number who navigate , view , analyse , segment , and interpret medical images . the analysis and visualization of the image stack received from the acquisition devices are difficult to evaluate due to the quantity of clinical data and the amount of noise existing in medical images due to the scanners itself . computerized analysis and automated information systems can offer help dealing with the large amounts of data , and new image processing techniques may help to denoise those images . multiresolution analysis ( mra ) [ 13 ] has been successfully used in image processing specially with image segmentation , wavelet - based features has been used in various applications including image compression , denoising , and classification . recently , the finite ridgelet and curvelet transforms have been introduced as a higher dimensional mra tool [ 7 , 8 ] . image segmentation requires extracting specific features from an image by distinguishing objects from the background . the process involves classifying each pixel of an image into a set of distinct classes , where the number of classes is much smaller . medical image segmentation aims to separate known anatomical structures from the background such cancer diagnosis , quantification of tissue volumes , radiotherapy treatment planning , and study of anatomical structures . segmentation can be manually performed by a human expert who simply examines an image , determines borders between regions , and classifies each region . this is perhaps the most reliable and accurate method of image segmentation , because the human visual system is immensely complex and well suited to the task . curvelet transform is a new extension of wavelet transform which aims to deal with interesting phenomena occurring along curved edges in 2d images . it is a high - dimensional generalization of the wavelet transform designed to represent images at different scales and different orientations ( angles ) . it is viewed as a multiscale pyramid with frame elements indexed by location , scale , and orientation parameters with needle - shaped elements at fine scales . curvelets have time - frequency localization properties of wavelets but also shows a very high degree of directionality and anisotropy , and its singularities can be well approximated with very few coefficients . this paper is focusing on a robust implementation of mra techniques for segmenting medical volumes using features derived from the wavelet , ridgelet , and curvelet transforms of medical images obtained from a ct scanner . the rest of this paper is organised as follow : section 2 illustrates the proposed medical image segmentation system using mra techniques . the mathematical background and the methodology for the proposed mra techniques have been explained in section 3 . the results and analysis of the implemented wavelet , ridgelet , and curvelet transforms for medical image segmentation are illustrated in section 4 . the main aim of this research is to facilitate the process of highlighting roi in medical images , which may be encapsulated within other objects or surrounded by noise that make the segmentation process not easy . wavelet , ridgelet , and curvelet transforms are applied on medical images with other pre- and postprocessing techniques to present segmented outputs and detected roi in an easier and more accurate way . image segmentation using mra such as wavelets has been widely used in recent years and provides better accuracy in segmenting different types of images . many recent developments in mra have taken place , while wavelets are suitable for dealing with objects with point singularities . wavelets can only capture limited directional information due to its poor orientation selectivity . by decomposing the image into a series of high - pass and low - pass filter bands , the wavelet transform extracts directional details that capture horizontal , vertical , and diagonal activity . however , these three linear directions are limiting and might not capture enough directional information in noisy images , such as medical ct scans , which do not have strong horizontal , vertical , or diagonal directional elements . ridgelet improves mra segmentation ; however , they capture structural information of an image based on multiple radial directions in the frequency domain . one limitation to use ridgelet in image segmentation is that ridgelet is most effective in detecting linear radial structures , which are not dominant in medical images . the curvelet transform is a recent extension of ridgelet transform that overcome ridgelet weaknesses in medical image segmentation . curvelet is proven to be particularly effective at detecting image activity along curves instead of radial directions which are the most comprising objects of medical images . in the last decade , wavelet transform has been recognized as a powerful tool in a wide range of applications , including image / video processing , numerical analysis , and telecommunication . the advantage of wavelet is that wavelet performs an mra of a signal with localization in both time and frequency [ 10 , 11 ] . in addition to this , functions with discontinuities and functions with sharp spikes require fewer wavelet basis vectors in the wavelet domain than sine cosine basis vectors to achieve a comparable approximation . wavelet operates by convolving the target function with wavelet kernels to obtain wavelet coefficients representing the contributions in the function at different scales and orientations . wavelet or multiresolution theory can be used alongside segmentation approaches , creating new systems which can provide a segmentation of superior quality to those segmentation approaches computed exclusively within the spatial domain . discrete wavelet transform ( dwt ) can be implemented as a set of high - pass and low - pass filter banks . in standard wavelet decomposition , the output from the low - pass filter can be then decomposed further , with the process continuing recursively in this manner . according to , dwt can be mathematically expressed by ( 1)aj(n)=i=0l1l(i)aj1(2ni ) , 0n < nj , dj(n)=i=0l1h(i)dj1(2ni ) , 0n < nj . the coefficients a(n ) and d(n ) refer to approximation and detailed components in the signal at decomposition level j , respectively . the l(i ) and h(i ) represent the coefficients of low - pass and high - pass filters , respectively . the wavelet decomposition of an image creates at each scale j a set of coefficient values wj with an overall mean of zero . wj contains the same number of voxels as the original image ; therefore , this wavelet transform is redundant [ 14 , 15 ] . for images , 1d - dwt can be readily extended into 2d . in standard 2d wavelet decomposition , the image rows are fully decomposed , with the output being fully decomposed columnwise . in nonstandard wavelet decomposition , all the rows are decomposed by one decomposition level followed by one decomposition level of the columns . wavelet uses a set of filters to decompose images depending on filter coefficients and the number of those coefficients . the most popular wavelet filter is haar wavelet filter ( hwf ) which takes the averages and differences from the low- and high - pass filters , respectively . figure 3 illustrates an example of applying 2d - dwt using hwf on an image for 2 levels of decompositions . in 1998 , donoho introduced the ridgelet transform continuous ridgelet transform ( crt ) can be defined from a 1d wavelet function oriented at constant lines and radial directions . ridgelet transform [ 1719 ] has been generating a lot of interest due to their superior performance over wavelets . while wavelets have been very successful in applications such as denoising and compact approximations of images containing zero dimensional or point singularities . wavelets do not isolate the smoothness along edges that occurs in images , and they are thus more appropriate for the reconstruction of sharp point singularities than lines or edges . these shortcomings of wavelet are well addressed by the ridgelet transform ; the functionality of wavelet has been extended to higher dimensional singularities and becomes an effective tool to perform sparse directional analysis [ 3 , 21 ] . generally speaking , wavelets detect objects with point singularities , while ridgelets are able to represent objects with line singularities . the finite ridgelet transform ( frit ) was computed in two steps : a calculation of discrete radon transform and an application of a wavelet transform . the finite radon transform ( frat ) is computed in two steps : a calculation of 2d fast fourier transform ( fft ) for the image and an application of a 1d inverse fast fourier transform ( ifft ) on each of the 32 radial directions of the radon projection . 1d wavelet is applied restricted to radial directions going through the origin for three levels of decompositions . applying frat on image can be presented as a set of projections of the image taken at different angles to map the image space to projection space . its computation is important in image processing and computer vision for problems such as pattern recognition and the reconstruction of medical images . for discrete images , a projection is computed by summation of all data points that lie within specified unit - width strips ; those lines are defined in a finite geometry . depending on , frat of a real function on the finite grid zp is defined in ( 2)rk[l]=fratf(k , l)=1p(i , j)l(k , l)f(i , j ) . here , l(k , l ) denotes the set of points that make up a line on the lattice zp as in ( 3)l(k , l)={(i , j):j = ki+l(mod p),izp } , 0k < p , l(p , l)={(l , j):jzp}. to compute the kth radon projection ( i.e. , the kth row in the array ) , all pixels of the original image need to be passed once and use p histogrammers : one for every pixel in the row . at the end , all p histogrammed values are divided by k to get the average values . according to alzu'bi and amira in , once the wavelet and radon transforms have been implemented , the ridgelet transform is straightforward . each output of the radon projection is simply passed through the wavelet transform before it reaches the output multiplier . as shown in figure 4 , ridgelets use frat as a basic building block , where frat maps a line singularity into point singularity , and the wavelet transform has used to effectively detect and segment the point singularity in radon domain . figure 5 shows a clinical chest slice from a ct scanner in the last step of ridgelet transform before image reconstruction at different block sizes . continuous ridgelet transform is similar to the continuous wavelet transform except that point parameters ( x , y ) in the cartesian grid ( figure 6(a ) ) which perform pixels in the image or an entry in a 2d matrix are now replaced by line parameters ( , ) , where is the intercept and is the angle . figure 6(b ) illustrates the radial grid in ridgelet transform ; however , straight lines evaluate the image in the frequency domain . medical images comprised from curves which are still not singularity points after applying radon transform . wavelet transform can not detect those singularities properly , since it still not singularity points , resulting that ridgelet transformation is not suitable for segmenting these images . ridgelet transform can be used in other applications , where images contain edges and straight lines . curvelet transform has been introduced to solve this problem ; it deals with higher singularities compared to wavelet and ridgelet transforms . it was first introduced in [ 25 , 26 ] by cands and donoho in 2000 , which used a complex series of steps involving the ridgelet analysis of the radon transform of an image . their performance was very slow ; hence , researchers developed a new version which is easier to use and understand . in this new method , the use of the ridgelet transform as a preprocessing step of curvelet was discarded , thus reducing the amount of redundancy in the transform and increasing the speed considerably . curvelet aims to deal with interesting phenomena occurring along curved edges in a 2d image . as illustrated in figure 7 , curvelet needs fewer coefficients for representation , and the edge produced from curvelet is smoother than wavelet edge . the newly constructed and improved version of curvelet transform is known as fast discrete curvelet transform ( fdct ) . this new technique is simpler , faster and less redundant than the original curvelet transform which based on ridgelets . according to candes et al . in , two implementations of fdct are proposed : unequally spaced fast fourier transforms ( usfft ) , both implementations of fdct differ mainly by the choice of spatial grid that used to translate curvelets at each scale and angle . both digital transformations return a table of digital curvelet coefficients indexed by a scale parameter , an orientation parameter , and a spatial location parameter . wrapping - based transform is based on wrapping a specially selected fourier samples , and it is easier to implement and understand . curvelet transform works in two dimensions with spatial variable x , frequency domain variable , and the frequency - domain polar coordinates r and . curvelet transform can be defined by a pair of windows , radial window { w(r ) } , and angular window { v(t ) } . as illustrated in ( 4 ) , these windows will always obey the admissibility conditions . ( 4)j=w2(2jr)=1 , r(34,32),j=v2(tl)=1 , t(12 , 12 ) . a polar wedge represented by uj is supported by the radial window { w(r ) } and angular window { v(r)}. equation ( 5 ) defines uj in the fourier domain ( 5)uj(r,)=23j/4w(2jr)v(2j/22 ) . equation ( 6 ) defines the curvelet transform as a function of { x = ( x1 , x2 ) } at scale 2 , orientation l and position xk(j , l ) , where r is the rotation in radians . figure 8 illustrates the induced tiling of the frequency plane and the spatial cartesian grid associated with a given scale and orientation , and shaded area represents the polar wedge by uj(6)j , l , k(x)=j(rl(xxk(j , l ) ) ) . the new implementation of curvelet transform based on wrapping of fourier samples takes a 2d image as an input in the form of a cartesian array f[m , n ] , where 0 m < m , 0 n < n where m and n are the dimensions of the array . as illustrated in ( 7 ) , the outputs will be a collection of curvelet coefficients c(j , l , k1k2 ) indexed by a scale j , an orientation l and spatial location parameters k1 and k2 . each j , l , k1k2 is a digital curvelet waveform , superscript d stands for digital . these approach implementations are the effective parabolic scaling law on the subbands in the frequency domain to capture curved edges within an image in more effective way . as mentioned earlier , wrapping based curvelet transform is a multiscale pyramid which consists of several subbands at different scales consisting of different orientations and positions in the frequency domain . at a high frequency level , curvelets are so fine and looks like a needle shaped element and they are non - directional coarse elements at low frequency level . figure 9 illustrates the whole image represented in spectral domain in the form of rectangular frequency tiling by combining all frequency responses of curvelets at different scales and orientations . it can be seen from figure 9 that curvelet becomes finer and smaller in the spatial domain and shows more sensitivity to curved edges as the resolution level is increased , thus allowing to effectively capturing the curves in an image , and curved singularities can be well - approximated with fewer coefficients . in order to achieve a higher level of efficiency , curvelet transform this means that a 2d fft is applied to the image . for each scale and orientation , a product of ujl wedge is obtained ; the result is then wrapped around the origin , and 2d ifft is then applied resulting in discrete curvelet coefficients . describe the discrete curvelet transform in as illustrated in ( 8)curvelet transform = ifft[fft(curvelet)fft(image ) ] . the difficulty behind this is that trapezoidal wedge does not fit in a rectangle of size 2 2 aligned with the axes in the frequency plane in which the 2d ifft could be applied to collect curvelet coefficients . wedge wrapping procedure proposed in uses a parallelogram with sides 2 and 2 to support the wedge data . the wrapping is done by periodic tiling of the spectrum inside the wedge and then collecting the rectangular coefficient area in the centre . the centre rectangle of size 2 2 successfully collects all the information in that parallelogram . figure 10 illustrates the process of wrapping wedge where the angle is in the range ( /4 , 3/4 ) and the rectangles have the same width and length as the parallelogram is centred at the origin . step 1apply the 2d fft to an image to obtain fourier samples ( 9)f^[m , n ] , n2m , n < n2 . apply the 2d fft to an image to obtain fourier samples ( 9)f^[m , n ] , n2m , n < n2 . step 2for each scale j and angle l , form the product ( 10)uj , l[m , n]f^[m , n ] . for each scale j and angle l , form the product ( 10)uj , l[m , n]f^[m , n ] . step 3wrap this product around the origin and obtain ( 11)fj , l[m , n]=w(uj , lf^)[m , n ] , where the range for m , n , and is now 0 m < 2 , 0 n < 2 , and /4 < /4 . wrap this product around the origin and obtain ( 11)fj , l[m , n]=w(uj , lf^)[m , n ] , where the range for m , n , and is now 0 m < 2 , 0 n < 2 , and /4 step 4apply ifft to each fj , l , hence collecting the discrete coefficients c(j , l , k1k2 ) . apply ifft to each fj , l , hence collecting the discrete coefficients c(j , l , k1k2 ) . the curvelet transform is a multiscale transform such as wavelet , with frame elements indexed by scale and location parameters . wavelets are only suitable for objects with point singularities , ridgelets are only suitable for objects with line singularities , while curvelets have directional parameters and its pyramid contains elements with a very high degree of directional specificity . the elements obey a special scaling law , where the length and the width of frame elements support are linked using ( 12)widthlength2 discrete curvelet transform in the spectral domain utilizes the advantages of fft . during fft , both image and curvelet at a given scale and orientation are transformed into the fourier domain . the convolution of the curvelet with the image in the spatial domain then becomes their product in the fourier domain . at the end of this computation process , a set of curvelet coefficients are obtained by applying ifft to the spectral product . ( a ) optimally sparse representation of objects with edgescurvelets provide optimally sparse representation of objects which display curve - punctuated smoothness except for discontinuity along a general curve with a bounded curvature . such representations are nearly as sparse as if the object were not singular and turn out to be far sparser than other transforms decomposition of the object . curvelets provide optimally sparse representation of objects which display curve - punctuated smoothness except for discontinuity along a general curve with a bounded curvature . such representations are nearly as sparse as if the object were not singular and turn out to be far sparser than other transforms decomposition of the object . ( b ) optimal image reconstruction in severely ill - posed problemscurvelets also have special microlocal features which make them especially adapted to certain reconstruction problems with missing data . for example , in many important medical applications , one wishes to reconstruct an object f(x1 , x2 ) from noisy and incomplete tomographic data . because of its relevance in biomedical imaging , this problem has been extensively studied , yet curvelets offer surprisingly new quantitative insights . for example , an application of the phase - space localization of the curvelet transform allows a very precise description of those features of the object of function ( f ) which can be reconstructed accurately from such data and how well , and of those features which can not be recovered . curvelets also have special microlocal features which make them especially adapted to certain reconstruction problems with missing data . for example , in many important medical applications , one wishes to reconstruct an object f(x1 , x2 ) from noisy and incomplete tomographic data . because of its relevance in biomedical imaging , this problem has been extensively studied , yet curvelets offer surprisingly new quantitative insights . for example , an application of the phase - space localization of the curvelet transform allows a very precise description of those features of the object of function ( f ) which can be reconstructed accurately from such data and how well , and of those features which can not be recovered . as illustrated in ( 8) , the data acquisition geometry separates the curvelet expansion of the object into two pieces as illustrated in ( 13)f=ngoodf,nn+ngoodf,nn . the first part of ( 13 ) what is interesting here is that one can provably reconstruct the recoverable part with an accuracy similar to that one would achieve even if one had complete data . there is indeed a quantitative theory showing that for some statistical models which allow for discontinuities in the object to be recovered , there are simple algorithms based on the shrinkage of curvelet biorthogonal decompositions , which achieve optimal statistical rates of convergence . figure 11 illustrates the frequency response of curvelets at different scales and orientations for some test images using curvelab ( version : 2.1.2 ) in both spatial and frequency domain . figures 11(a ) , 11(c ) , 11(e ) , 11(g ) , and 11(i ) are the spatial representation of curvelet at scales 1 to 5 . and figures 11(b ) , 11(d ) , 11(f ) , 11(h ) , and 11(j ) are the frequency domain representation of curvelet that is modulus of fft . figure 12 illustrates a clinical data for human chest from ct scanner in spatial domain and its curvelet coefficients . in figure 12 , the low frequency coefficients ( coarse scale ) the concentric coronae ( formed by black strips ) show coefficients at different scales and the outer coronae correspond to higher frequencies . each corona has four strips further subdivided in angular panels ; each panel represents coefficients at a specified scale and orientation suggested by the position of the panel . wedge wrapping is done for all the wedges at each scale in the frequency domain to obtain a set of subbands or wedges at each curvelet decomposition level , and these subbands are the collection of discrete curvelet coefficients . the aim is to identify the most effective texture descriptor for medical images to capture edge information more accurately . the discrete curvelet transform can be calculated to various resolutions or scales and angles ; the maximum number of resolution depends on the original image size and the angles . number of angles at the second coarsest level must be at least eight and a multiple of four ; that is , 512 512 image has five maximum possible resolution levels containing structural information of the image . figure 13 illustrates how curvelet - based edge reconstruction in medical imaging differs from other transforms methods . the end users of the proposed system are the radiologists and specialists who analyse medical images for cancer diagnosis . after several meetings with those people in the radiology departments in some hospitals , the main goal that they are working is to detect the accurate cancer size in medical images with the least error . this process may be affected by the noise surrounding roi , which make the process of measuring the exact dimensions of the lesion so hard . different datasets have been carried out with the proposed system to validate it for clinical applications . the first one is nema iec body phantom which consists of an elliptical water filled cavity with six spherical inserts suspended by plastic rods of inner diameters : 10 , 13 , 17 , 22 , 28 , and 37 mm [ 25 , 26 ] . real clinical human images acquired by a ct scanner have also been used to experiment the proposed approaches , this data has been previously analysed by the radiologists and the provided reports explains that the patients are diagnosed by cancer . table 1 illustrates the snr values of extracted features from nema iec data set in spatial domain , different levels of decomposition of wavelet domain and at different block sizes in ridgelet domain . it can be seen from table 1 that small values of snr have been obtained for all techniques ; this is due to the noise from the acquisition systems itself . this noise will be a part of the medical image after the reconstruction of all slices . relatively , better snr values can be achieved with the second level of wavelet decomposition and as the block size ( p ) is getting bigger with the ridgelet transform , where the transformed image is getting more similar to the original image . this can be assigned to the major limitation of using ridgelet transformation in medical image segmentation , where ridges rarely exist in such data . thresholding technique has been applied as a preprocessing step on the original images at threshold value ( t = 35 ) to remove as much artificial spam sequel produced from the scanners . the transform then applied to effectively represent objects with edges which are the contours of the medical images followed by another thresholding at ( t = 7 ) to remove most of the remaining noise and facilitate the measurement process . figures 14(a ) and 14(c ) illustrate the original images from a ct scanner , and figures 14(b ) and 14(d ) illustrate the segmented phantom image and real chest image , respectively , using curvelet transform . as illustrated in figure 15 , results of the proposed segmentation technique are vary in terms of smooth reconstruction of the spheres . curvelet transform segments the input image and removes artifacts from the image to exhibit smooth and optimal segmentation of nema phantom . ridgelet transform detect roi but does not give promising segmentation results due to the lack of ridges or straight lines in the tested data set . wavelet quadrants are varying also in their quality ; relatively , the best results have been achieved with the ll - filter output . table 2 illustrates nema spheres diameters error percentages measured using different multiresolution analysis techniques and compared to previously implemented techniques . ed has been used to measure the spheres diameters and calculate the error percentages for each technique , and sphere diameter error percentages have been calculated as follows : ( 14)error % = measured diameteractual diameteractual diameter100% . from table 2 , in the case of k - means clustering , tumor volumes are underestimated by approximately 5%-6% in most cases ; however , for the two smaller spherical inserts , with diameter of 10 mm and 13 mm , respectively , these underestimations are significantly greater . for the smallest sphere , more than a 13% volume discrepancy is recorded , with the k - means algorithm finding it difficult to quantify the tumor accurately . k - means clustering , mrfm tends to overestimate the volumes of the spherical inserts , with the exception of spheres 1 and 2 . three decomposition levels of dwt have been applied on nema phantom [ 25 , 26 ] using two different filters ( haar , daubechies ) , and the measured diameters were doubled at each level to produce a fair comparison with the other available techniques . it can be seen that most of the error percentages were decreasing while the spheres diameter increasing ; it is worth mentioning that there is no upper bound of the spheres diameters to keep the errors decreasing , because the roi becomes clearer and easier to be detected and measured properly . but tumours in real life are usually very small in the early stage cancer , and the problem is to detect those turnouts ' tumours as soon as possible . the two smallest spherical inserts are still underestimated in most of the techniques and got the largest error percentages . the large volumetric errors encountered using this acquisition exist as a consequence of the poor slice thickness setting selected for the scan . the 4.25 mm slice thickness causes large fluctuations in transaxial tumour areas to occur between image slices . this problematic characteristic occurs most notably with the smallest spherical inserts , where single voxel reallocation causes a large deviation in percentage error . in figure 16 , the percentage error computed between the actual sphere volume and the volumes obtained using all methodologies for each of the six tumours inserts is plotted . it can be seen that all techniques are settled down according to the error percentages as the sphere diameters increased . it can be also seen from table 2 that acceptable error percentages have been achieved using ridgelet transform for the big spheres ( 22 mm , 28 mm , and 37 mm ) , where the curves are not sharp and ridgelet detect it accurately as it close to be ridges . but for the small spheres , ridgelet weakness for medical image segmentation start appears clearly . curvelet transform overcomes the weakness of wavelet for segmenting sharp curves and detect the small spheres accurately with error percentages ( 0.82%2.65% ) . for the big spheres , errors achieved using wavelet transform are still better than those achieved using curvelet transform due to the sharpness of that spheres . but still very good results using curvelet transform and acceptable for clinical applications . psnr and mse have been also used to evaluate the quality of the proposed techniques . the original image has been contaminated with gaussian white noise at = 20% of the maximum intensity . table 3 illustrates a comparison study of curvelet transform with the other traditional transforms , and comparison terms psnr and mse have been used to test the quality of the transformed image . from table 3 , it can be seen that the best results according to both psnr and mse have been achieved using curvelet transform . figure 17 illustrates two noisy images and the denoised outputs using both wavelet and curvelet . according to a study done by dettori and semler , the ridgelet - based descriptors had significantly higher performance measures in comparison to wavelet - based descriptors , with accuracy rates higher than any other wavelet - based feature set for all individual organs . this is not surprising given the fact that the ridgelet transform is able to capture multidirectional features , as opposed to the wavelet transform which focus mainly on horizontal , vertical , and diagonal features . this can be generalized to most of the images except for medical scanners , where the weakness of wavelet is not dominant in such images . curvelet - based descriptors had an even higher performance in comparison to both the wavelet and ridgelet , with accuracy rates higher , respectively . the accuracy rate using curvelet transform is better ; this is expected , since the curvelet transform is able to capture multidirectional features in wedges , as opposed to lines or points as in the ridgelet or wavelet transform . the multidirectional features in curvelets are very effective in extracting the important features from medical images and then segmented accurately . as illustrated in the previous tables and figures , it can be seen that more efficient and smooth image reconstruction is achieved using curvelet transform . in terms of optimal reconstruction of the objects with edges and curves , the algorithm presented in this chapter is able to classify normal tissues in ct scans with high accuracy rates . these hypotheses will be further tested and validated on different predefined clinical data sets in chapter 8 of this thesis . segmentation using curvelet transform has been chosen for experimenting the pet scanner sensitivity variables , curvelet was applied in parallel with multithresholding and classification techniques to classify the spheres in a separate class from the other comprising objects at least noise included . the experiment was evaluated based on the ratio between the spheres area to the other area of the scanned slice . the actual spheres area can be calculated according to ( 15 ) , given that the spheres diameters are 10 , 13 , 17 , 22 , 28 , 37 mm ( 15)soriginal=12r{a}r2 , { a}={10,13,17,22,28,37 } , where soriginal is the actual area of all six spheres together . the scan resolution can be acquired from amide software where each pixel size is 4.6875 4.6875 mm and each slice size is 128 128 pixels . the overall slice area and the ratio between both areas can be calculated according to ( 16 ) , respectively , where sbr is the spheres to background ratio ( 16)aoriginal=1281284.68754.6875=360000 mm2,sbr = soriginalaoriginalsoriginal100%=0.702%.table 4 illustrates the results for a sample data provided from the collaborator for different scanner variables . it can be seen that the sbr percentages varies based on the scanner variables used . to explain the effects of those variables on the output image , figure 18 illustrates the changes in the quality of the segmented image based on the scanners variables . it can be seen from figure 18 that 3d scans produce closer sbr percentages than 2d for all iterations except at it = 1 . it can be noticed that the area evaluating the spheres decreases as the it value increases for both 2d and 3d and for all bed section scanning times . a predefined clinical dataset comprised of 217 slices , with slice thickness of 3.0 mm has been tested on the proposed system . based on the provided report , the patient is affected by multiple bilateral renal cortical cysts ; the largest one is seen in the lower pole of the right kidney , measuring about 47 45 mm . a snapshot taken for a dicom viewer window and the roi has been located by the red lines in three different orientations of the patient 's body scan ( figure 20 ) . it can be seen that roi appears more clearly in its biggest illustration in slice 198 ; this slice is illustrated in figure 21 , and the roi ( kidney cancer ) has been highlighted by red colour . mra have been applied on the medical image to segment it and detect roi . the performance of the proposed techniques for segmenting the illustrated slice in figure 21 is explained in table 5 . the area of the cancer has been measured and compared to the provided report and then used to qualify the performance of each technique as well as mse , data loss , and psnr . the clinical datasets have been segmented also using 3d segmentation techniques , and the lesions were detected accurately . curvelet transform has been used before 3d segmentation to achieve a denoised ct output and ensure smoother edges . patient data which includes lesions in liver , kidney and lung has been segmented and visualized in figures 23 , 24 , and 25 , where the oois are located . due to the changing shapes of organs in medical images , segmentation process using multiresolution analysis combined with thresholding as pre- and postprocessing step allows accurate detection of rois . multiresolution analysis such as wavelet transform is extensively used in medical image segmentation and provides better accuracy in results . curvelet and ridgelet transforms are new extension of the wavelet transform that aims to deal with interesting phenomena occurring along higher dimensional singularities . though wavelets are well suited to point singularities , they have limitations with orientation selectivity hence do not represent changing geometric features along edges effectively . curvelet transform exhibits good reconstruction of the edge data by incorporating a directional component to the traditional wavelet transform . experimental study in this report has shown that curvelet - based segmentation of the medical images not only provide good - quality reconstruction of detected roi , promising results are also achieved in terms of accurately detecting roi and denoising process . curvelet transform is a new tool and utilization of this technique ; it is far from sufficient in the medical image processing area . the future work related to this is the implementation of 3d mra transform which can be applied directly on medical volumes to detect obstacle and objects of interest .

the experimental study presented in this paper is aimed at the development of an automatic image segmentation system for classifying region of interest ( roi ) in medical images which are obtained from different medical scanners such as pet , ct , or mri . multiresolution analysis ( mra ) using wavelet , ridgelet , and curvelet transforms has been used in the proposed segmentation system . it is particularly a challenging task to classify cancers in human organs in scanners output using shape or gray - level information ; organs shape changes throw different slices in medical stack and the gray - level intensity overlap in soft tissues . curvelet transform is a new extension of wavelet and ridgelet transforms which aims to deal with interesting phenomena occurring along curves . curvelet transforms has been tested on medical data sets , and results are compared with those obtained from the other transforms . tests indicate that using curvelet significantly improves the classification of abnormal tissues in the scans and reduce the surrounding noise .

1. Introduction 2. Proposed Medical Image Segmentation System 3. MethodologyMultiresolution Analysis 4. Results and Analysis 5. Conclusion

in the last decade , the use of 3d image processing has been increased especially for medical applications ; this leads to increase the qualified radiologists ' number who navigate , view , analyse , segment , and interpret medical images . the analysis and visualization of the image stack received from the acquisition devices are difficult to evaluate due to the quantity of clinical data and the amount of noise existing in medical images due to the scanners itself . computerized analysis and automated information systems can offer help dealing with the large amounts of data , and new image processing techniques may help to denoise those images . multiresolution analysis ( mra ) [ 13 ] has been successfully used in image processing specially with image segmentation , wavelet - based features has been used in various applications including image compression , denoising , and classification . recently , the finite ridgelet and curvelet transforms have been introduced as a higher dimensional mra tool [ 7 , 8 ] . image segmentation requires extracting specific features from an image by distinguishing objects from the background . the process involves classifying each pixel of an image into a set of distinct classes , where the number of classes is much smaller . medical image segmentation aims to separate known anatomical structures from the background such cancer diagnosis , quantification of tissue volumes , radiotherapy treatment planning , and study of anatomical structures . curvelet transform is a new extension of wavelet transform which aims to deal with interesting phenomena occurring along curved edges in 2d images . it is a high - dimensional generalization of the wavelet transform designed to represent images at different scales and different orientations ( angles ) . it is viewed as a multiscale pyramid with frame elements indexed by location , scale , and orientation parameters with needle - shaped elements at fine scales . curvelets have time - frequency localization properties of wavelets but also shows a very high degree of directionality and anisotropy , and its singularities can be well approximated with very few coefficients . this paper is focusing on a robust implementation of mra techniques for segmenting medical volumes using features derived from the wavelet , ridgelet , and curvelet transforms of medical images obtained from a ct scanner . the rest of this paper is organised as follow : section 2 illustrates the proposed medical image segmentation system using mra techniques . the mathematical background and the methodology for the proposed mra techniques have been explained in section 3 . the results and analysis of the implemented wavelet , ridgelet , and curvelet transforms for medical image segmentation are illustrated in section 4 . the main aim of this research is to facilitate the process of highlighting roi in medical images , which may be encapsulated within other objects or surrounded by noise that make the segmentation process not easy . wavelet , ridgelet , and curvelet transforms are applied on medical images with other pre- and postprocessing techniques to present segmented outputs and detected roi in an easier and more accurate way . image segmentation using mra such as wavelets has been widely used in recent years and provides better accuracy in segmenting different types of images . by decomposing the image into a series of high - pass and low - pass filter bands , the wavelet transform extracts directional details that capture horizontal , vertical , and diagonal activity . however , these three linear directions are limiting and might not capture enough directional information in noisy images , such as medical ct scans , which do not have strong horizontal , vertical , or diagonal directional elements . ridgelet improves mra segmentation ; however , they capture structural information of an image based on multiple radial directions in the frequency domain . one limitation to use ridgelet in image segmentation is that ridgelet is most effective in detecting linear radial structures , which are not dominant in medical images . the curvelet transform is a recent extension of ridgelet transform that overcome ridgelet weaknesses in medical image segmentation . curvelet is proven to be particularly effective at detecting image activity along curves instead of radial directions which are the most comprising objects of medical images . in the last decade , wavelet transform has been recognized as a powerful tool in a wide range of applications , including image / video processing , numerical analysis , and telecommunication . in addition to this , functions with discontinuities and functions with sharp spikes require fewer wavelet basis vectors in the wavelet domain than sine cosine basis vectors to achieve a comparable approximation . in standard wavelet decomposition , the output from the low - pass filter can be then decomposed further , with the process continuing recursively in this manner . the wavelet decomposition of an image creates at each scale j a set of coefficient values wj with an overall mean of zero . wj contains the same number of voxels as the original image ; therefore , this wavelet transform is redundant [ 14 , 15 ] . ridgelet transform [ 1719 ] has been generating a lot of interest due to their superior performance over wavelets . these shortcomings of wavelet are well addressed by the ridgelet transform ; the functionality of wavelet has been extended to higher dimensional singularities and becomes an effective tool to perform sparse directional analysis [ 3 , 21 ] . its computation is important in image processing and computer vision for problems such as pattern recognition and the reconstruction of medical images . according to alzu'bi and amira in , once the wavelet and radon transforms have been implemented , the ridgelet transform is straightforward . as shown in figure 4 , ridgelets use frat as a basic building block , where frat maps a line singularity into point singularity , and the wavelet transform has used to effectively detect and segment the point singularity in radon domain . continuous ridgelet transform is similar to the continuous wavelet transform except that point parameters ( x , y ) in the cartesian grid ( figure 6(a ) ) which perform pixels in the image or an entry in a 2d matrix are now replaced by line parameters ( , ) , where is the intercept and is the angle . figure 6(b ) illustrates the radial grid in ridgelet transform ; however , straight lines evaluate the image in the frequency domain . medical images comprised from curves which are still not singularity points after applying radon transform . curvelet transform has been introduced to solve this problem ; it deals with higher singularities compared to wavelet and ridgelet transforms . in this new method , the use of the ridgelet transform as a preprocessing step of curvelet was discarded , thus reducing the amount of redundancy in the transform and increasing the speed considerably . curvelet aims to deal with interesting phenomena occurring along curved edges in a 2d image . as illustrated in figure 7 , curvelet needs fewer coefficients for representation , and the edge produced from curvelet is smoother than wavelet edge . the newly constructed and improved version of curvelet transform is known as fast discrete curvelet transform ( fdct ) . this new technique is simpler , faster and less redundant than the original curvelet transform which based on ridgelets . wrapping - based transform is based on wrapping a specially selected fourier samples , and it is easier to implement and understand . curvelet transform works in two dimensions with spatial variable x , frequency domain variable , and the frequency - domain polar coordinates r and . curvelet transform can be defined by a pair of windows , radial window { w(r ) } , and angular window { v(t ) } . figure 8 illustrates the induced tiling of the frequency plane and the spatial cartesian grid associated with a given scale and orientation , and shaded area represents the polar wedge by uj(6)j , l , k(x)=j(rl(xxk(j , l ) ) ) . the new implementation of curvelet transform based on wrapping of fourier samples takes a 2d image as an input in the form of a cartesian array f[m , n ] , where 0 m < m , 0 n < n where m and n are the dimensions of the array . as mentioned earlier , wrapping based curvelet transform is a multiscale pyramid which consists of several subbands at different scales consisting of different orientations and positions in the frequency domain . it can be seen from figure 9 that curvelet becomes finer and smaller in the spatial domain and shows more sensitivity to curved edges as the resolution level is increased , thus allowing to effectively capturing the curves in an image , and curved singularities can be well - approximated with fewer coefficients . for each scale and orientation , a product of ujl wedge is obtained ; the result is then wrapped around the origin , and 2d ifft is then applied resulting in discrete curvelet coefficients . describe the discrete curvelet transform in as illustrated in ( 8)curvelet transform = ifft[fft(curvelet)fft(image ) ] . figure 10 illustrates the process of wrapping wedge where the angle is in the range ( /4 , 3/4 ) and the rectangles have the same width and length as the parallelogram is centred at the origin . wrap this product around the origin and obtain ( 11)fj , l[m , n]=w(uj , lf^)[m , n ] , where the range for m , n , and is now 0 m < 2 , 0 n < 2 , and /4 step 4apply ifft to each fj , l , hence collecting the discrete coefficients c(j , l , k1k2 ) . the curvelet transform is a multiscale transform such as wavelet , with frame elements indexed by scale and location parameters . the elements obey a special scaling law , where the length and the width of frame elements support are linked using ( 12)widthlength2 discrete curvelet transform in the spectral domain utilizes the advantages of fft . at the end of this computation process , a set of curvelet coefficients are obtained by applying ifft to the spectral product . such representations are nearly as sparse as if the object were not singular and turn out to be far sparser than other transforms decomposition of the object . for example , an application of the phase - space localization of the curvelet transform allows a very precise description of those features of the object of function ( f ) which can be reconstructed accurately from such data and how well , and of those features which can not be recovered . for example , an application of the phase - space localization of the curvelet transform allows a very precise description of those features of the object of function ( f ) which can be reconstructed accurately from such data and how well , and of those features which can not be recovered . there is indeed a quantitative theory showing that for some statistical models which allow for discontinuities in the object to be recovered , there are simple algorithms based on the shrinkage of curvelet biorthogonal decompositions , which achieve optimal statistical rates of convergence . in figure 12 , the low frequency coefficients ( coarse scale ) the concentric coronae ( formed by black strips ) show coefficients at different scales and the outer coronae correspond to higher frequencies . wedge wrapping is done for all the wedges at each scale in the frequency domain to obtain a set of subbands or wedges at each curvelet decomposition level , and these subbands are the collection of discrete curvelet coefficients . the discrete curvelet transform can be calculated to various resolutions or scales and angles ; the maximum number of resolution depends on the original image size and the angles . figure 13 illustrates how curvelet - based edge reconstruction in medical imaging differs from other transforms methods . the end users of the proposed system are the radiologists and specialists who analyse medical images for cancer diagnosis . after several meetings with those people in the radiology departments in some hospitals , the main goal that they are working is to detect the accurate cancer size in medical images with the least error . the first one is nema iec body phantom which consists of an elliptical water filled cavity with six spherical inserts suspended by plastic rods of inner diameters : 10 , 13 , 17 , 22 , 28 , and 37 mm [ 25 , 26 ] . real clinical human images acquired by a ct scanner have also been used to experiment the proposed approaches , this data has been previously analysed by the radiologists and the provided reports explains that the patients are diagnosed by cancer . it can be seen from table 1 that small values of snr have been obtained for all techniques ; this is due to the noise from the acquisition systems itself . this can be assigned to the major limitation of using ridgelet transformation in medical image segmentation , where ridges rarely exist in such data . thresholding technique has been applied as a preprocessing step on the original images at threshold value ( t = 35 ) to remove as much artificial spam sequel produced from the scanners . the transform then applied to effectively represent objects with edges which are the contours of the medical images followed by another thresholding at ( t = 7 ) to remove most of the remaining noise and facilitate the measurement process . figures 14(a ) and 14(c ) illustrate the original images from a ct scanner , and figures 14(b ) and 14(d ) illustrate the segmented phantom image and real chest image , respectively , using curvelet transform . as illustrated in figure 15 , results of the proposed segmentation technique are vary in terms of smooth reconstruction of the spheres . curvelet transform segments the input image and removes artifacts from the image to exhibit smooth and optimal segmentation of nema phantom . table 2 illustrates nema spheres diameters error percentages measured using different multiresolution analysis techniques and compared to previously implemented techniques . ed has been used to measure the spheres diameters and calculate the error percentages for each technique , and sphere diameter error percentages have been calculated as follows : ( 14)error % = measured diameteractual diameteractual diameter100% . three decomposition levels of dwt have been applied on nema phantom [ 25 , 26 ] using two different filters ( haar , daubechies ) , and the measured diameters were doubled at each level to produce a fair comparison with the other available techniques . it can be seen that most of the error percentages were decreasing while the spheres diameter increasing ; it is worth mentioning that there is no upper bound of the spheres diameters to keep the errors decreasing , because the roi becomes clearer and easier to be detected and measured properly . but tumours in real life are usually very small in the early stage cancer , and the problem is to detect those turnouts ' tumours as soon as possible . it can be also seen from table 2 that acceptable error percentages have been achieved using ridgelet transform for the big spheres ( 22 mm , 28 mm , and 37 mm ) , where the curves are not sharp and ridgelet detect it accurately as it close to be ridges . but for the small spheres , ridgelet weakness for medical image segmentation start appears clearly . curvelet transform overcomes the weakness of wavelet for segmenting sharp curves and detect the small spheres accurately with error percentages ( 0.82%2.65% ) . for the big spheres , errors achieved using wavelet transform are still better than those achieved using curvelet transform due to the sharpness of that spheres . but still very good results using curvelet transform and acceptable for clinical applications . table 3 illustrates a comparison study of curvelet transform with the other traditional transforms , and comparison terms psnr and mse have been used to test the quality of the transformed image . from table 3 , it can be seen that the best results according to both psnr and mse have been achieved using curvelet transform . figure 17 illustrates two noisy images and the denoised outputs using both wavelet and curvelet . this is not surprising given the fact that the ridgelet transform is able to capture multidirectional features , as opposed to the wavelet transform which focus mainly on horizontal , vertical , and diagonal features . this can be generalized to most of the images except for medical scanners , where the weakness of wavelet is not dominant in such images . curvelet - based descriptors had an even higher performance in comparison to both the wavelet and ridgelet , with accuracy rates higher , respectively . the accuracy rate using curvelet transform is better ; this is expected , since the curvelet transform is able to capture multidirectional features in wedges , as opposed to lines or points as in the ridgelet or wavelet transform . as illustrated in the previous tables and figures , it can be seen that more efficient and smooth image reconstruction is achieved using curvelet transform . in terms of optimal reconstruction of the objects with edges and curves , the algorithm presented in this chapter is able to classify normal tissues in ct scans with high accuracy rates . segmentation using curvelet transform has been chosen for experimenting the pet scanner sensitivity variables , curvelet was applied in parallel with multithresholding and classification techniques to classify the spheres in a separate class from the other comprising objects at least noise included . the experiment was evaluated based on the ratio between the spheres area to the other area of the scanned slice . the overall slice area and the ratio between both areas can be calculated according to ( 16 ) , respectively , where sbr is the spheres to background ratio ( 16)aoriginal=1281284.68754.6875=360000 mm2,sbr = soriginalaoriginalsoriginal100%=0.702%.table 4 illustrates the results for a sample data provided from the collaborator for different scanner variables . to explain the effects of those variables on the output image , figure 18 illustrates the changes in the quality of the segmented image based on the scanners variables . a predefined clinical dataset comprised of 217 slices , with slice thickness of 3.0 mm has been tested on the proposed system . a snapshot taken for a dicom viewer window and the roi has been located by the red lines in three different orientations of the patient 's body scan ( figure 20 ) . it can be seen that roi appears more clearly in its biggest illustration in slice 198 ; this slice is illustrated in figure 21 , and the roi ( kidney cancer ) has been highlighted by red colour . the area of the cancer has been measured and compared to the provided report and then used to qualify the performance of each technique as well as mse , data loss , and psnr . the clinical datasets have been segmented also using 3d segmentation techniques , and the lesions were detected accurately . curvelet transform has been used before 3d segmentation to achieve a denoised ct output and ensure smoother edges . patient data which includes lesions in liver , kidney and lung has been segmented and visualized in figures 23 , 24 , and 25 , where the oois are located . due to the changing shapes of organs in medical images , segmentation process using multiresolution analysis combined with thresholding as pre- and postprocessing step allows accurate detection of rois . multiresolution analysis such as wavelet transform is extensively used in medical image segmentation and provides better accuracy in results . curvelet and ridgelet transforms are new extension of the wavelet transform that aims to deal with interesting phenomena occurring along higher dimensional singularities . experimental study in this report has shown that curvelet - based segmentation of the medical images not only provide good - quality reconstruction of detected roi , promising results are also achieved in terms of accurately detecting roi and denoising process . curvelet transform is a new tool and utilization of this technique ; it is far from sufficient in the medical image processing area . the future work related to this is the implementation of 3d mra transform which can be applied directly on medical volumes to detect obstacle and objects of interest .

it is usually used to treat cervical spondylotic myelopathy , ossification of the posterior longitudinal ligament , and cervical disk herniation associated with developmental spinal canal stenosis13 ) . open - door laminoplasty is one of the most popular surgical methods that provides enough decompression of the spinal canal over multiple segments and reconstructs the stable laminar arch with sufficient room for the spinal cord27121517 ) . the basic principle of this technique is elastic deformation of thinned lamina , which allows complete healing . when the procedure was relatively new , hirabayashi used a wire for lamina fixation ; however , open lamina reclosure or fracture was a problem23 ) . o'brien et al.8 ) modified open - door type laminoplasty by augmenting the procedure with titanium plates to maintain patency of the deformed lamina . these so - called hinge fractures ( hfs ) could happen intra - operatively while manipulating the hinge but also post - operatively in recovery stage , which are defined as delayed hfs10 ) . however , only few surgeons have investigated about hfs because the lamina is well fused in the majority of cases , and most surgeons believe that hfs do not exert much influence over clinical outcomes . some recent studies have indicated that hfs can cause axial pain or palsy that may be chronic21014 ) . our previous study showed that patients with 3 or more hfs experienced significant worsening of visual analog scales for neck ( vasn ) and neck disability index ( ndi ) compared with patients with 2 hfs or less in multi - level open door laminoplasty cases10 ) . nevertheless , risk factors related to this complication remain unclear . therefore , we radiographically evaluated hinge status and determined risk factors for delayed hfs following plate - only open - door cervical laminoplasty . from april 2005 to january 2012 , 119 patients with cervical myelopathy underwent open - door laminoplasty in our hospitals . among them , 80 patients who had postoperative computed tomography ( ct ) scans within 2 years were enrolled in this study . all of the 80 patients suffered from myelopathic symptoms , such as motor weakness , sensory disturbance , or voiding difficulties . there were 45 patients with spondylotic myelopathy , 32 with symptomatic ossification of the posterior longitudinal ligament , and 3 with developmental stenosis . the mean age of the patients was 58 years ( range : 3278 years ) . the male : female ratio was 49 : 31 . among the patients , 17 were smokers . all surgeries were performed by a senior surgeon who used a modification of the classic hirabayashi open - door technique , securing the opened lamina with a titanium miniplate ( medtronic , memphis , tn , usa)23 ) . open side and hinge side gutters were made using a cutting burr and a kerrison rongeur , respectively . a green - stick deformation of the hinge was produced and laminar elevation and miniplate fixation were performed . one 7 mm screw was placed into the lateral mass through the plate and one 5 mm screw was placed through the plate into the cut lamina in 65 patients . in 16 patients , ct scanning was performed with a 16-multidetector ct ( mdct ) scanner ( sensation ; siemens , erlangen , germany ) . images were reconstructed using 2-mm - thick sections with no intervals and were optimized for evaluation of bony structures ( width , 1500 ; center , 450 ) . postoperative axial ct scans were independently evaluated by three different reviewers ( three neurosurgeons ) . the initial status of the hinge gutter and delayed hfs were evaluated as described previously ( table 2)10 ) . briefly , hinges were defined as deformed if the ventral cortices were bent but not broken ( non - hf ) . the number of slices with a broken or disconnected inner cortex was counted among six or seven thin slices of ct scans obtained from a single lamina ( dx ) which indicates poor hinge status as disconnected number increases . if all of the inner cortices were broken , it was defined as fractured , and if the inner cortices had moved inside , it was defined as migrated and these are all considered as hfs . in later observations , primary union ( pu ) was considered as non - hf . contrastively , nonunion , callus formation , and secondary union ( su ) which demonstrates healing process occurred after fracture were considered as hf . the healing status of each lamina was determined by consensus of two or more of the three reviewers . 1 shows radiographic measurements of hinge - side gutter and open - side gutter location , open angle , and hinge width . kappa coefficients were used to check the reliability of initial and delayed hinge status assessments among observers . continuous variables ( age , radiographic measurements , number of screws ) are shown as meansstandard deviations , whereas categorical variables ( hinge status , surgical level , sex , and smoking ) are expressed as ratios or percentages . pearson 's correlation was used for continuous variables and spearman 's correlation was used for categorical variables . to identify variables independently related to hfs , all tests were two - sided , and a value of p<0.05 was considered statistically significant . all analysis were performed with spss software ( version 20.0 ; spss , chicago , il , usa ) . all surgeries were performed by a senior surgeon who used a modification of the classic hirabayashi open - door technique , securing the opened lamina with a titanium miniplate ( medtronic , memphis , tn , usa)23 ) . open side and hinge side gutters were made using a cutting burr and a kerrison rongeur , respectively . a green - stick deformation of the hinge was produced and laminar elevation and miniplate fixation were performed . one 7 mm screw was placed into the lateral mass through the plate and one 5 mm screw was placed through the plate into the cut lamina in 65 patients . in 16 patients , ct scanning was performed with a 16-multidetector ct ( mdct ) scanner ( sensation ; siemens , erlangen , germany ) . images were reconstructed using 2-mm - thick sections with no intervals and were optimized for evaluation of bony structures ( width , 1500 ; center , 450 ) . postoperative axial ct scans were independently evaluated by three different reviewers ( three neurosurgeons ) . the initial status of the hinge gutter and delayed hfs were evaluated as described previously ( table 2)10 ) . briefly , hinges were defined as deformed if the ventral cortices were bent but not broken ( non - hf ) . the number of slices with a broken or disconnected inner cortex was counted among six or seven thin slices of ct scans obtained from a single lamina ( dx ) which indicates poor hinge status as disconnected number increases . if all of the inner cortices were broken , it was defined as fractured , and if the inner cortices had moved inside , it was defined as migrated and these are all considered as hfs . in later observations , primary union ( pu ) was considered as non - hf . contrastively , nonunion , callus formation , and secondary union ( su ) which demonstrates healing process occurred after fracture were considered as hf . the healing status of each lamina was determined by consensus of two or more of the three reviewers . 1 shows radiographic measurements of hinge - side gutter and open - side gutter location , open angle , and hinge width . kappa coefficients were used to check the reliability of initial and delayed hinge status assessments among observers . continuous variables ( age , radiographic measurements , number of screws ) are shown as meansstandard deviations , whereas categorical variables ( hinge status , surgical level , sex , and smoking ) are expressed as ratios or percentages . pearson 's correlation was used for continuous variables and spearman 's correlation was used for categorical variables . to identify variables independently related to hfs , all tests were two - sided , and a value of p<0.05 was considered statistically significant . all analysis were performed with spss software ( version 20.0 ; spss , chicago , il , usa ) . a total of 114 ct scans from 80 patients were available for the assessment of hinge status : 38 patients at 1 week after surgery , and 76 patients at 4 months after surgery or later up until 2 years . the mean number of operated levels was 4.5 ( range : 37 ) and the mean number of laminoplasties was 3.3 ( range : 25 ) . the cts from 80 patients and a total of 270 laminae levels were evaluated for radiographic measurements . the mean hinge open angle was 304 degrees ( range 2139 degrees ) , which was a narrow range . the average hinge and open locations were 1.50.9 mm ( range 04.1 mm ) and 0.50.7 mm ( range 03.5 mm ) , respectively . initial hinge gutter status ( dx ) was evaluated with cts taken from 38 patients 1 week after surgery , and 10% ( 12 of 115 ) of hinges were found to be in danger of fracture or overtly fractured ( d6 or worse ) ( table 3 ) . however , cts taken at 4 months or later postoperatively ( 76 patients ) showed an increased number of hfs ( 117 of 254 , 46% ) which indicates that majority of hfs occurred at delayed period rather than perioperative period . the ct scans of 34 patients , who had both 1-week and later observations , were evaluated to verify the relationship between the independent variables including initial hinge status , open angle , operated level , number of screws used , and hinge - location . initial hinge status and the open angle , operated level , number of screws used showed high correlation ( p<0.05 ) . univariate and multivariate logistic regression analysis were both used to identify hf risk factors among several variables within 80 patients ( 38 patients for initial hinge status ) . hinge width , sex , age , and smoking were statistically nonsignificant ( p>0.05 ) . poor initial hinge status [ odds ratio ( or)=2.025 , 95% ci=1.5252.689 ] , medially placed hinge gutter ( or=1.807 , 95% ci=1.2852.542 ) , double screw fixation on the elevated lamina ( or=3.096 , 95% ci=1.5966.005 ) , upper surgical level ( or=0.633 , 95% ci=0.4710.849 ) , and wide open angle ( or=1.107 , 95% ci=1.0231.198 ) were identified as predictors of hfs by univariate logistic analysis ( p<0.05 ) . among them , the multivariate logistic regression showed that poor initial hinge status ( or=1.982 , 95% ci=1.4132.780 ) , medially placed hinge gutter ( or=2.376 , 95% ci=1.2714.440 ) , and surgical level ( or=0.367 , 95% ci=0.2040.662 ) were risk factors for hfs after adjustment for other confounding factors , but double screw fixation was excluded . however , because the multivariate analysis results seemed to be biased due to colinearity between initial status and other variables ( table 4 ) , additional multivariate analysis without initial hinge status was performed . medially placed hinge gutter ( or=1.85 , 95% ci=1.2862.698 ) , double screw fixation on the elevated lamina ( or=3.177 , 95% ci=1.5286.605 ) , upper surgical level ( or=0.555 , 95% ci=0.3990.772 ) , and wide open angle ( or=1.085 , 95% ci=0.9961.182 ) had very similar ors to univariate logistic regression analysis and was statistically meaningful ( p<0.05 ) except open angle ( p=0.063 ) . overall results are shown in table 5 . the kappa coefficients for hinge status at each observation ( 1 week , the cts from 80 patients and a total of 270 laminae levels were evaluated for radiographic measurements . the mean hinge open angle was 304 degrees ( range 2139 degrees ) , which was a narrow range . the average hinge and open locations were 1.50.9 mm ( range 04.1 mm ) and 0.50.7 mm ( range 03.5 mm ) , respectively . initial hinge gutter status ( dx ) was evaluated with cts taken from 38 patients 1 week after surgery , and 10% ( 12 of 115 ) of hinges were found to be in danger of fracture or overtly fractured ( d6 or worse ) ( table 3 ) . however , cts taken at 4 months or later postoperatively ( 76 patients ) showed an increased number of hfs ( 117 of 254 , 46% ) which indicates that majority of hfs occurred at delayed period rather than perioperative period . the ct scans of 34 patients , who had both 1-week and later observations , were evaluated to verify the relationship between the independent variables including initial hinge status , open angle , operated level , number of screws used , and hinge - location . initial hinge status and the open angle , operated level , number of screws used showed high correlation ( p<0.05 ) . univariate and multivariate logistic regression analysis were both used to identify hf risk factors among several variables within 80 patients ( 38 patients for initial hinge status ) . hinge width , sex , age , and smoking were statistically nonsignificant ( p>0.05 ) . poor initial hinge status [ odds ratio ( or)=2.025 , 95% ci=1.5252.689 ] , medially placed hinge gutter ( or=1.807 , 95% ci=1.2852.542 ) , double screw fixation on the elevated lamina ( or=3.096 , 95% ci=1.5966.005 ) , upper surgical level ( or=0.633 , 95% ci=0.4710.849 ) , and wide open angle ( or=1.107 , 95% ci=1.0231.198 ) were identified as predictors of hfs by univariate logistic analysis ( p<0.05 ) . among them , the multivariate logistic regression showed that poor initial hinge status ( or=1.982 , 95% ci=1.4132.780 ) , medially placed hinge gutter ( or=2.376 , 95% ci=1.2714.440 ) , and surgical level ( or=0.367 , 95% ci=0.2040.662 ) were risk factors for hfs after adjustment for other confounding factors , but double screw fixation was excluded . however , because the multivariate analysis results seemed to be biased due to colinearity between initial status and other variables ( table 4 ) , additional multivariate analysis without initial hinge status was performed . medially placed hinge gutter ( or=1.85 , 95% ci=1.2862.698 ) , double screw fixation on the elevated lamina ( or=3.177 , 95% ci=1.5286.605 ) , upper surgical level ( or=0.555 , 95% ci=0.3990.772 ) , and wide open angle ( or=1.085 , 95% ci=0.9961.182 ) had very similar ors to univariate logistic regression analysis and was statistically meaningful ( p<0.05 ) except open angle ( p=0.063 ) . the kappa coefficients for hinge status at each observation ( 1 week , 4 months or later ) were 0.61 and 0.78 , respectively . laminoplasty typically achieves good operative results . however , there are still some postoperative complications despite many preventative efforts . c5 palsy is a representative complication of laminoplasty , with an average occurrence of 4.6% ( range 030% ) patients after laminoplasty13 ) . many studies have attempted to determine the pathologic mechanism of c5 palsy , which is still unclear . satomi et al.15 ) reviewed postoperative ct scans of patients with weakness on the hinge side , which implicated the lamina dropping into the spinal canal as the cause of weakness in 4 patients among 16 . this suggests that the downward displacement of the elevated lamina into the spinal canal might injure the nerve root131415 ) . since hosono et al.4 ) first reported axial symptoms in 1996 , there have been many clinical reports of persistent axial neck and shoulder pain after laminoplasty9 ) . such pain is currently one of the most well - known complications of cervical laminoplasty . possible sources of neck pain include dissected nuchal muscles , destroyed zygapophysial joints , and injured nerve roots24 ) . recently , some authors have postulated that hf may be a source of postoperative pain21014 ) . hosono et al.4 ) suggested that the gutter on the hinge side causes shoulder pain until bony union is completed in this area . tsuji16 ) reported that hinge gutter shows radiologic union within 1 year and postoperative shoulder pain also subsides within this period . our previous study also showed that multiple fractures of laminae can aggravate postoperative vasn and ndi.10 ) as hf may increase c5 palsy and axial pain occurrence , hf prevention could be important for favorable post - operative outcome . however , to the best of our knowledge , there are no existing studies that determined hf risk factors ; this study is the first attempt . in the present study , multivariate logistic regression analysis indicated that poor initial hinge status , medially located hinge gutter , and upper cervical level are risk factors of hfs after laminoplasty . double screws used for a single lamina are also thought to be a risk factor following interpretation of the multivariate logistic regression analysis excluding initial hinge status . the prevalence of delayed hf was highly correlated with initial hinge status . as the count of disconnected inner cortex ct slices increased ( i.e. , d6 or worse ) , occurrence rate of delayed hf increased simultaneously . the variation in univariate and multivariate logistic regression or was very small ( 2.025 vs. 1.982 ) . consequently , as a confounding factor on multivariate logistic regression , initial hinge status could distort the results of other independent factors . furthermore , poor initial hinge status is highly correlated with other independent factors , thus the real correlation of other factors with hf could be devalued . therefore , only hinge location , double screws , level , and open angle were sorted out as independent variables and additionally analyzed using multivariate logistic regression . this also has additional meaning to exclude the incomplete data , because initial hinge status data were collected from 38 patients instead of 80 . hinge gutter location has also been proposed to be an important variable2 ) . in this study , medially placed hinge gutters had a high or and significant p - value in both univariate and multivariate logistic regression analyses . specifically , comparing univariate logistic regression to multivariate logistic regression without initial status , or ( 1.807 vs. 1.85 ) was very similar and both p - values ( 0.001 , 0.002 ) were also highly statistically significant . first , the medial lamina is thinner than the lateral lamina and a thin gutter would be fragile . second , even with the same length of elevation , open angle increases with medial gutter placement . however , it is difficult to say if a more lateral hinge gutter always results in a good outcome . because a lateral hinge gutter is a possible risk factor for c5 palsy and axial pain , some clinicians insist that the outcome of making a medial hinge gutter is better than making a lateral one6 ) . although there were no significant differences between single screw and double screw use in initial multivariate logistic regression analysis ( with initial hinge status ) , it seems to be a predictor of hf . first , because of the formulaic technique , constant open angle was a prerequisite between two groups ( 304 in single , 293 in double ) . however , there was a significant difference in the initial status average between the two groups ( p=0.0163 , mann - whitney u test ; median = d0 in single , d2 in double screw ) . this revealed that the use of two screws to fill in the lamina may worsen its status . it may be that when a screw is tightened in a clockwise rotatory motion , the hinge 's unidirectional shear stress might increase . therefore , second multivariate analysis ( without initial hinge status ) seemed to be more reliable . 2 shows representative cases . comparing c3 & c4 with c5 , or c5 & c6 and c7 , the upper level had a high or compared to the adjacent lower level . the remote level ( c3 & c4 and c6 & c7 ) showed a more significant difference . we hypothesize that it could be because of anatomic predisposition that the upper lamina is usually thinner than the lower . in addition , when elevation has been accomplished , the reformed lamina moves backward and becomes twisted slightly cranially so that its rostral end becomes closer to the upper intact lamina . on the contrary , the lower end lamina moves backward and away from the intact lamina below . hence , the lower end laminae obtain more room to move freely . thus , on neck extension , the upper lamina is more likely to contact the intact lamina10 ) . rhee et al.11 ) claimed that c6 and c7 tended to have high union rates and healing speed compared to c4 and c5 . univariate logistic regression analysis indicated wide open angle to be a predictor of hf but multivariate analysis did not . our surgical technique for hinge opening was standardized , thus the open angle varied within a narrow range ( 304 degrees ) . alternatively , open angle and initial hinge status may essentially be correlated with each other . using a load deformation curve ( young modulus ) as reference , if open angle becomes larger , it will pass by the elastic range and plastic range ( deformation ) , and mechanical failure could occur . in this case , hinge status could be poor at the first week postoperative ct evaluation . therefore , open angle and hinge status may be colinear , which could distort the multivariate analysis results . in fact , initial hinge status and open angle were positively correlated ( r statistic=0.25 , p=0.01 ) , as determined by 115 hinges . multivariate analysis without initial hinge status had a better p - value ( 0.063 vs. 0.171 ) , but it was still greater than 0.05 . combining two hypotheses more force should be exerted on a narrow hinge within an equivalent area ; thus , we expected that narrow hinge width would be a risk factor for hf , but we did not find a significant correlation between the two . age was not significantly different in either univariate or multivariate logistic regression analysis . for sex , although it was statistically nonsignificant ( p=0.146 ) , females ( f : m , or=0.639 ) had a trend of fewer hfs . satomi et al.14 ) recommended using careful surgical technique to avoid hf , such as making the hinge gutter more lateral than on the open side and detaching the open side before drilling the hinge side , with frequent hinge stability checks during elevation by pushing the spinous process . kawaguchi et al.5 ) suggested a 3 mm diameter drill and an acorn - type bone - cutting bit for making the gutter . in addition , we suggest meticulous screwing on the hinge side and avoiding an unnecessary wide open angle so as not to deform the hinge . laminoplasty typically achieves good operative results . however , there are still some postoperative complications despite many preventative efforts . c5 palsy is a representative complication of laminoplasty , with an average occurrence of 4.6% ( range 030% ) patients after laminoplasty13 ) . many studies have attempted to determine the pathologic mechanism of c5 palsy , which is still unclear . satomi et al.15 ) reviewed postoperative ct scans of patients with weakness on the hinge side , which implicated the lamina dropping into the spinal canal as the cause of weakness in 4 patients among 16 . this suggests that the downward displacement of the elevated lamina into the spinal canal might injure the nerve root131415 ) . since hosono et al.4 ) first reported axial symptoms in 1996 , there have been many clinical reports of persistent axial neck and shoulder pain after laminoplasty9 ) . such pain is currently one of the most well - known complications of cervical laminoplasty . possible sources of neck pain include dissected nuchal muscles , destroyed zygapophysial joints , and injured nerve roots24 ) . recently , some authors have postulated that hf may be a source of postoperative pain21014 ) . hosono et al.4 ) suggested that the gutter on the hinge side causes shoulder pain until bony union is completed in this area . tsuji16 ) reported that hinge gutter shows radiologic union within 1 year and postoperative shoulder pain also subsides within this period . our previous study also showed that multiple fractures of laminae can aggravate postoperative vasn and ndi.10 ) as hf may increase c5 palsy and axial pain occurrence , hf prevention could be important for favorable post - operative outcome . however , to the best of our knowledge , there are no existing studies that determined hf risk factors ; this study is the first attempt . in the present study , multivariate logistic regression analysis indicated that poor initial hinge status , medially located hinge gutter , and upper cervical level are risk factors of hfs after laminoplasty . double screws used for a single lamina are also thought to be a risk factor following interpretation of the multivariate logistic regression analysis excluding initial hinge status . the prevalence of delayed hf was highly correlated with initial hinge status . as the count of disconnected inner cortex ct slices increased ( i.e. , d6 or worse ) , occurrence rate of delayed hf increased simultaneously . the variation in univariate and multivariate logistic regression or was very small ( 2.025 vs. 1.982 ) . consequently , as a confounding factor on multivariate logistic regression , initial hinge status could distort the results of other independent factors . furthermore , poor initial hinge status is highly correlated with other independent factors , thus the real correlation of other factors with hf could be devalued . therefore , only hinge location , double screws , level , and open angle were sorted out as independent variables and additionally analyzed using multivariate logistic regression . this also has additional meaning to exclude the incomplete data , because initial hinge status data were collected from 38 patients instead of 80 . hinge gutter location has also been proposed to be an important variable2 ) . in this study , medially placed hinge gutters had a high or and significant p - value in both univariate and multivariate logistic regression analyses . specifically , comparing univariate logistic regression to multivariate logistic regression without initial status , or ( 1.807 vs. 1.85 ) was very similar and both p - values ( 0.001 , 0.002 ) were also highly statistically significant . first , the medial lamina is thinner than the lateral lamina and a thin gutter would be fragile . second , even with the same length of elevation , open angle increases with medial gutter placement . however , it is difficult to say if a more lateral hinge gutter always results in a good outcome . because a lateral hinge gutter is a possible risk factor for c5 palsy and axial pain , some clinicians insist that the outcome of making a medial hinge gutter is better than making a lateral one6 ) . although there were no significant differences between single screw and double screw use in initial multivariate logistic regression analysis ( with initial hinge status ) , it seems to be a predictor of hf . first , because of the formulaic technique , constant open angle was a prerequisite between two groups ( 304 in single , 293 in double ) . however , there was a significant difference in the initial status average between the two groups ( p=0.0163 , mann - whitney u test ; median = d0 in single , d2 in double screw ) . this revealed that the use of two screws to fill in the lamina may worsen its status . it may be that when a screw is tightened in a clockwise rotatory motion , the hinge 's unidirectional shear stress might increase . therefore , second multivariate analysis ( without initial hinge status ) seemed to be more reliable . 2 shows representative cases . comparing c3 & c4 with c5 , or c5 & c6 and c7 , the upper level had a high or compared to the adjacent lower level . the remote level ( c3 & c4 and c6 & c7 ) showed a more significant difference . we hypothesize that it could be because of anatomic predisposition that the upper lamina is usually thinner than the lower . in addition , when elevation has been accomplished , the reformed lamina moves backward and becomes twisted slightly cranially so that its rostral end becomes closer to the upper intact lamina . for this reason , undercutting of the cranial lamina is usually needed . on the contrary , the lower end lamina moves backward and away from the intact lamina below . thus , on neck extension , the upper lamina is more likely to contact the intact lamina10 ) . rhee et al.11 ) claimed that c6 and c7 tended to have high union rates and healing speed compared to c4 and c5 . univariate logistic regression analysis indicated wide open angle to be a predictor of hf but multivariate analysis did not . our surgical technique for hinge opening was standardized , thus the open angle varied within a narrow range ( 304 degrees ) . alternatively , open angle and initial hinge status may essentially be correlated with each other . using a load deformation curve ( young modulus ) as reference , if open angle becomes larger , it will pass by the elastic range and plastic range ( deformation ) , and mechanical failure could occur . in this case therefore , open angle and hinge status may be colinear , which could distort the multivariate analysis results . in fact , initial hinge status and open angle were positively correlated ( r statistic=0.25 , p=0.01 ) , as determined by 115 hinges . multivariate analysis without initial hinge status had a better p - value ( 0.063 vs. 0.171 ) , but it was still greater than 0.05 . combining two hypotheses more force should be exerted on a narrow hinge within an equivalent area ; thus , we expected that narrow hinge width would be a risk factor for hf , but we did not find a significant correlation between the two . age was not significantly different in either univariate or multivariate logistic regression analysis . for sex , although it was statistically nonsignificant ( p=0.146 ) , females ( f : m , or=0.639 ) had a trend of fewer hfs . satomi et al.14 ) recommended using careful surgical technique to avoid hf , such as making the hinge gutter more lateral than on the open side and detaching the open side before drilling the hinge side , with frequent hinge stability checks during elevation by pushing the spinous process . kawaguchi et al.5 ) suggested a 3 mm diameter drill and an acorn - type bone - cutting bit for making the gutter . in addition , we suggest meticulous screwing on the hinge side and avoiding an unnecessary wide open angle so as not to deform the hinge . we determined the risk factors for delayed hf following plate - only open - door laminoplasty , which could be a source of axial pain or c5 palsy . poor initial hinge status , medially placed hinge gutter , double screwed lamina , and upper cervical level seemed to be firm risk factors .

objectivedelayed hinge fracture ( hf ) that develops after cervical open door laminoplasty can be a source of postoperative complications such as axial pain . however , risk factors related to this complication remain unclear . we performed a retrospective clinical series to determine risk factors for delayed hf following plate - only open - door cervical laminoplasty.methodspatients who underwent plate - only open - door laminoplasty and had available postoperative computed tomography ( ct ) scans ( 80 patients with 270 laminae ) were enrolled . hinge status , hinge gutter location , open location , hinge width , number of screws used , operation level , and open angle were observed in the ct to determine radiographic outcome . demographic data were collected as well . radiographic and clinical parameters were analyzed using univariate and multivariate logistic regression analysis to determine the risk factors for hf.resultsunivariate logistic regression analysis results indicated poor initial hinge status , medially placed hinge gutter , double screw fixation on the elevated lamina , upper surgical level , and wide open angle as predictors for hf ( p<0.05 ) . initial hinge status seemed to be the most powerful risk factor for hf ( p=0.000 ) and thus was collinear with other variables . therefore , multivariate logistic regression analysis was performed excluding initial hinge status , and the results indicated that medially placed hinge gutter , double screw fixation on the elevated lamina , and upper surgical level were risk factors for hf after adjustment for other confounding factors.conclusionto prevent hf and to draw a successful postoperative outcome after cervical laminoplasty , surgical and clinical precautions should be considered .

INTRODUCTION MATERIALS AND METHODS Surgical technique Radiographic measurement Statistical analysis RESULTS Radiographic outcomes Correlation coefficients between variables Logistic regression analysis Interobserver agreement DISCUSSION HF and related complications Risk factors for HF CONCLUSION

open - door laminoplasty is one of the most popular surgical methods that provides enough decompression of the spinal canal over multiple segments and reconstructs the stable laminar arch with sufficient room for the spinal cord27121517 ) . when the procedure was relatively new , hirabayashi used a wire for lamina fixation ; however , open lamina reclosure or fracture was a problem23 ) . o'brien et al.8 ) modified open - door type laminoplasty by augmenting the procedure with titanium plates to maintain patency of the deformed lamina . however , only few surgeons have investigated about hfs because the lamina is well fused in the majority of cases , and most surgeons believe that hfs do not exert much influence over clinical outcomes . some recent studies have indicated that hfs can cause axial pain or palsy that may be chronic21014 ) . our previous study showed that patients with 3 or more hfs experienced significant worsening of visual analog scales for neck ( vasn ) and neck disability index ( ndi ) compared with patients with 2 hfs or less in multi - level open door laminoplasty cases10 ) . nevertheless , risk factors related to this complication remain unclear . therefore , we radiographically evaluated hinge status and determined risk factors for delayed hfs following plate - only open - door cervical laminoplasty . from april 2005 to january 2012 , 119 patients with cervical myelopathy underwent open - door laminoplasty in our hospitals . among them , 80 patients who had postoperative computed tomography ( ct ) scans within 2 years were enrolled in this study . all of the 80 patients suffered from myelopathic symptoms , such as motor weakness , sensory disturbance , or voiding difficulties . all surgeries were performed by a senior surgeon who used a modification of the classic hirabayashi open - door technique , securing the opened lamina with a titanium miniplate ( medtronic , memphis , tn , usa)23 ) . the number of slices with a broken or disconnected inner cortex was counted among six or seven thin slices of ct scans obtained from a single lamina ( dx ) which indicates poor hinge status as disconnected number increases . 1 shows radiographic measurements of hinge - side gutter and open - side gutter location , open angle , and hinge width . continuous variables ( age , radiographic measurements , number of screws ) are shown as meansstandard deviations , whereas categorical variables ( hinge status , surgical level , sex , and smoking ) are expressed as ratios or percentages . to identify variables independently related to hfs , all tests were two - sided , and a value of p<0.05 was considered statistically significant . all surgeries were performed by a senior surgeon who used a modification of the classic hirabayashi open - door technique , securing the opened lamina with a titanium miniplate ( medtronic , memphis , tn , usa)23 ) . the number of slices with a broken or disconnected inner cortex was counted among six or seven thin slices of ct scans obtained from a single lamina ( dx ) which indicates poor hinge status as disconnected number increases . 1 shows radiographic measurements of hinge - side gutter and open - side gutter location , open angle , and hinge width . continuous variables ( age , radiographic measurements , number of screws ) are shown as meansstandard deviations , whereas categorical variables ( hinge status , surgical level , sex , and smoking ) are expressed as ratios or percentages . to identify variables independently related to hfs , all tests were two - sided , and a value of p<0.05 was considered statistically significant . a total of 114 ct scans from 80 patients were available for the assessment of hinge status : 38 patients at 1 week after surgery , and 76 patients at 4 months after surgery or later up until 2 years . the mean number of operated levels was 4.5 ( range : 37 ) and the mean number of laminoplasties was 3.3 ( range : 25 ) . the cts from 80 patients and a total of 270 laminae levels were evaluated for radiographic measurements . the average hinge and open locations were 1.50.9 mm ( range 04.1 mm ) and 0.50.7 mm ( range 03.5 mm ) , respectively . initial hinge gutter status ( dx ) was evaluated with cts taken from 38 patients 1 week after surgery , and 10% ( 12 of 115 ) of hinges were found to be in danger of fracture or overtly fractured ( d6 or worse ) ( table 3 ) . the ct scans of 34 patients , who had both 1-week and later observations , were evaluated to verify the relationship between the independent variables including initial hinge status , open angle , operated level , number of screws used , and hinge - location . initial hinge status and the open angle , operated level , number of screws used showed high correlation ( p<0.05 ) . univariate and multivariate logistic regression analysis were both used to identify hf risk factors among several variables within 80 patients ( 38 patients for initial hinge status ) . hinge width , sex , age , and smoking were statistically nonsignificant ( p>0.05 ) . poor initial hinge status [ odds ratio ( or)=2.025 , 95% ci=1.5252.689 ] , medially placed hinge gutter ( or=1.807 , 95% ci=1.2852.542 ) , double screw fixation on the elevated lamina ( or=3.096 , 95% ci=1.5966.005 ) , upper surgical level ( or=0.633 , 95% ci=0.4710.849 ) , and wide open angle ( or=1.107 , 95% ci=1.0231.198 ) were identified as predictors of hfs by univariate logistic analysis ( p<0.05 ) . among them , the multivariate logistic regression showed that poor initial hinge status ( or=1.982 , 95% ci=1.4132.780 ) , medially placed hinge gutter ( or=2.376 , 95% ci=1.2714.440 ) , and surgical level ( or=0.367 , 95% ci=0.2040.662 ) were risk factors for hfs after adjustment for other confounding factors , but double screw fixation was excluded . however , because the multivariate analysis results seemed to be biased due to colinearity between initial status and other variables ( table 4 ) , additional multivariate analysis without initial hinge status was performed . medially placed hinge gutter ( or=1.85 , 95% ci=1.2862.698 ) , double screw fixation on the elevated lamina ( or=3.177 , 95% ci=1.5286.605 ) , upper surgical level ( or=0.555 , 95% ci=0.3990.772 ) , and wide open angle ( or=1.085 , 95% ci=0.9961.182 ) had very similar ors to univariate logistic regression analysis and was statistically meaningful ( p<0.05 ) except open angle ( p=0.063 ) . the kappa coefficients for hinge status at each observation ( 1 week , the cts from 80 patients and a total of 270 laminae levels were evaluated for radiographic measurements . the average hinge and open locations were 1.50.9 mm ( range 04.1 mm ) and 0.50.7 mm ( range 03.5 mm ) , respectively . initial hinge gutter status ( dx ) was evaluated with cts taken from 38 patients 1 week after surgery , and 10% ( 12 of 115 ) of hinges were found to be in danger of fracture or overtly fractured ( d6 or worse ) ( table 3 ) . the ct scans of 34 patients , who had both 1-week and later observations , were evaluated to verify the relationship between the independent variables including initial hinge status , open angle , operated level , number of screws used , and hinge - location . initial hinge status and the open angle , operated level , number of screws used showed high correlation ( p<0.05 ) . univariate and multivariate logistic regression analysis were both used to identify hf risk factors among several variables within 80 patients ( 38 patients for initial hinge status ) . hinge width , sex , age , and smoking were statistically nonsignificant ( p>0.05 ) . poor initial hinge status [ odds ratio ( or)=2.025 , 95% ci=1.5252.689 ] , medially placed hinge gutter ( or=1.807 , 95% ci=1.2852.542 ) , double screw fixation on the elevated lamina ( or=3.096 , 95% ci=1.5966.005 ) , upper surgical level ( or=0.633 , 95% ci=0.4710.849 ) , and wide open angle ( or=1.107 , 95% ci=1.0231.198 ) were identified as predictors of hfs by univariate logistic analysis ( p<0.05 ) . among them , the multivariate logistic regression showed that poor initial hinge status ( or=1.982 , 95% ci=1.4132.780 ) , medially placed hinge gutter ( or=2.376 , 95% ci=1.2714.440 ) , and surgical level ( or=0.367 , 95% ci=0.2040.662 ) were risk factors for hfs after adjustment for other confounding factors , but double screw fixation was excluded . however , because the multivariate analysis results seemed to be biased due to colinearity between initial status and other variables ( table 4 ) , additional multivariate analysis without initial hinge status was performed . medially placed hinge gutter ( or=1.85 , 95% ci=1.2862.698 ) , double screw fixation on the elevated lamina ( or=3.177 , 95% ci=1.5286.605 ) , upper surgical level ( or=0.555 , 95% ci=0.3990.772 ) , and wide open angle ( or=1.085 , 95% ci=0.9961.182 ) had very similar ors to univariate logistic regression analysis and was statistically meaningful ( p<0.05 ) except open angle ( p=0.063 ) . the kappa coefficients for hinge status at each observation ( 1 week , 4 months or later ) were 0.61 and 0.78 , respectively . however , there are still some postoperative complications despite many preventative efforts . many studies have attempted to determine the pathologic mechanism of c5 palsy , which is still unclear . this suggests that the downward displacement of the elevated lamina into the spinal canal might injure the nerve root131415 ) . recently , some authors have postulated that hf may be a source of postoperative pain21014 ) . however , to the best of our knowledge , there are no existing studies that determined hf risk factors ; this study is the first attempt . in the present study , multivariate logistic regression analysis indicated that poor initial hinge status , medially located hinge gutter , and upper cervical level are risk factors of hfs after laminoplasty . double screws used for a single lamina are also thought to be a risk factor following interpretation of the multivariate logistic regression analysis excluding initial hinge status . the prevalence of delayed hf was highly correlated with initial hinge status . the variation in univariate and multivariate logistic regression or was very small ( 2.025 vs. 1.982 ) . consequently , as a confounding factor on multivariate logistic regression , initial hinge status could distort the results of other independent factors . furthermore , poor initial hinge status is highly correlated with other independent factors , thus the real correlation of other factors with hf could be devalued . therefore , only hinge location , double screws , level , and open angle were sorted out as independent variables and additionally analyzed using multivariate logistic regression . this also has additional meaning to exclude the incomplete data , because initial hinge status data were collected from 38 patients instead of 80 . hinge gutter location has also been proposed to be an important variable2 ) . in this study , medially placed hinge gutters had a high or and significant p - value in both univariate and multivariate logistic regression analyses . specifically , comparing univariate logistic regression to multivariate logistic regression without initial status , or ( 1.807 vs. 1.85 ) was very similar and both p - values ( 0.001 , 0.002 ) were also highly statistically significant . second , even with the same length of elevation , open angle increases with medial gutter placement . however , it is difficult to say if a more lateral hinge gutter always results in a good outcome . because a lateral hinge gutter is a possible risk factor for c5 palsy and axial pain , some clinicians insist that the outcome of making a medial hinge gutter is better than making a lateral one6 ) . although there were no significant differences between single screw and double screw use in initial multivariate logistic regression analysis ( with initial hinge status ) , it seems to be a predictor of hf . however , there was a significant difference in the initial status average between the two groups ( p=0.0163 , mann - whitney u test ; median = d0 in single , d2 in double screw ) . therefore , second multivariate analysis ( without initial hinge status ) seemed to be more reliable . univariate logistic regression analysis indicated wide open angle to be a predictor of hf but multivariate analysis did not . alternatively , open angle and initial hinge status may essentially be correlated with each other . therefore , open angle and hinge status may be colinear , which could distort the multivariate analysis results . in fact , initial hinge status and open angle were positively correlated ( r statistic=0.25 , p=0.01 ) , as determined by 115 hinges . combining two hypotheses more force should be exerted on a narrow hinge within an equivalent area ; thus , we expected that narrow hinge width would be a risk factor for hf , but we did not find a significant correlation between the two . age was not significantly different in either univariate or multivariate logistic regression analysis . satomi et al.14 ) recommended using careful surgical technique to avoid hf , such as making the hinge gutter more lateral than on the open side and detaching the open side before drilling the hinge side , with frequent hinge stability checks during elevation by pushing the spinous process . in addition , we suggest meticulous screwing on the hinge side and avoiding an unnecessary wide open angle so as not to deform the hinge . however , there are still some postoperative complications despite many preventative efforts . this suggests that the downward displacement of the elevated lamina into the spinal canal might injure the nerve root131415 ) . such pain is currently one of the most well - known complications of cervical laminoplasty . recently , some authors have postulated that hf may be a source of postoperative pain21014 ) . however , to the best of our knowledge , there are no existing studies that determined hf risk factors ; this study is the first attempt . in the present study , multivariate logistic regression analysis indicated that poor initial hinge status , medially located hinge gutter , and upper cervical level are risk factors of hfs after laminoplasty . double screws used for a single lamina are also thought to be a risk factor following interpretation of the multivariate logistic regression analysis excluding initial hinge status . the prevalence of delayed hf was highly correlated with initial hinge status . the variation in univariate and multivariate logistic regression or was very small ( 2.025 vs. 1.982 ) . consequently , as a confounding factor on multivariate logistic regression , initial hinge status could distort the results of other independent factors . furthermore , poor initial hinge status is highly correlated with other independent factors , thus the real correlation of other factors with hf could be devalued . therefore , only hinge location , double screws , level , and open angle were sorted out as independent variables and additionally analyzed using multivariate logistic regression . this also has additional meaning to exclude the incomplete data , because initial hinge status data were collected from 38 patients instead of 80 . hinge gutter location has also been proposed to be an important variable2 ) . in this study , medially placed hinge gutters had a high or and significant p - value in both univariate and multivariate logistic regression analyses . specifically , comparing univariate logistic regression to multivariate logistic regression without initial status , or ( 1.807 vs. 1.85 ) was very similar and both p - values ( 0.001 , 0.002 ) were also highly statistically significant . second , even with the same length of elevation , open angle increases with medial gutter placement . however , it is difficult to say if a more lateral hinge gutter always results in a good outcome . because a lateral hinge gutter is a possible risk factor for c5 palsy and axial pain , some clinicians insist that the outcome of making a medial hinge gutter is better than making a lateral one6 ) . although there were no significant differences between single screw and double screw use in initial multivariate logistic regression analysis ( with initial hinge status ) , it seems to be a predictor of hf . however , there was a significant difference in the initial status average between the two groups ( p=0.0163 , mann - whitney u test ; median = d0 in single , d2 in double screw ) . therefore , second multivariate analysis ( without initial hinge status ) seemed to be more reliable . univariate logistic regression analysis indicated wide open angle to be a predictor of hf but multivariate analysis did not . alternatively , open angle and initial hinge status may essentially be correlated with each other . using a load deformation curve ( young modulus ) as reference , if open angle becomes larger , it will pass by the elastic range and plastic range ( deformation ) , and mechanical failure could occur . in this case therefore , open angle and hinge status may be colinear , which could distort the multivariate analysis results . in fact , initial hinge status and open angle were positively correlated ( r statistic=0.25 , p=0.01 ) , as determined by 115 hinges . combining two hypotheses more force should be exerted on a narrow hinge within an equivalent area ; thus , we expected that narrow hinge width would be a risk factor for hf , but we did not find a significant correlation between the two . age was not significantly different in either univariate or multivariate logistic regression analysis . satomi et al.14 ) recommended using careful surgical technique to avoid hf , such as making the hinge gutter more lateral than on the open side and detaching the open side before drilling the hinge side , with frequent hinge stability checks during elevation by pushing the spinous process . in addition , we suggest meticulous screwing on the hinge side and avoiding an unnecessary wide open angle so as not to deform the hinge . we determined the risk factors for delayed hf following plate - only open - door laminoplasty , which could be a source of axial pain or c5 palsy . poor initial hinge status , medially placed hinge gutter , double screwed lamina , and upper cervical level seemed to be firm risk factors .

. however , several precorneal factors such as lacrimal secretion ( tear turn over and reflux tearing ) , tear protein binding , ph , shorter contact time , and other corneal constraints limit drug penetration . moreover , presence of various uptake and efflux transporters in cornea also reposted to further complicate the ocular bioavailability . therefore conventionally , the drugs intended for oral administrations have been developed in consideration of their physicochemical properties . this enables to enhance the pharmacokinetic properties , efficacy and reduces the toxicity of lead compound . in drug discovery exploitation of in silico techniques to expedite the process of lead optimization in drug discovery is rampant . among such techniques , quantitative structure property relationship ( qspr ) approach correlates the biological activity of a molecule with its physicochemical properties through variety of descriptors . in recent years , qspr approach has been exploited for the development of models to predict penetration of drugs across physiological barriers such as cns , blood , and intestinal . the drugs used for ophthalmic indications are arbitrarily developed from the oral or systemic indications , rather than systematically ocular specific studies . to date , very few drugs have been designed , developed , and studied for ocular specific use . however , the ocular pharmacokinetics of a drug is known to be different and complicated compared to any other indications . thus , in this context a drug intended for oral use is expected to behave differently when used empirically for the topical application . as physicochemical properties such as lipophilicity , molecular size , charge , degree of ionization , solubility , and ph , affect the rate and extent of corneal permeability . in ophthalmology these agents were initially discovered , designed , and developed for systemic infections and further extended for ophthalmic use . their broad - spectrum activity , bactericidal property , better ocular penetration , and relative safety embark its use in ophthalmology . most of the fluoroquinolones for topical use lack regress understanding of their corneal penetration , residence time , required spectrum , optimum frequency of usage , and corneal toxicity despite of their widespread use in ophthalmics . therefore , an emphasis on qspr strategy to optimize ocular pharmacokinetic properties along with antimicrobial efficacy is desirable . previously reported qspr models to predict corneal permeability for congeneric and noncongeneric drugs have been developed based upon in vitro studies . a weak correlation is reported to exist between the in vitro and in vivo studies [ 8 , 9 ] . eventually their applicability of these models to predict in vivo corneal penetration remains unclear . in present study we developed novel qspr based in silico model to predict corneal permeability for fluoroquinolones based upon in vivo the applicability of previously reported models based upon in vitro data to predict corneal permeability for fluoroquinolone was also studied . furthermore , the generated qspr model was evaluated for its aptness using the other training sets ( -blockers ) . pure samples of norfloxacin , ciprofloxacin hcl , gatifloxacin , and lomefloxacin hcl were generously gifted by dr . reddy labs ( hyderabad , india ) , lupin labs ( pune , india ) , sun pharmaceuticals ( mumbai , india ) , and organics ltd . , ( hyderabad , india ) , respectively . gratis samples of ofloxacin , pefloxacin mesylate , and sparfloxacin were obtained from cipla ltd . , ( mumbai , india ) . moxifloxacin and levofloxacin were obtained as a generous gift from capital pharma ( baddi , india ) . other chemicals used in the study were of analytical grade and procured from standard drug companies . a total of nine fluoroquinolones ( table 1 ) were randomly divided into 2 groups wherein , group a consists of ofloxacin , sparfloxacin , pefloxacin mesylate , and gatifloxacin and group b consists of norfloxacin , ciprofloxacin hcl , lomefloxacin hcl , levofloxacin , and moxifloxacin . in both groups , thus , the total fluoroquinolones concentration in group a and group b were 0.4% and 0.5% , respectively . sterile boric acid ( 1.9% w / v ) in water was used as the aqueous media and the final formulations were filtered using 0.22 filter and autoclaved further before use . study protocol and experimental procedures were approved by the institutional animal ethics committee of all india institute of medical sciences ( aiims ) , new delhi , india . new zealand albino rabbits of either sex ( 1.52.0 kg ) were obtained from central animal facility , aiims . animals were housed at standard laboratory conditions temperature - controlled room at 24 2c and humidity 55 15% and given food and water ad libitum . all experiments were performed in accordance to the association for research in vision and ophthalmology ( arvo ) statement for the use of animals in ophthalmic and vision research . the sterile cocktail formulations of group a and group b were individually instilled ( 50 l ) on the lower fornix of rabbit eyes ( n = 4 ) with the help of a calibrated micropipette . aqueous paracentesis was performed under the influence of topical anesthesia at 5 , 15 , 30 , 60 , 120 , and 240 min after the instillation of cocktail formulation . for each time point , a volume amounting to 50 l of aqueous humor was aspirated through the corneal surface . a thermo finnigan ( thermo electron corporation , usa ) hplc equipped with degasser , quaternary pump , autosampler , and pda detector was employed for quantification of fluoroquinolones in samples . in the chromatographic quantification , analytical separation was performed using a c18 symmetry shield column ( 4.6 150 mm , 5 m , waters , usa ) under a gradient flow of methanol , acetonitrile , and potassium phosphate buffer ( 20 mm , ph 2.5 ) at different ratio and time . for each spiked compound an external calibration curve was plotted and the analyte 's spectra was assessed by matching them with custom made pda spectra of inbuilt library of chromquest version 4 ( thermo electron corporation , usa ) . samples were deproteinized with pure acetonitrile ( ratio of 1 : 2 v / v ) , vortexed , and centrifuged at 3500 g for 10 min . the dried concentrate was reconstituted with 100 l mixture of water and acetonitrile ( 1 : 1 ) , and 20 l of the obtained supernatant was injected for quantification . available literature reveals existence of two models correlating the corneal permeability of compounds with their physiochemical properties . therefore , present study evaluated the suitability of these reported models to predict in vivo corneal penetration for fluoroquinolones . the first model reported by yoshida and topliss was based upon two molecular descriptors , log p and log d to predict corneal permeability coefficient ( logpc ) . algorithm ( 1 ) is proposed to predict corneal permeability for noncongeneric compounds ( 1)logpc=0.404( 0.114)log p+0.141( 0.090)log d 3.862( 0.451 ) , wherein logpc denotes permeability coefficient , log p expresses the difference between the octanol - water partition coefficients ( log poctanol ) and alkane - water partition coefficients ( log palkane ) , and log d denotes dissociation constant as reported , log p was calculated using logpstar software in training sets of 32 diverse noncongeneric compounds including steroids and -blockers . since logpstar was currently unavailable and obsolete . log p was derived as the difference in log p(o / w ) to log p(cyclohexane / water ) by using shake flask method given in oecd guidelines of chemical testing ( no . the other molecular descriptor log d was calculated using ( 2)log d(ph)=log plog ( 1 + 10(pkaph)).log molecular descriptors ( log p and log d ) were derived for the studied fluoroquinolones ( norfloxacin , ciprofloxacin , lomefloxacin , ofloxacin , levofloxacin , sparfloxacin , pefloxacin , gatifloxacin , and moxifloxacin ) and logpc was determined using algorithm ( 1 ) . the derived logpc from algorithm ( 1 ) was correlated with that obtained from controlled in vivo experiment in rabbits . model 2 evaluated reported by fu and liang was based upon charge and molecular volume as the molecular descriptors to predict logpc . algorithm ( 3 ) was reported to predict corneal permeability for noncongeneric compounds ( 3)logpc=5.566qh2 + 3.027qh0.155qo , n 9.413104 v4.278 wherein logpc denotes permeability coefficient , qh is sum of the absolute values of net atomic charge of hydrogen atom , and qo , n is sum of the absolute values of the net atomic charges of oxygen and nitrogen atoms . the charge ( qh , qh , qo , n ) for all fluoroquinolones was calculated by using gamess software and molecular volume by drug design software developed at super computing facility , indian institute of technology , new delhi . structures of all fluoroquinolones were drawn hyperchem version 11.0 and optimized the geometry to lowest energy state . the net charge on each atom was calculated as the sum of all atoms present in the particular structure . the molecular descriptors used in algorithm ( 3 ) were derived for the studied nine fluoroquinolones to predict logpc . the derived logpc from algorithm ( 3 ) was also correlated with in vivo experimentally obtained logpc . a novel qspr model was generated using pooled molecular descriptors determined using in vitro , in vivo , and in silico approaches for fluoroquinolones . in vitro datapartition coefficient ( log p ) 107 and 117 ) using biphasic system of octanol / water at 25c and 37c . partition coefficient ( log p ) was determined using oecd shake flask method for chemical testing ( no . 107 and 117 ) using biphasic system of octanol / water at 25c and 37c . in vivo datapermeability coefficients ( logpc ) were obtained from controlled in vivo experiment conducted in rabbits using cassette dosing ( n - in - one ) approach . permeability coefficients ( logpc ) were obtained from controlled in vivo experiment conducted in rabbits using cassette dosing ( n - in - one ) approach . in silico datadifferent molecular descriptors were generated using softwares like cache scientific ( fujitsu version 6.1.12.33 , japan ) , acd / chemsketch ( freeware version 10 ) , chemdraw and chem biodraw ultra from cambridge soft ( trial version ) , calculator plugins of chem axon 's marvin version 5.2.5.1 , chem axon ltd . all fluoroquinolone structures were drawn in respective workspace and standard molecular mechanics were run before geometry optimization . the descriptors like molecular weight , log p , topological polar surface area ( tpsa ) , molar refractivity , number of h-bond donors / acceptor , dipole moment , lowest unoccupied molecular orbitals ( lumo ) , highest unoccupied molecular orbital ( humo ) , gap , molecular volume , connolly accessible area , connolly molecular area , principal moment , dipole moment , molecular weight , wiener index , melting point , polar surface area , number of rotatable bonds and molar refractivity , molar volume , polarizibility , parachor , index of refraction , surface tension , density , monoisotopic mass , nominal mass , average mass , apka , and charge on the each atom , that is , qn , qo , n , qh , qf and qc were extracted using different kind of softwares . different molecular descriptors were generated using softwares like cache scientific ( fujitsu version 6.1.12.33 , japan ) , acd / chemsketch ( freeware version 10 ) , chemdraw and chem biodraw ultra from cambridge soft ( trial version ) , calculator plugins of chem axon 's marvin version 5.2.5.1 , chem axon ltd . all fluoroquinolone structures were drawn in respective workspace and standard molecular mechanics were run before geometry optimization . the descriptors like molecular weight , log p , topological polar surface area ( tpsa ) , molar refractivity , number of h-bond donors / acceptor , dipole moment , lowest unoccupied molecular orbitals ( lumo ) , highest unoccupied molecular orbital ( humo ) , gap , molecular volume , connolly accessible area , connolly molecular area , principal moment , dipole moment , molecular weight , wiener index , melting point , polar surface area , number of rotatable bonds and molar refractivity , molar volume , polarizibility , parachor , index of refraction , surface tension , density , monoisotopic mass , nominal mass , average mass , apka , and charge on the each atom , that is , qn , qo , n , qh , qf and qc were extracted using different kind of softwares . to ensure the suitability of newly developed qspr model , the congeneric compounds used by yoshida and topliss and fu and liang were pooled . all the congeneric compounds ( -blockers , n = 15 ) were pooled and molecular descriptors used in newly developed qspr model were extracted . the -blockers pooled include acebutolol , alprenolol , atenolol , betaxolol , bevantolol , bufuralol , levobunolol , metoprolol , nadolol , oxprenolol , penbutolol , pindolol , propanolol , sotalol , and timolol . the logpc derived using newly developed qspr model was correlated with reported logpc for all the -blockers . two different hplc - pda method were developed and validated to elute all fluoroquinolones in group a and b. for the analysis of group a ( ofloxacin , sparfloxacin , pefloxacin , and gatifloxacin ) the gradient mobile phase consisted of different ratios of methanol , acetonitrile , and potassium phosphate buffer ( 20 mm , ph 2.5 ) over the period of 12 min ( table 2 ) . for the analysis of fluoroquinolones in group b ( norfloxacin , ciprofloxacin , lomefloxacin , levofloxacin , and gatifloxacin ) the gradient mobile phase consisted of different ratios of methanol , acetonitrile , and potassium phosphate buffer ( 20 mm , ph 2.5 ) over the period of 10 min ( table 2 ) . the representative chromatograms of all fluoroquinolones eluted in group a and group b are shown in figures 1(a ) and 1(b ) . the validation parameters regarding the accuracy and precision were found to be within the allowable limits according to ich guidelines for hplc in bioanalysis . the mean concentration versus time plot for all fluoroquinolones ( group a and b ) the derived pharmacokinetics parameters like cmax , tmax , auc , logpc at 30 min and 240 min generated for all fluoroquinolones are tabulated in table 3 . both molecular descriptors ( logp and logd ) derived for fluoroquinolones in algorithm ( 1 ) to derive logpc showed a weak spearman correlation ( 0.133 ) with that obtained from in vivo experiment ( table 4 ) . the weak correlation was observed at 30 min ( r = 0.0699 ) and 240 min ( r = 0.0137 ) . the statistically insignificant correlation suggests that algorithm ( 1 ) is unable to appropriately predict corneal permeability for fluoroquinolones ( figure 3(a ) ) . algorithm ( 3 ) reported by fu and liang was employed to derive logpc for all studied fluoroquinolones ( table 4 ) . a very weak spearman correlation of 0.134 was observed between the logpc derived experimentally at both 30 min and 240 min with that logpc derived using algorithm ( 3 ) ( figure 3(b ) ) . the statistically insignificant correlation suggests that algorithm ( 3 ) is unable to appropriately predict corneal permeability for fluoroquinolones . a total of 72 molecular descriptors were extracted for all nine topically studied fluoroquinolones ( norfloxacin , ciprofloxacin , lomefloxacin , ofloxacin , levofloxacin , sparfloxacin , pefloxacin , gatifloxacin and moxifloxacin ) using in vitro , in vivo , and in silico approaches . all sets of data were subjected to multilinear regression ( mlr ) statistical analysis by sigma stat software ( version 3.5 , germany ) . for the generation of algorithm the in vivo data of logpc calculated for absorption phase ( 30 min ) and elimination phase ( 240 min ) was used . the algorithms developed with log p and apka either with gap ( algorithms ( 4 ) and ( 5 ) ) and or with tpsa ( algorithms ( 6 ) and ( 7 ) ) exhibited good correlation ( 4)logpc 30 min = 13.972+(1.529log p ) (1.375apka)+(1.308gap),r=0.908 rsqr=0.825 adj rsqr=0.720 , ( 5)logpc 240 min = 9.946+(1.368log p ) (1.429apka)+(0.952gap)r=0.940 rsqr=0.883 adj rsqr=0.813 , wherein logpc denotes logarithm of permeability coefficient , log p is partition coefficient at water 25 1c for 30 min , gap is the difference in humo and lumo energy levels , and apka is acid dissociation constant . algorithms ( 6 ) and ( 7 ) were developed using logp , tpsa , and apka as molecular descriptor at 30 min and 240 min . ( 6)log pc 30 min = 1.453+(1.726log p ) (0.708apka)+(0.0104tpsa)r=0.934 rsqr=0.872 adj rsqr=0.796 , ( 7)log pc 240 min = 2.726+(1.439log p ) (0.672apka)+(0.00421tpsa)r=0.937 rsqr=0.877 adj rsqr=0.803 , wherein logpc denotes logarithm of permeability coefficient , log p as partition coefficient at water 36 1c for 5 min , tpsa as topological polar surface area , apka is acid dissociation constant . figure 4 shows the pictorial representation of newly developed qspr model using fluoroquinolone as model group . in the four newly developed algorithms ( algorithms ( 4 ) , ( 5 ) , ( 6 ) , and ( 7 ) ) log p and apka are common molecular descriptors , signifying the key descriptors affecting penetration of fluoroquinolones across the cornea . algorithms ( 4 ) , ( 5 ) , ( 6 ) , and ( 7 ) showed a positive correlation with log p and inverse correlation with apka . tpsa and gap also showed a positive correlation with permeability coefficient in all newly developed algorithms . newly developed qspr model showed a positive spearman correlation of 0.831 and 0.887 between the newly developed algorithms ( 4 ) and ( 5 ) with already reported logpc ( figure 5(a ) ) . a positive spearman correlation of 0.881 and 0.803 between the logpc generated for -blockers using newly developed algorithms ( 6 ) and ( 7 ) and reported logpc ( figure 5(b ) ) . conventionally , antimicrobial drugs developed and approved for systemic infections are extended for ocular infections . an antimicrobial agent having good corneal penetration and efficacy is desired in preventing sight threatening infections . it is well known that less than 5% of the topically applied drug penetrates through the cornea . therefore , there is an urgent need to understand the constraints exerted by the eye for the development of an ocular specific antimicrobial agent . in present study , qspr approach has been used to comprehend the physicochemical factors affecting corneal permeability for topically applied drugs . an attempt was made in present study to evaluate the suitability of in silico models in predicting the in vivo corneal permeability of fluoroquinolones . to ascertain the obtained results , additionally , in vivo corneal permeability was determined in rabbits employing cassette dosing approach , a high throughput pharmacokinetic screening technique which has been exploited for various indication [ 1214 ] . so far this approach has been limited to in vitro and few in vivo models in ophthalmic drug research [ 1517 ] . however , for the first time , we employed cassette dosing technique to study the in vivo pharmacokinetic profile of topically applied fluoroquinolones and to derive a controlled in vivo permeability coefficient nine topically used fluoroquinolones were dissolved in two cassettes : group a with four and group b with five fluoroquinolones . being weakly basic in nature fluoroquinolones are expected to exist in ionized form at ph 4.5 . therefore , ph of both formulations was maintained at 5.0 and osmolarity as 307 mosm / l using boric acid . boric acid ( 1.9% ) has been reported to be an appropriate vehicle for the preparation of ophthalmic solutions having basic nature . in addition , it is reported to avoid any interaction between fluoroquinolones and di / monovalent cations . in the literature the first model was reported by yoshida and topliss based upon log p and log d as molecular descriptors . in this model , log p mainly correlates with the solute hydrogen bonding acidity or basicity and to lesser extend dipolarity or polarizability . the second model was reported by fu and liang was based upon charge and molecular volume as the molecular descriptors . unfortunately , both the earlier reported models were unsuitable to predict in vivo corneal permeability of fluoroquinolones . a poor correlation between the permeability coefficient derived using reported models [ 10 , 11 ] with that derived from present in vivo study was observed . this may be due to the fact that reported models were developed based upon permeability coefficients ( logpc ) pooled from various in vitro studies . the in vitro conditions denote a static system rather than actual dynamic conditions which exists in eye . moreover , the involvement of various drug transporters and precorneal factors is not considered in such studies and are also believed to have major role . thus , reported models [ 10 , 11 ] based upon in vitro corneal permeability are unable to correlate with in vivo corneal permeability for fluoroquinolones . therefore , a new in silico model based upon in vivo permeability coefficient data was felt desirable . also the in silico model should be able to correlate with parameters like ionizing property , transporter susceptibility , and lipophilicity , precorneal ph of compound . a novel qspr model was developed based upon in vivo corneal permeability along with molecular descriptors like log p , apka , gap , and tpsa . civiale and coworkers also reported that corneal permeation process can be confined at the ocular epithelium layer . studies have been reported that in corneal epithelium the drugs are expected to undergo transcellular pathway [ 21 , 22 ] . whereas the penetrating molecules are expected in hydrophilic form as a result of ph or after hydrolysis due to enzymes ( by prodrug attempt ) to cross hydrophilic stroma . the dissociation constant ( pka ) of the compound determines the hlb ( hydrophilic / lipophilic balance ) in the cornea . based upon the presence of ionizable / functional groups molecules can posses more than one pka . an assumption was made in the newly developed qspr model that for fluoroquinolones apka has more implication on the availability of the species . other studies also report that corneal penetration can be enhanced by selecting the drug molecule with appropriate pka and offering optimal lipid solubility [ 23 , 24 ] . in general , adjusting ph so that a drug is mostly in the unionized form increases its lipophilicity and thus , its transcellular permeability , and ocular absorption . another molecular descriptor gap , which is difference in energy between ehumo and elumo , has also reported to be an important stability index and related to transporters susceptibility . the ehumo measures the electron donating and elumo measures electron accepting property of the molecule . a humo and lumo energy separation has been used as a conventional measure of kinetic stability for various -electron systems . a large gap has been reported to relate with high stability for a molecule in the sense of its lower sensitivity in chemical reaction [ 26 , 27 ] . tpsa is defined as the sum of surfaces of polar atoms in a molecule and known to correlate with transporter susceptibility . tpsa makes use of functional group based upon large database of structures that avoids the need to calculate ligand 3d structure or to decide which one is relevant biological conformation . therefore , in this analysis , tpsa has been taken into account to define the transcorneal penetration of congeneric compounds . fernandes and coworkers suggested that compounds with high tpsa are transported , while those with low tpsa are not . moreover , conjugation to compounds like gsh is reported to increase the tpsa values as a favoring transport mechanism . the algorithm developed either with tpsa or gap proved to predict the intraocular penetration appropriately as compared with other molecular descriptors tried with highest degree of correlation ( r > 0.9 ) the applicability of the newly developed algorithms ( 4 ) , ( 5 ) , ( 6 ) , and ( 7 ) on other sets of compounds apart from fluoroquinolones was also tested . the congeneric compound trial sets taken for this analysis belong to the category of -blockers where the ioniziability and ph - dependent changes in the lipophilicity are not much of concern . their structures predominately lacking primary amino groups or carboxylic acids ( except atenolol having primary amine ) therefore , ph - induced changes are not expected to play a major role . this study further evaluated the applicability of the developed algorithm on the noncongeneric compounds using similar training sets . the newly developed in silico model is based upon in vivo data and hence may predict corneal permeability for congeneric compounds other than fluoroquinolone more appropriately . the previously reported algorithms based on in vitro data failed to predict in vivo corneal permeability for fluoroquinolones . a novel qspr model consisting of four new algorithms were developed using gap , tpsa , logp , and apka as molecular descriptors to predict in vivo corneal permeability . the hypothesis generated showed high degree of applicability to predict transcorneal penetration as it was based upon the in vivo corneal permeability coefficients data . moreover , the developed model was also found to predict corneal permeability of the congeneric -blockers ( r > 0.6 ) reported in the literature . further studies are in progress to evaluate its utility in large number of other congeneric compounds .

this study was undertaken to determine in vivo permeability coefficients for fluoroquinolones and to assess its correlation with the permeability derived using reported models in the literature . further , the aim was to develop novel qspr model to predict corneal permeability for fluoroquinolones and test its suitability on other training sets . the in vivo permeability coefficient was determined using cassette dosing ( n - in - one ) approach for nine fluoroquinolones ( norfloxacin , ciprofloxacin , lomefloxacin , ofloxacin , levofloxacin , sparfloxacin , pefloxacin , gatifloxacin , and moxifloxacin ) in rabbits . the correlation between corneal permeability derived using in vivo studies with that derived from reported models was determined . novel qspr - based model was developed using in vivo corneal permeability along with other molecular descriptors . the suitability of developed model was tested on -blockers ( n = 15 ) . the model showed better prediction of corneal permeability for fluoroquinolones ( r2 > 0.9 ) as well as -blockers ( r2 > 0.6 ) . the newly developed qspr model based upon in vivo generated data was found suitable to predict corneal permeability for fluoroquinolones as well as other sets of compounds .

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

however , several precorneal factors such as lacrimal secretion ( tear turn over and reflux tearing ) , tear protein binding , ph , shorter contact time , and other corneal constraints limit drug penetration . therefore conventionally , the drugs intended for oral administrations have been developed in consideration of their physicochemical properties . among such techniques , quantitative structure property relationship ( qspr ) approach correlates the biological activity of a molecule with its physicochemical properties through variety of descriptors . in recent years , qspr approach has been exploited for the development of models to predict penetration of drugs across physiological barriers such as cns , blood , and intestinal . to date , very few drugs have been designed , developed , and studied for ocular specific use . however , the ocular pharmacokinetics of a drug is known to be different and complicated compared to any other indications . as physicochemical properties such as lipophilicity , molecular size , charge , degree of ionization , solubility , and ph , affect the rate and extent of corneal permeability . their broad - spectrum activity , bactericidal property , better ocular penetration , and relative safety embark its use in ophthalmology . most of the fluoroquinolones for topical use lack regress understanding of their corneal penetration , residence time , required spectrum , optimum frequency of usage , and corneal toxicity despite of their widespread use in ophthalmics . previously reported qspr models to predict corneal permeability for congeneric and noncongeneric drugs have been developed based upon in vitro studies . a weak correlation is reported to exist between the in vitro and in vivo studies [ 8 , 9 ] . eventually their applicability of these models to predict in vivo corneal penetration remains unclear . in present study we developed novel qspr based in silico model to predict corneal permeability for fluoroquinolones based upon in vivo the applicability of previously reported models based upon in vitro data to predict corneal permeability for fluoroquinolone was also studied . furthermore , the generated qspr model was evaluated for its aptness using the other training sets ( -blockers ) . pure samples of norfloxacin , ciprofloxacin hcl , gatifloxacin , and lomefloxacin hcl were generously gifted by dr . reddy labs ( hyderabad , india ) , lupin labs ( pune , india ) , sun pharmaceuticals ( mumbai , india ) , and organics ltd . gratis samples of ofloxacin , pefloxacin mesylate , and sparfloxacin were obtained from cipla ltd . a total of nine fluoroquinolones ( table 1 ) were randomly divided into 2 groups wherein , group a consists of ofloxacin , sparfloxacin , pefloxacin mesylate , and gatifloxacin and group b consists of norfloxacin , ciprofloxacin hcl , lomefloxacin hcl , levofloxacin , and moxifloxacin . in both groups , thus , the total fluoroquinolones concentration in group a and group b were 0.4% and 0.5% , respectively . sterile boric acid ( 1.9% w / v ) in water was used as the aqueous media and the final formulations were filtered using 0.22 filter and autoclaved further before use . the sterile cocktail formulations of group a and group b were individually instilled ( 50 l ) on the lower fornix of rabbit eyes ( n = 4 ) with the help of a calibrated micropipette . aqueous paracentesis was performed under the influence of topical anesthesia at 5 , 15 , 30 , 60 , 120 , and 240 min after the instillation of cocktail formulation . a thermo finnigan ( thermo electron corporation , usa ) hplc equipped with degasser , quaternary pump , autosampler , and pda detector was employed for quantification of fluoroquinolones in samples . in the chromatographic quantification , analytical separation was performed using a c18 symmetry shield column ( 4.6 150 mm , 5 m , waters , usa ) under a gradient flow of methanol , acetonitrile , and potassium phosphate buffer ( 20 mm , ph 2.5 ) at different ratio and time . the dried concentrate was reconstituted with 100 l mixture of water and acetonitrile ( 1 : 1 ) , and 20 l of the obtained supernatant was injected for quantification . available literature reveals existence of two models correlating the corneal permeability of compounds with their physiochemical properties . therefore , present study evaluated the suitability of these reported models to predict in vivo corneal penetration for fluoroquinolones . the first model reported by yoshida and topliss was based upon two molecular descriptors , log p and log d to predict corneal permeability coefficient ( logpc ) . algorithm ( 1 ) is proposed to predict corneal permeability for noncongeneric compounds ( 1)logpc=0.404( 0.114)log p+0.141( 0.090)log d 3.862( 0.451 ) , wherein logpc denotes permeability coefficient , log p expresses the difference between the octanol - water partition coefficients ( log poctanol ) and alkane - water partition coefficients ( log palkane ) , and log d denotes dissociation constant as reported , log p was calculated using logpstar software in training sets of 32 diverse noncongeneric compounds including steroids and -blockers . the other molecular descriptor log d was calculated using ( 2)log d(ph)=log plog ( 1 + 10(pkaph)).log molecular descriptors ( log p and log d ) were derived for the studied fluoroquinolones ( norfloxacin , ciprofloxacin , lomefloxacin , ofloxacin , levofloxacin , sparfloxacin , pefloxacin , gatifloxacin , and moxifloxacin ) and logpc was determined using algorithm ( 1 ) . the derived logpc from algorithm ( 1 ) was correlated with that obtained from controlled in vivo experiment in rabbits . model 2 evaluated reported by fu and liang was based upon charge and molecular volume as the molecular descriptors to predict logpc . algorithm ( 3 ) was reported to predict corneal permeability for noncongeneric compounds ( 3)logpc=5.566qh2 + 3.027qh0.155qo , n 9.413104 v4.278 wherein logpc denotes permeability coefficient , qh is sum of the absolute values of net atomic charge of hydrogen atom , and qo , n is sum of the absolute values of the net atomic charges of oxygen and nitrogen atoms . the net charge on each atom was calculated as the sum of all atoms present in the particular structure . the molecular descriptors used in algorithm ( 3 ) were derived for the studied nine fluoroquinolones to predict logpc . a novel qspr model was generated using pooled molecular descriptors determined using in vitro , in vivo , and in silico approaches for fluoroquinolones . partition coefficient ( log p ) was determined using oecd shake flask method for chemical testing ( no . in vivo datapermeability coefficients ( logpc ) were obtained from controlled in vivo experiment conducted in rabbits using cassette dosing ( n - in - one ) approach . permeability coefficients ( logpc ) were obtained from controlled in vivo experiment conducted in rabbits using cassette dosing ( n - in - one ) approach . different molecular descriptors were generated using softwares like cache scientific ( fujitsu version 6.1.12.33 , japan ) , acd / chemsketch ( freeware version 10 ) , chemdraw and chem biodraw ultra from cambridge soft ( trial version ) , calculator plugins of chem axon 's marvin version 5.2.5.1 , chem axon ltd . to ensure the suitability of newly developed qspr model , the congeneric compounds used by yoshida and topliss and fu and liang were pooled . all the congeneric compounds ( -blockers , n = 15 ) were pooled and molecular descriptors used in newly developed qspr model were extracted . the logpc derived using newly developed qspr model was correlated with reported logpc for all the -blockers . two different hplc - pda method were developed and validated to elute all fluoroquinolones in group a and b. for the analysis of group a ( ofloxacin , sparfloxacin , pefloxacin , and gatifloxacin ) the gradient mobile phase consisted of different ratios of methanol , acetonitrile , and potassium phosphate buffer ( 20 mm , ph 2.5 ) over the period of 12 min ( table 2 ) . for the analysis of fluoroquinolones in group b ( norfloxacin , ciprofloxacin , lomefloxacin , levofloxacin , and gatifloxacin ) the gradient mobile phase consisted of different ratios of methanol , acetonitrile , and potassium phosphate buffer ( 20 mm , ph 2.5 ) over the period of 10 min ( table 2 ) . the mean concentration versus time plot for all fluoroquinolones ( group a and b ) the derived pharmacokinetics parameters like cmax , tmax , auc , logpc at 30 min and 240 min generated for all fluoroquinolones are tabulated in table 3 . both molecular descriptors ( logp and logd ) derived for fluoroquinolones in algorithm ( 1 ) to derive logpc showed a weak spearman correlation ( 0.133 ) with that obtained from in vivo experiment ( table 4 ) . the statistically insignificant correlation suggests that algorithm ( 1 ) is unable to appropriately predict corneal permeability for fluoroquinolones ( figure 3(a ) ) . a very weak spearman correlation of 0.134 was observed between the logpc derived experimentally at both 30 min and 240 min with that logpc derived using algorithm ( 3 ) ( figure 3(b ) ) . the statistically insignificant correlation suggests that algorithm ( 3 ) is unable to appropriately predict corneal permeability for fluoroquinolones . a total of 72 molecular descriptors were extracted for all nine topically studied fluoroquinolones ( norfloxacin , ciprofloxacin , lomefloxacin , ofloxacin , levofloxacin , sparfloxacin , pefloxacin , gatifloxacin and moxifloxacin ) using in vitro , in vivo , and in silico approaches . for the generation of algorithm the in vivo data of logpc calculated for absorption phase ( 30 min ) and elimination phase ( 240 min ) was used . the algorithms developed with log p and apka either with gap ( algorithms ( 4 ) and ( 5 ) ) and or with tpsa ( algorithms ( 6 ) and ( 7 ) ) exhibited good correlation ( 4)logpc 30 min = 13.972+(1.529log p ) (1.375apka)+(1.308gap),r=0.908 rsqr=0.825 adj rsqr=0.720 , ( 5)logpc 240 min = 9.946+(1.368log p ) (1.429apka)+(0.952gap)r=0.940 rsqr=0.883 adj rsqr=0.813 , wherein logpc denotes logarithm of permeability coefficient , log p is partition coefficient at water 25 1c for 30 min , gap is the difference in humo and lumo energy levels , and apka is acid dissociation constant . algorithms ( 6 ) and ( 7 ) were developed using logp , tpsa , and apka as molecular descriptor at 30 min and 240 min . ( 6)log pc 30 min = 1.453+(1.726log p ) (0.708apka)+(0.0104tpsa)r=0.934 rsqr=0.872 adj rsqr=0.796 , ( 7)log pc 240 min = 2.726+(1.439log p ) (0.672apka)+(0.00421tpsa)r=0.937 rsqr=0.877 adj rsqr=0.803 , wherein logpc denotes logarithm of permeability coefficient , log p as partition coefficient at water 36 1c for 5 min , tpsa as topological polar surface area , apka is acid dissociation constant . figure 4 shows the pictorial representation of newly developed qspr model using fluoroquinolone as model group . in the four newly developed algorithms ( algorithms ( 4 ) , ( 5 ) , ( 6 ) , and ( 7 ) ) log p and apka are common molecular descriptors , signifying the key descriptors affecting penetration of fluoroquinolones across the cornea . algorithms ( 4 ) , ( 5 ) , ( 6 ) , and ( 7 ) showed a positive correlation with log p and inverse correlation with apka . tpsa and gap also showed a positive correlation with permeability coefficient in all newly developed algorithms . newly developed qspr model showed a positive spearman correlation of 0.831 and 0.887 between the newly developed algorithms ( 4 ) and ( 5 ) with already reported logpc ( figure 5(a ) ) . a positive spearman correlation of 0.881 and 0.803 between the logpc generated for -blockers using newly developed algorithms ( 6 ) and ( 7 ) and reported logpc ( figure 5(b ) ) . in present study , qspr approach has been used to comprehend the physicochemical factors affecting corneal permeability for topically applied drugs . an attempt was made in present study to evaluate the suitability of in silico models in predicting the in vivo corneal permeability of fluoroquinolones . to ascertain the obtained results , additionally , in vivo corneal permeability was determined in rabbits employing cassette dosing approach , a high throughput pharmacokinetic screening technique which has been exploited for various indication [ 1214 ] . so far this approach has been limited to in vitro and few in vivo models in ophthalmic drug research [ 1517 ] . however , for the first time , we employed cassette dosing technique to study the in vivo pharmacokinetic profile of topically applied fluoroquinolones and to derive a controlled in vivo permeability coefficient nine topically used fluoroquinolones were dissolved in two cassettes : group a with four and group b with five fluoroquinolones . in the literature the first model was reported by yoshida and topliss based upon log p and log d as molecular descriptors . in this model , log p mainly correlates with the solute hydrogen bonding acidity or basicity and to lesser extend dipolarity or polarizability . the second model was reported by fu and liang was based upon charge and molecular volume as the molecular descriptors . unfortunately , both the earlier reported models were unsuitable to predict in vivo corneal permeability of fluoroquinolones . a poor correlation between the permeability coefficient derived using reported models [ 10 , 11 ] with that derived from present in vivo study was observed . this may be due to the fact that reported models were developed based upon permeability coefficients ( logpc ) pooled from various in vitro studies . the in vitro conditions denote a static system rather than actual dynamic conditions which exists in eye . thus , reported models [ 10 , 11 ] based upon in vitro corneal permeability are unable to correlate with in vivo corneal permeability for fluoroquinolones . therefore , a new in silico model based upon in vivo permeability coefficient data was felt desirable . also the in silico model should be able to correlate with parameters like ionizing property , transporter susceptibility , and lipophilicity , precorneal ph of compound . a novel qspr model was developed based upon in vivo corneal permeability along with molecular descriptors like log p , apka , gap , and tpsa . the dissociation constant ( pka ) of the compound determines the hlb ( hydrophilic / lipophilic balance ) in the cornea . based upon the presence of ionizable / functional groups molecules can posses more than one pka . an assumption was made in the newly developed qspr model that for fluoroquinolones apka has more implication on the availability of the species . in general , adjusting ph so that a drug is mostly in the unionized form increases its lipophilicity and thus , its transcellular permeability , and ocular absorption . tpsa makes use of functional group based upon large database of structures that avoids the need to calculate ligand 3d structure or to decide which one is relevant biological conformation . the algorithm developed either with tpsa or gap proved to predict the intraocular penetration appropriately as compared with other molecular descriptors tried with highest degree of correlation ( r > 0.9 ) the applicability of the newly developed algorithms ( 4 ) , ( 5 ) , ( 6 ) , and ( 7 ) on other sets of compounds apart from fluoroquinolones was also tested . the congeneric compound trial sets taken for this analysis belong to the category of -blockers where the ioniziability and ph - dependent changes in the lipophilicity are not much of concern . this study further evaluated the applicability of the developed algorithm on the noncongeneric compounds using similar training sets . the newly developed in silico model is based upon in vivo data and hence may predict corneal permeability for congeneric compounds other than fluoroquinolone more appropriately . the previously reported algorithms based on in vitro data failed to predict in vivo corneal permeability for fluoroquinolones . a novel qspr model consisting of four new algorithms were developed using gap , tpsa , logp , and apka as molecular descriptors to predict in vivo corneal permeability . the hypothesis generated showed high degree of applicability to predict transcorneal penetration as it was based upon the in vivo corneal permeability coefficients data . moreover , the developed model was also found to predict corneal permeability of the congeneric -blockers ( r > 0.6 ) reported in the literature .

key language abilities , such as perception and production , are governed by multiple regions in the brain . these abilities can quickly become jeopardized in people with brain tumors , epilepsy , or other structural abnormalities . many of these patients require resection of pathological tissue near eloquent language areas to prolong or improve quality of life . thus , functional language mapping for precise localization of eloquent language areas is necessary for achieving optimal surgical outcomes in such patients . functional language mapping for perioperative planning in individual patients is of utmost importance given the high variability in structural anatomy and function across individuals . most typically , language mapping is achieved using electrical cortical stimulation ( ecs ) mapping . while ecs is widely considered the gold standard , , it does have noteworthy limitations . second , ecs increases the risk of after - discharges or seizures that result from active stimulation of the cortex using electrical impulses . finally , ecs can be difficult to accomplish in the subset of pediatric patients and patients with psychiatric and cognitive comorbidities . these issues have prompted recent and increasingly encouraging investigations suggesting that passive methodologies , such as electrocorticography ( ecog ) or functional magnetic resonance imaging ( fmri ) , may prove useful for functional mapping and may have distinct advantages in efficiency , morbidity , or the range of patients that can benefit from it , , , , , , , , . unfortunately , traditional mapping of expressive language function with any of these existing techniques carries the additional requirement that patients actually speak , i.e. , fully participate in specific tasks such as verb generation , object naming , or counting . this requirement currently precludes the use of these techniques in many patients , such as those with aphasia or cognitive deficits or very young patients . together , these limitations and requirements preclude or greatly impede functional mapping of expressive language areas in certain clinical circumstances ( such as during awake craniotomies ) or with certain populations ( such as pediatric patients ) . hence , it is desirable to have access to a technique that does not electrically stimulate the brain and that eliminates or reduces the requirement for patient participation . such a technique may eventually reduce ecs mapping time by guiding the clinician with a preliminary map of eloquent expressive language cortex . this would not only diminish the risks of patient morbidity , discomfort , and iatrogenic seizures but would also increase the number of patients who could be eligible for functional mapping of expressive language areas . identification of eloquent expressive language cortex without requiring the patient to speak is supported by several findings . previous fmri studies reported activations of the left , and bilateral , , , inferior frontal gyri while subjects listened to speech stimuli but did not perform any overt speaking task . in addition , suarez et al . demonstrated using fmri that a passive listening task recruited similar cortical areas as a verb generation task in a cohort of 15 pediatric patients . however , fmri is still expensive and requires substantial expertise that is not available in all centers , and its reliability in the context of functional mapping is still uncertain , . electrocorticographic recordings also provide opportunities for functional mapping in the context of mapping of motor , , , , or language , function , in pediatric patients , and in the operating room , . together , these studies demonstrated that ecog - based mapping can be achieved in real time ( i.e. , while signals are being recorded ) , does not require expertise in signal analysis , and can produce clinically useful results that can readily be compared with ecs results in a few minutes . however , evidence for its utility in identifying expressive language without subject participation was lacking . indeed , only two previous neuroscientific ecog studies reported activations in the inferior frontal cortex during a passive listening task , , but they did not determine whether these activations could be identified using a common analysis approach , establish the concordance between locations resulting from ecog- and ecs - based mapping , or discuss the feasibility of such passive mapping ecog protocol in the context of presurgical or intraoperative mapping . all three subjects were patients at albany medical center ( albany , new york ) . subject a was diagnosed with a low - grade glioma in the left frontal lobe after presenting with new - onset seizures . all subjects underwent temporary placement of subdural electrode grids to localize seizure foci and eloquent cortex prior to surgical resection . the electrode grids were approved for human use ( ad - tech medical corp . , racine , wi and pmt corp . , chanhassen , mn ) and covered different areas within frontal , temporal , and parietal lobes of the left hemisphere . most importantly , all three subjects had coverage of frontal lobe language areas , and two of the three ( subjects b and c ) also had coverage of temporal lobe language areas . electrodes consisted of platinum iridium discs ( 4 mm in diameter , 2.33 mm exposed ) , were embedded in silicone , and were spaced 610 mm apart . the total number of implanted electrodes was 61 , 98 , and 134 in subjects a c , respectively . following subdural grid implantation , each subject had postoperative anterior posterior and lateral radiographs , as well as computer tomography ( ct ) scans to verify grid location . preoperative language lateralization ( ll ) had been assessed previously with fmri in subject a and with wada testing in subjects b and c. based on these evaluations , language was lateralized to the left hemisphere in all three subjects . all subjects signed informed consent to participate in the study , which was approved by the institutional review board of albany medical college and the human research protections office of the us army medical research and materiel command . once subjects recovered postoperatively , we recorded ecog signals at the bedside using general - purpose bci2000 software , , which controlled eight 16-channel g.usbamp biosignal acquisition devices ( g.tec , graz , austria ) . to ensure integrity of clinical data collection , a connector split the electrode cables into two separate sets one set was connected to the clinical monitoring system , and another set was connected to the g.usbamp acquisition devices . the ecog signals were amplified , digitized at 1200 hz , and stored by bci2000 . we used electrode contacts distant from epileptogenic foci and areas of interest for reference and ground . we created 3d cortical brain models for each subject by submitting preoperative high - resolution magnetic resonance imaging ( mri ) scans to freesurfer software ( http://surfer.nmr.mgh.harvard.edu/ ) . we coregistered mri scans with postoperative ct images using spm software ( http://www.fil.ion.ucl.ac.uk/spm/ ) and identified the stereotactic coordinates of each grid electrode using custom matlab scripts ( the mathworks inc . , natick , ma ) . finally , we visualized the cortical surface of each subject and ecog grid locations using neuralact software . in our study , we asked the subjects to listen to four short stories narrated by a male voice ( stimulus duration : 17.1535.70 s ; interstimulus interval ( isi ) of 10 s ) which were part of the boston aphasia battery . the stimuli were digitized at 44.1 khz in waveform audio file format and binaurally presented to each subject using in - ear monitoring earphones ( 12 to 23.5 khz audio bandwidth , 20 db isolation from environmental noise ) . the subjects did not perform any overt task ( such as repeating words and generating verbs in response to the words they heard ) . we identified ecog activations by detecting task - related changes in the broadband gamma ( 70170 hz ) band . activity in this band has been shown to be related to the average firing rate of neuronal populations directly underneath an electrode , , . a large number of studies have shown that broadband gamma activity increases reliably in task - related cortical areas , , including locations traditionally thought to be active during speech perception , , . to identify those locations that responded to auditory stimulation , we first removed channels that did not contain clear ecog signals ( e.g. , ground / reference channels , channels with broken connections , or channels corrupted by environmental artifacts or interictal activity ) . of a total of 61 , 98 , and 134 channels , this left 59 , 79 , and 132 channels for subjects a c , respectively , which we submitted to subsequent analyses . in these analyses , we high - pass filtered the signals at 0.1 hz to remove drifts and re - referenced the signals to a common average reference ( car ) montage . we band - pass filtered the results in the broadband gamma band using a butterworth filter of order 16 . we then obtained the power of these signals by computing the square of the analytical signal of the hilbert transform , followed by a low - pass filter at 4 hz and down - sampling to 120 hz . finally , we normalized the resulting broadband gamma power estimates by subtracting from them the signal mean calculated from a baseline period ( 6 to 0.5 s prior to the onset of the auditory stimulus ) and by dividing them by the standard deviation of the signal during the baseline period . we determined those locations whose ecog broadband gamma activity following onset of the auditory stimulus ( i.e. , the response period ) was different from that during the baseline period . several studies have shown that , in receptive auditory areas , broadband gamma activity reliably tracks the time course of the envelope of the intensity of the auditory stimulus , or speech stimulus . a few isolated reports documented discrete and brief broadband gamma activations in inferior frontal cortex after the onset of an auditory speech stimulus , that occurred after the activations in receptive auditory areas . based on these reports , we defined the response period as 250750 ms following the onset of the auditory stimulus . then , for each location , we determined the magnitude of the change in ecog broadband gamma power that was related to auditory stimulation by calculating the coefficient of determination ( pearson 's r value ) . finally , we determined the statistical significance of each r value , i.e. , the probability that ecog broadband gamma samples differed in amplitude between the response and baseline periods , using a permutation test . in this test , we cut the ecog broadband gamma power time courses into blocks of 500 ms ( thereby preserving the autocorrelation of the signal ) , randomly permutated the resulting blocks , and finally calculated the corresponding random r value . we repeated the permutation step 1000 times , thus generating a distribution of 1000 random r values at each location . we considered r values to be significant at the 95th percentile of that distribution ( p = 0.05 , bonferroni - corrected for the total number of electrodes in each subject ) . the result of this procedure was a set of locations whose ecog broadband gamma activity was significantly different between the baseline and the response periods and , hence , responded to the speech stimuli . among the resulting locations , we identified those that were situated within inferior frontal cortex . this included all electrodes whose talairach coordinate was within x 28 to 55 , y 8 to + 34 , and z 0 to 28 , consistent with previous observations . the subjects took part in two simple tasks commonly used for this purpose : a picture - naming task , during which subjects were asked to verbally name sequentially presented pictures of simple objects and a verb generation task , during which subjects had to verbally generate verbs associated with simple nouns presented auditorily . different electrode pairs were stimulated to establish whether a given pair induced a disruption of expressive language function , e.g. , speech arrest or hesitation . stimulation intensity typically started at 2 ma and was increased in incremental steps of 2 ma until the neurologist observed clinical effects or after - discharges or reached the 10 ma threshold . all three subjects were patients at albany medical center ( albany , new york ) . subject a was diagnosed with a low - grade glioma in the left frontal lobe after presenting with new - onset seizures . all subjects underwent temporary placement of subdural electrode grids to localize seizure foci and eloquent cortex prior to surgical resection . the electrode grids were approved for human use ( ad - tech medical corp . , racine , wi and pmt corp . , chanhassen , mn ) and covered different areas within frontal , temporal , and parietal lobes of the left hemisphere . most importantly , all three subjects had coverage of frontal lobe language areas , and two of the three ( subjects b and c ) also had coverage of temporal lobe language areas . electrodes consisted of platinum iridium discs ( 4 mm in diameter , 2.33 mm exposed ) , were embedded in silicone , and were spaced 610 mm apart . the total number of implanted electrodes was 61 , 98 , and 134 in subjects a c , respectively . following subdural grid implantation , each subject had postoperative anterior posterior and lateral radiographs , as well as computer tomography ( ct ) scans to verify grid location . preoperative language lateralization ( ll ) had been assessed previously with fmri in subject a and with wada testing in subjects b and c. based on these evaluations , language was lateralized to the left hemisphere in all three subjects . all subjects signed informed consent to participate in the study , which was approved by the institutional review board of albany medical college and the human research protections office of the us army medical research and materiel command . once subjects recovered postoperatively , we recorded ecog signals at the bedside using general - purpose bci2000 software , , which controlled eight 16-channel g.usbamp biosignal acquisition devices ( g.tec , graz , austria ) . to ensure integrity of clinical data collection , a connector split the electrode cables into two separate sets . one set was connected to the clinical monitoring system , and another set was connected to the g.usbamp acquisition devices . the ecog signals were amplified , digitized at 1200 hz , and stored by bci2000 . we used electrode contacts distant from epileptogenic foci and areas of interest for reference and ground . we created 3d cortical brain models for each subject by submitting preoperative high - resolution magnetic resonance imaging ( mri ) scans to freesurfer software ( http://surfer.nmr.mgh.harvard.edu/ ) . we coregistered mri scans with postoperative ct images using spm software ( http://www.fil.ion.ucl.ac.uk/spm/ ) and identified the stereotactic coordinates of each grid electrode using custom matlab scripts ( the mathworks inc . , finally , we visualized the cortical surface of each subject and ecog grid locations using neuralact software . in our study , we asked the subjects to listen to four short stories narrated by a male voice ( stimulus duration : 17.1535.70 s ; interstimulus interval ( isi ) of 10 s ) which were part of the boston aphasia battery . the stimuli were digitized at 44.1 khz in waveform audio file format and binaurally presented to each subject using in - ear monitoring earphones ( 12 to 23.5 khz audio bandwidth , 20 db isolation from environmental noise ) . the subjects did not perform any overt task ( such as repeating words and generating verbs in response to the words they heard ) . we identified ecog activations by detecting task - related changes in the broadband gamma ( 70170 hz ) band . activity in this band has been shown to be related to the average firing rate of neuronal populations directly underneath an electrode , , . a large number of studies have shown that broadband gamma activity increases reliably in task - related cortical areas , , including locations traditionally thought to be active during speech perception , , . to identify those locations that responded to auditory stimulation , we first removed channels that did not contain clear ecog signals ( e.g. , ground / reference channels , channels with broken connections , or channels corrupted by environmental artifacts or interictal activity ) . of a total of 61 , 98 , and 134 channels , this left 59 , 79 , and 132 channels for subjects a c , respectively , which we submitted to subsequent analyses . in these analyses , we high - pass filtered the signals at 0.1 hz to remove drifts and re - referenced the signals to a common average reference ( car ) montage . we band - pass filtered the results in the broadband gamma band using a butterworth filter of order 16 . we then obtained the power of these signals by computing the square of the analytical signal of the hilbert transform , followed by a low - pass filter at 4 hz and down - sampling to 120 hz . finally , we normalized the resulting broadband gamma power estimates by subtracting from them the signal mean calculated from a baseline period ( 6 to 0.5 s prior to the onset of the auditory stimulus ) and by dividing them by the standard deviation of the signal during the baseline period . we determined those locations whose ecog broadband gamma activity following onset of the auditory stimulus ( i.e. , the response period ) was different from that during the baseline period . several studies have shown that , in receptive auditory areas , broadband gamma activity reliably tracks the time course of the envelope of the intensity of the auditory stimulus , or speech stimulus . a few isolated reports documented discrete and brief broadband gamma activations in inferior frontal cortex after the onset of an auditory speech stimulus , that occurred after the activations in receptive auditory areas . based on these reports , we defined the response period as 250750 ms following the onset of the auditory stimulus . then , for each location , we determined the magnitude of the change in ecog broadband gamma power that was related to auditory stimulation by calculating the coefficient of determination ( pearson 's r value ) . finally , we determined the statistical significance of each r value , i.e. , the probability that ecog broadband gamma samples differed in amplitude between the response and baseline periods , using a permutation test . in this test , we cut the ecog broadband gamma power time courses into blocks of 500 ms ( thereby preserving the autocorrelation of the signal ) , randomly permutated the resulting blocks , and finally calculated the corresponding random r value . we repeated the permutation step 1000 times , thus generating a distribution of 1000 random r values at each location . we considered r values to be significant at the 95th percentile of that distribution ( p = 0.05 , bonferroni - corrected for the total number of electrodes in each subject ) . the result of this procedure was a set of locations whose ecog broadband gamma activity was significantly different between the baseline and the response periods and , hence , responded to the speech stimuli . among the resulting locations , we identified those that were situated within inferior frontal cortex . this included all electrodes whose talairach coordinate was within x 28 to 55 , y 8 to + 34 , and z 0 to 28 , consistent with previous observations . the subjects took part in two simple tasks commonly used for this purpose : a picture - naming task , during which subjects were asked to verbally name sequentially presented pictures of simple objects and a verb generation task , during which subjects had to verbally generate verbs associated with simple nouns presented auditorily . different electrode pairs were stimulated to establish whether a given pair induced a disruption of expressive language function , e.g. , speech arrest or hesitation . stimulation intensity typically started at 2 ma and was increased in incremental steps of 2 ma until the neurologist observed clinical effects or after - discharges or reached the 10 ma threshold . 1 . this figure highlights those locations that were identified by our analyses of the ecog signals corresponding to the presentation of the speech stimuli ( filled circles ) and locations that produced arrest of expressive language function using ecs mapping ( yellow circles ) . locations identified by ecog mapping included the expected locations ( highlighted by gray - filled circles ) in superior temporal gyrus and/or perisylvian areas ( all subjects ) as well as in premotor and/or supplementary motor areas ( subjects a and c ) . consistent with previous observations ( see fig . 8 in ) , our method also identified responsive locations on or close to superior precentral gyrus ( patient c ) . most relevant in the context of the present study , our ecog - based mapping identified locations ( highlighted by blue - filled circles ) in inferior frontal cortex ( pars triangularis and/or pars opercularis ) in all three subjects . 1c also presents exemplary time courses of ecog broadband gamma activity in patient c. electrical cortical stimulation mapping identified 12 locations in which stimulation produced expressive language arrest in each subject ( yellow circles ) . the ecs - positive sites overlapped with the sites identified using ecog or were located no more than one contact away . in our study of three patients with chronically implanted subdural electrode grids , we provide initial evidence that it is possible to use passively recorded ecog in response to presentation of speech stimuli to identify not only locations in the receptive language network that are located primarily in the temporal lobe but also locations within the expressive language network in the inferior frontal cortex . with further refinement of the protocol and validation in more subjects , the passive mapping approach described here could lead to a mapping method that may have important clinical implications . the ability to map expressive language cortex with greatly reduced needs for patient participation expands the utility of functional language mapping . specifically , it enables functional mapping of expressive language in patients who are unable to cooperate productively such as pediatric populations or patients suffering from aphasia or psychiatric and cognitive comorbidities . we envision passive language mapping using ecog to either complement existing ecs or fmri mapping protocols or provide an alternative when other expressive language mapping techniques are inadequate . the ecog passive mapping may also have distinct advantages in the time - limited settings of the intraoperative environment . a preliminary map of eloquent expressive language cortex could inform ecs mapping , likely resulting in reduced ecs mapping time and thereby diminishing the risks of patient morbidity , discomfort , and iatrogenic seizures . recent studies already demonstrated the feasibility of intraoperative real - time mapping of motor , and language , , mapping using acutely placed subdural grids . one important general question that remains to be answered is the reason for the variable congruency between ecs and passive ecog in the context of language mapping . using traditional ecog - based mapping tasks ( such as verb generation , picture naming , and passive listening ) , previously reported concordance rates between ecog and ecs range from 38%89% in sensitivity and from 48%92% in specificity , , , . other potential explanations for the discrepancies between ecs and ecog involve the statistical issues that necessarily result from the comparison of the single - electrode ecog method with the pair - wise ecs method , and the fundamental difference between a lesion - based model approach versus a task - based physiologic approach , : while ecog should identify all locations at which neuronal populations subserve the specific function , ecs will only identify the ( potentially small ) subset of those locations that completely disrupt function . thus , ecog can be expected to define a larger area for preservation and underestimate the margin for safe resection . in this context , it is worth noting that patients have been reported to have postoperative language deficits after resection of an ecog(+)/ecs( ) node , , , , , . in a study of 77 patients , postoperative language deficits could be predicted by the number of ecog(+ ) language nodes resected . at present , most resections are based primarily on ecs results even though ecs has never been validated in randomized , clinical trials . this reality implies that , with continued refinement and validation , the ecog method may play an even larger role in presurgical functional mapping in the future . at the same time , without additional information , we currently do not suggest replacing exhaustive ecs mapping but rather argue that ecog - based mapping provides useful and complementary information . another critical question raised by the present study is to what extent the encouraging results presented here generalize to a larger number of patients . for two reasons , we are optimistic that the results in a larger number of patients will echo the initial results reported here . first , several groups have reported activation of the inferior frontal gyrus in response to presentation of passive speech stimuli , , , , , . mazoyer et al . first demonstrated activation of the left inferior frontal gyrus on positron emission tomography ( pet ) scans in 16 subjects while listening to lists of words and stories . several fmri , , , , and ecog , studies have replicated these results using similar tasks . furthermore , it is well known that the blood - oxygen level - dependent ( bold ) signal changes detected using fmri correlate very well with the broadband gamma increases in ecog , , , , , , , which are the basis of ecog - based functional mapping . second , recent evidence indicates that ecog - based mapping can identify locations in expressive language areas when sites in receptive language areas are stimulated using electrical stimulation ( corticocortical evoked potentials ( cceps ) ) , , , . for example , matsumoto et al . described the technique of delivering a single pulse electrical stimulation in the inferior frontal language area and recording a cortical evoked potential in the temporal parietal area , establishing structural neuronal connectivity between the two functional regions . in a smaller subset of patients , they were able to elicit cceps in the inferior frontal and basal temporal regions with stimulation of the temporo parietal language area . this bidirectional connectivity is likely mediated at least in part by the arcuate fasciculus , although the anatomical distribution of the arcuate fasciculus may be more complex than historically assumed , , , . more generally , the language network connectivity model appears to be much more complex than initially believed , with an interplay of numerous cortical regions and white matter tracts , , , . while our initial results are encouraging , different circumstances could temper the significant positive implications of expressive language mapping using passive stimuli . when applied in intraoperative scenarios , different surgical realities ( such as intermittent irrigation on the subdural grid , cable adjustment , variable clinical or cognitive status of the patient ) may lead to lower signal - to - noise ratio and a resultant decrease in ability to detect task - related ecog changes . the duration of mapping may be increased if the grid requires repositioning with reinitiation of tasks . furthermore , it is possible that passive engagement of expressive language function may not elucidate the whole expressive language network . finally , the current study is only reporting results for 3 subjects . further investigation in a larger number of patients is required to assess the potential benefit of our findings to resective neurosurgical planning . furthermore , while our method successfully identified expressive language sites in a patient diagnosed with a left frontal tumor in close proximity to broca 's area ( subject a ) , it is not possible to predict how our method would generalize to patient populations with different types of distorting pathologies . it would also be valuable to determine if our method can identify eloquent expressive language cortex in patients with aphasia . one important general question that remains to be answered is the reason for the variable congruency between ecs and passive ecog in the context of language mapping . using traditional ecog - based mapping tasks ( such as verb generation , picture naming , and passive listening ) , previously reported concordance rates between ecog and ecs range from 38%89% in sensitivity and from 48%92% in specificity , , , . other potential explanations for the discrepancies between ecs and ecog involve the statistical issues that necessarily result from the comparison of the single - electrode ecog method with the pair - wise ecs method , and the fundamental difference between a lesion - based model approach versus a task - based physiologic approach , : while ecog should identify all locations at which neuronal populations subserve the specific function , ecs will only identify the ( potentially small ) subset of those locations that completely disrupt function . thus , ecog can be expected to define a larger area for preservation and underestimate the margin for safe resection . in this context , it is worth noting that patients have been reported to have postoperative language deficits after resection of an ecog(+)/ecs( ) node , , , , , . in a study of 77 patients , postoperative language deficits could be predicted by the number of ecog(+ ) language nodes resected . at present , most resections are based primarily on ecs results even though ecs has never been validated in randomized , clinical trials . this reality implies that , with continued refinement and validation , the ecog method may play an even larger role in presurgical functional mapping in the future . at the same time , without additional information , we currently do not suggest replacing exhaustive ecs mapping but rather argue that ecog - based mapping provides useful and complementary information . another critical question raised by the present study is to what extent the encouraging results presented here generalize to a larger number of patients . for two reasons , we are optimistic that the results in a larger number of patients will echo the initial results reported here . first , several groups have reported activation of the inferior frontal gyrus in response to presentation of passive speech stimuli , , , , , . mazoyer et al . first demonstrated activation of the left inferior frontal gyrus on positron emission tomography ( pet ) scans in 16 subjects while listening to lists of words and stories . several fmri , , , , and ecog , studies have replicated these results using similar tasks . furthermore , it is well known that the blood - oxygen level - dependent ( bold ) signal changes detected using fmri correlate very well with the broadband gamma increases in ecog , , , , , , , which are the basis of ecog - based functional mapping . second , recent evidence indicates that ecog - based mapping can identify locations in expressive language areas when sites in receptive language areas are stimulated using electrical stimulation ( corticocortical evoked potentials ( cceps ) ) , , , . for example , matsumoto et al . described the technique of delivering a single pulse electrical stimulation in the inferior frontal language area and recording a cortical evoked potential in the temporal parietal area , establishing structural neuronal connectivity between the two functional regions . in a smaller subset of patients , they were able to elicit cceps in the inferior frontal and basal temporal regions with stimulation of the temporo this bidirectional connectivity is likely mediated at least in part by the arcuate fasciculus , although the anatomical distribution of the arcuate fasciculus may be more complex than historically assumed , , , . more generally , the language network connectivity model appears to be much more complex than initially believed , with an interplay of numerous cortical regions and white matter tracts , , , . while our initial results are encouraging , different circumstances could temper the significant positive implications of expressive language mapping using passive stimuli . when applied in intraoperative scenarios , different surgical realities ( such as intermittent irrigation on the subdural grid , cable adjustment , variable clinical or cognitive status of the patient ) may lead to lower signal - to - noise ratio and a resultant decrease in ability to detect task - related ecog changes . the duration of mapping may be increased if the grid requires repositioning with reinitiation of tasks . furthermore , it is possible that passive engagement of expressive language function may not elucidate the whole expressive language network . finally , the current study is only reporting results for 3 subjects . further investigation in a larger number of patients is required to assess the potential benefit of our findings to resective neurosurgical planning . furthermore , while our method successfully identified expressive language sites in a patient diagnosed with a left frontal tumor in close proximity to broca 's area ( subject a ) , it is not possible to predict how our method would generalize to patient populations with different types of distorting pathologies . it would also be valuable to determine if our method can identify eloquent expressive language cortex in patients with aphasia . in this paper , we report initial results of an approach to functional mapping of expressive language function that could greatly reduce the need for subject participation . with further refinement and validation , the approach described here may lead to a simple , easy - to - use protocol that would simultaneously identify receptive and expressive language areas for surgical planning . this protocol would be widely applicable in a significantly greater number of patients . finally , because our approach does not require the patient to speak , it opens up the possibility for applying it to patients under general anesthesia . thus , this approach has the potential to completely revolutionize functional language mapping in neurosurgery ; the initial results presented here clearly encourage further investigation . the authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper .

objectivepatients requiring resective brain surgery often undergo functional brain mapping during perioperative planning to localize expressive language areas . currently , all established protocols to perform such mapping require substantial time and patient participation during verb generation or similar tasks . these issues can make language mapping impractical in certain clinical circumstances ( e.g. , during awake craniotomies ) or with certain populations ( e.g. , pediatric patients ) . thus , it is important to develop new techniques that reduce mapping time and the requirement for active patient participation . several neuroscientific studies reported that the mere auditory presentation of speech stimuli can engage not only receptive but also expressive language areas . here , we tested the hypothesis that submission of electrocorticographic ( ecog ) recordings during a short speech listening task to an appropriate analysis procedure can identify eloquent expressive language cortex without requiring the patient to speak.methodsthree patients undergoing temporary placement of subdural electrode grids passively listened to stories while we recorded their ecog activity . we identified those sites whose activity in the broadband gamma range ( 70170 hz ) changed immediately after presentation of the speech stimuli with respect to a prestimulus baseline.resultsour analyses revealed increased broadband gamma activity at distinct locations in the inferior frontal cortex , superior temporal gyrus , and/or perisylvian areas in all three patients and premotor and/or supplementary motor areas in two patients . the sites in the inferior frontal cortex that we identified with our procedure were either on or immediately adjacent to locations identified using electrical cortical stimulation ( ecs ) mapping.conclusionsthe results of this study provide encouraging preliminary evidence that it may be possible that a brief and practical protocol can identify expressive language areas without requiring the patient to speak . this protocol could provide the clinician with a map of expressive language cortex within a few minutes . this may be useful as an adjunct to ecs interrogation or as an alternative to mapping using functional magnetic resonance imaging ( fmri ) . in conclusion , with further development and validation in more subjects , the approach presented here could help in identifying expressive language areas in situations where patients can not speak in response to task instructions .

Introduction Methods Patients Data collection Anatomical mapping Task and stimuli Feature extraction ECoG-based mapping of expressive language cortex ECS-based mapping of expressive language cortex Results Discussion Variable congruency between ECS and ECoG Additional evidence from other studies supports the mapping of expressive language function without requiring the patient to speak Study limitations Conclusions Disclosure

thus , functional language mapping for precise localization of eloquent language areas is necessary for achieving optimal surgical outcomes in such patients . most typically , language mapping is achieved using electrical cortical stimulation ( ecs ) mapping . second , ecs increases the risk of after - discharges or seizures that result from active stimulation of the cortex using electrical impulses . finally , ecs can be difficult to accomplish in the subset of pediatric patients and patients with psychiatric and cognitive comorbidities . these issues have prompted recent and increasingly encouraging investigations suggesting that passive methodologies , such as electrocorticography ( ecog ) or functional magnetic resonance imaging ( fmri ) , may prove useful for functional mapping and may have distinct advantages in efficiency , morbidity , or the range of patients that can benefit from it , , , , , , , , . unfortunately , traditional mapping of expressive language function with any of these existing techniques carries the additional requirement that patients actually speak , i.e. together , these limitations and requirements preclude or greatly impede functional mapping of expressive language areas in certain clinical circumstances ( such as during awake craniotomies ) or with certain populations ( such as pediatric patients ) . hence , it is desirable to have access to a technique that does not electrically stimulate the brain and that eliminates or reduces the requirement for patient participation . such a technique may eventually reduce ecs mapping time by guiding the clinician with a preliminary map of eloquent expressive language cortex . this would not only diminish the risks of patient morbidity , discomfort , and iatrogenic seizures but would also increase the number of patients who could be eligible for functional mapping of expressive language areas . identification of eloquent expressive language cortex without requiring the patient to speak is supported by several findings . previous fmri studies reported activations of the left , and bilateral , , , inferior frontal gyri while subjects listened to speech stimuli but did not perform any overt speaking task . demonstrated using fmri that a passive listening task recruited similar cortical areas as a verb generation task in a cohort of 15 pediatric patients . however , fmri is still expensive and requires substantial expertise that is not available in all centers , and its reliability in the context of functional mapping is still uncertain , . electrocorticographic recordings also provide opportunities for functional mapping in the context of mapping of motor , , , , or language , function , in pediatric patients , and in the operating room , . , while signals are being recorded ) , does not require expertise in signal analysis , and can produce clinically useful results that can readily be compared with ecs results in a few minutes . however , evidence for its utility in identifying expressive language without subject participation was lacking . indeed , only two previous neuroscientific ecog studies reported activations in the inferior frontal cortex during a passive listening task , , but they did not determine whether these activations could be identified using a common analysis approach , establish the concordance between locations resulting from ecog- and ecs - based mapping , or discuss the feasibility of such passive mapping ecog protocol in the context of presurgical or intraoperative mapping . all subjects underwent temporary placement of subdural electrode grids to localize seizure foci and eloquent cortex prior to surgical resection . most importantly , all three subjects had coverage of frontal lobe language areas , and two of the three ( subjects b and c ) also had coverage of temporal lobe language areas . all subjects signed informed consent to participate in the study , which was approved by the institutional review board of albany medical college and the human research protections office of the us army medical research and materiel command . once subjects recovered postoperatively , we recorded ecog signals at the bedside using general - purpose bci2000 software , , which controlled eight 16-channel g.usbamp biosignal acquisition devices ( g.tec , graz , austria ) . we created 3d cortical brain models for each subject by submitting preoperative high - resolution magnetic resonance imaging ( mri ) scans to freesurfer software ( http://surfer.nmr.mgh.harvard.edu/ ) . in our study , we asked the subjects to listen to four short stories narrated by a male voice ( stimulus duration : 17.1535.70 s ; interstimulus interval ( isi ) of 10 s ) which were part of the boston aphasia battery . we identified ecog activations by detecting task - related changes in the broadband gamma ( 70170 hz ) band . to identify those locations that responded to auditory stimulation , we first removed channels that did not contain clear ecog signals ( e.g. in these analyses , we high - pass filtered the signals at 0.1 hz to remove drifts and re - referenced the signals to a common average reference ( car ) montage . we band - pass filtered the results in the broadband gamma band using a butterworth filter of order 16 . finally , we normalized the resulting broadband gamma power estimates by subtracting from them the signal mean calculated from a baseline period ( 6 to 0.5 s prior to the onset of the auditory stimulus ) and by dividing them by the standard deviation of the signal during the baseline period . we determined those locations whose ecog broadband gamma activity following onset of the auditory stimulus ( i.e. several studies have shown that , in receptive auditory areas , broadband gamma activity reliably tracks the time course of the envelope of the intensity of the auditory stimulus , or speech stimulus . a few isolated reports documented discrete and brief broadband gamma activations in inferior frontal cortex after the onset of an auditory speech stimulus , that occurred after the activations in receptive auditory areas . based on these reports , we defined the response period as 250750 ms following the onset of the auditory stimulus . then , for each location , we determined the magnitude of the change in ecog broadband gamma power that was related to auditory stimulation by calculating the coefficient of determination ( pearson 's r value ) . in this test , we cut the ecog broadband gamma power time courses into blocks of 500 ms ( thereby preserving the autocorrelation of the signal ) , randomly permutated the resulting blocks , and finally calculated the corresponding random r value . the result of this procedure was a set of locations whose ecog broadband gamma activity was significantly different between the baseline and the response periods and , hence , responded to the speech stimuli . among the resulting locations , we identified those that were situated within inferior frontal cortex . the subjects took part in two simple tasks commonly used for this purpose : a picture - naming task , during which subjects were asked to verbally name sequentially presented pictures of simple objects and a verb generation task , during which subjects had to verbally generate verbs associated with simple nouns presented auditorily . different electrode pairs were stimulated to establish whether a given pair induced a disruption of expressive language function , e.g. all subjects underwent temporary placement of subdural electrode grids to localize seizure foci and eloquent cortex prior to surgical resection . most importantly , all three subjects had coverage of frontal lobe language areas , and two of the three ( subjects b and c ) also had coverage of temporal lobe language areas . all subjects signed informed consent to participate in the study , which was approved by the institutional review board of albany medical college and the human research protections office of the us army medical research and materiel command . once subjects recovered postoperatively , we recorded ecog signals at the bedside using general - purpose bci2000 software , , which controlled eight 16-channel g.usbamp biosignal acquisition devices ( g.tec , graz , austria ) . we created 3d cortical brain models for each subject by submitting preoperative high - resolution magnetic resonance imaging ( mri ) scans to freesurfer software ( http://surfer.nmr.mgh.harvard.edu/ ) . in our study , we asked the subjects to listen to four short stories narrated by a male voice ( stimulus duration : 17.1535.70 s ; interstimulus interval ( isi ) of 10 s ) which were part of the boston aphasia battery . the subjects did not perform any overt task ( such as repeating words and generating verbs in response to the words they heard ) . we identified ecog activations by detecting task - related changes in the broadband gamma ( 70170 hz ) band . a large number of studies have shown that broadband gamma activity increases reliably in task - related cortical areas , , including locations traditionally thought to be active during speech perception , , . to identify those locations that responded to auditory stimulation , we first removed channels that did not contain clear ecog signals ( e.g. in these analyses , we high - pass filtered the signals at 0.1 hz to remove drifts and re - referenced the signals to a common average reference ( car ) montage . we band - pass filtered the results in the broadband gamma band using a butterworth filter of order 16 . finally , we normalized the resulting broadband gamma power estimates by subtracting from them the signal mean calculated from a baseline period ( 6 to 0.5 s prior to the onset of the auditory stimulus ) and by dividing them by the standard deviation of the signal during the baseline period . we determined those locations whose ecog broadband gamma activity following onset of the auditory stimulus ( i.e. several studies have shown that , in receptive auditory areas , broadband gamma activity reliably tracks the time course of the envelope of the intensity of the auditory stimulus , or speech stimulus . a few isolated reports documented discrete and brief broadband gamma activations in inferior frontal cortex after the onset of an auditory speech stimulus , that occurred after the activations in receptive auditory areas . based on these reports , we defined the response period as 250750 ms following the onset of the auditory stimulus . then , for each location , we determined the magnitude of the change in ecog broadband gamma power that was related to auditory stimulation by calculating the coefficient of determination ( pearson 's r value ) . , the probability that ecog broadband gamma samples differed in amplitude between the response and baseline periods , using a permutation test . in this test , we cut the ecog broadband gamma power time courses into blocks of 500 ms ( thereby preserving the autocorrelation of the signal ) , randomly permutated the resulting blocks , and finally calculated the corresponding random r value . the result of this procedure was a set of locations whose ecog broadband gamma activity was significantly different between the baseline and the response periods and , hence , responded to the speech stimuli . among the resulting locations , we identified those that were situated within inferior frontal cortex . the subjects took part in two simple tasks commonly used for this purpose : a picture - naming task , during which subjects were asked to verbally name sequentially presented pictures of simple objects and a verb generation task , during which subjects had to verbally generate verbs associated with simple nouns presented auditorily . different electrode pairs were stimulated to establish whether a given pair induced a disruption of expressive language function , e.g. this figure highlights those locations that were identified by our analyses of the ecog signals corresponding to the presentation of the speech stimuli ( filled circles ) and locations that produced arrest of expressive language function using ecs mapping ( yellow circles ) . locations identified by ecog mapping included the expected locations ( highlighted by gray - filled circles ) in superior temporal gyrus and/or perisylvian areas ( all subjects ) as well as in premotor and/or supplementary motor areas ( subjects a and c ) . most relevant in the context of the present study , our ecog - based mapping identified locations ( highlighted by blue - filled circles ) in inferior frontal cortex ( pars triangularis and/or pars opercularis ) in all three subjects . 1c also presents exemplary time courses of ecog broadband gamma activity in patient c. electrical cortical stimulation mapping identified 12 locations in which stimulation produced expressive language arrest in each subject ( yellow circles ) . the ecs - positive sites overlapped with the sites identified using ecog or were located no more than one contact away . in our study of three patients with chronically implanted subdural electrode grids , we provide initial evidence that it is possible to use passively recorded ecog in response to presentation of speech stimuli to identify not only locations in the receptive language network that are located primarily in the temporal lobe but also locations within the expressive language network in the inferior frontal cortex . with further refinement of the protocol and validation in more subjects , the passive mapping approach described here could lead to a mapping method that may have important clinical implications . the ability to map expressive language cortex with greatly reduced needs for patient participation expands the utility of functional language mapping . specifically , it enables functional mapping of expressive language in patients who are unable to cooperate productively such as pediatric populations or patients suffering from aphasia or psychiatric and cognitive comorbidities . we envision passive language mapping using ecog to either complement existing ecs or fmri mapping protocols or provide an alternative when other expressive language mapping techniques are inadequate . the ecog passive mapping may also have distinct advantages in the time - limited settings of the intraoperative environment . a preliminary map of eloquent expressive language cortex could inform ecs mapping , likely resulting in reduced ecs mapping time and thereby diminishing the risks of patient morbidity , discomfort , and iatrogenic seizures . one important general question that remains to be answered is the reason for the variable congruency between ecs and passive ecog in the context of language mapping . in this context , it is worth noting that patients have been reported to have postoperative language deficits after resection of an ecog(+)/ecs( ) node , , , , , . this reality implies that , with continued refinement and validation , the ecog method may play an even larger role in presurgical functional mapping in the future . another critical question raised by the present study is to what extent the encouraging results presented here generalize to a larger number of patients . for two reasons , we are optimistic that the results in a larger number of patients will echo the initial results reported here . first , several groups have reported activation of the inferior frontal gyrus in response to presentation of passive speech stimuli , , , , , . furthermore , it is well known that the blood - oxygen level - dependent ( bold ) signal changes detected using fmri correlate very well with the broadband gamma increases in ecog , , , , , , , which are the basis of ecog - based functional mapping . second , recent evidence indicates that ecog - based mapping can identify locations in expressive language areas when sites in receptive language areas are stimulated using electrical stimulation ( corticocortical evoked potentials ( cceps ) ) , , , . described the technique of delivering a single pulse electrical stimulation in the inferior frontal language area and recording a cortical evoked potential in the temporal parietal area , establishing structural neuronal connectivity between the two functional regions . in a smaller subset of patients , they were able to elicit cceps in the inferior frontal and basal temporal regions with stimulation of the temporo parietal language area . more generally , the language network connectivity model appears to be much more complex than initially believed , with an interplay of numerous cortical regions and white matter tracts , , , . while our initial results are encouraging , different circumstances could temper the significant positive implications of expressive language mapping using passive stimuli . when applied in intraoperative scenarios , different surgical realities ( such as intermittent irrigation on the subdural grid , cable adjustment , variable clinical or cognitive status of the patient ) may lead to lower signal - to - noise ratio and a resultant decrease in ability to detect task - related ecog changes . furthermore , it is possible that passive engagement of expressive language function may not elucidate the whole expressive language network . furthermore , while our method successfully identified expressive language sites in a patient diagnosed with a left frontal tumor in close proximity to broca 's area ( subject a ) , it is not possible to predict how our method would generalize to patient populations with different types of distorting pathologies . it would also be valuable to determine if our method can identify eloquent expressive language cortex in patients with aphasia . other potential explanations for the discrepancies between ecs and ecog involve the statistical issues that necessarily result from the comparison of the single - electrode ecog method with the pair - wise ecs method , and the fundamental difference between a lesion - based model approach versus a task - based physiologic approach , : while ecog should identify all locations at which neuronal populations subserve the specific function , ecs will only identify the ( potentially small ) subset of those locations that completely disrupt function . this reality implies that , with continued refinement and validation , the ecog method may play an even larger role in presurgical functional mapping in the future . for two reasons , we are optimistic that the results in a larger number of patients will echo the initial results reported here . first , several groups have reported activation of the inferior frontal gyrus in response to presentation of passive speech stimuli , , , , , . first demonstrated activation of the left inferior frontal gyrus on positron emission tomography ( pet ) scans in 16 subjects while listening to lists of words and stories . furthermore , it is well known that the blood - oxygen level - dependent ( bold ) signal changes detected using fmri correlate very well with the broadband gamma increases in ecog , , , , , , , which are the basis of ecog - based functional mapping . second , recent evidence indicates that ecog - based mapping can identify locations in expressive language areas when sites in receptive language areas are stimulated using electrical stimulation ( corticocortical evoked potentials ( cceps ) ) , , , . described the technique of delivering a single pulse electrical stimulation in the inferior frontal language area and recording a cortical evoked potential in the temporal parietal area , establishing structural neuronal connectivity between the two functional regions . in a smaller subset of patients , they were able to elicit cceps in the inferior frontal and basal temporal regions with stimulation of the temporo this bidirectional connectivity is likely mediated at least in part by the arcuate fasciculus , although the anatomical distribution of the arcuate fasciculus may be more complex than historically assumed , , , . more generally , the language network connectivity model appears to be much more complex than initially believed , with an interplay of numerous cortical regions and white matter tracts , , , . while our initial results are encouraging , different circumstances could temper the significant positive implications of expressive language mapping using passive stimuli . when applied in intraoperative scenarios , different surgical realities ( such as intermittent irrigation on the subdural grid , cable adjustment , variable clinical or cognitive status of the patient ) may lead to lower signal - to - noise ratio and a resultant decrease in ability to detect task - related ecog changes . furthermore , it is possible that passive engagement of expressive language function may not elucidate the whole expressive language network . furthermore , while our method successfully identified expressive language sites in a patient diagnosed with a left frontal tumor in close proximity to broca 's area ( subject a ) , it is not possible to predict how our method would generalize to patient populations with different types of distorting pathologies . it would also be valuable to determine if our method can identify eloquent expressive language cortex in patients with aphasia . in this paper , we report initial results of an approach to functional mapping of expressive language function that could greatly reduce the need for subject participation . with further refinement and validation , the approach described here may lead to a simple , easy - to - use protocol that would simultaneously identify receptive and expressive language areas for surgical planning . finally , because our approach does not require the patient to speak , it opens up the possibility for applying it to patients under general anesthesia . thus , this approach has the potential to completely revolutionize functional language mapping in neurosurgery ; the initial results presented here clearly encourage further investigation .

health issues of migrant populations are an important public health concern . the term migrant covers both emigrants and immigrants . in some of the literature including this paper , it is used synonymously with immigrant defined as a person with two foreign born parents . . the healthcare needs of migrant women have been an area of concern both nationally and internationally , with particular focus on issues of sexual and maternal health including access to maternity care as well as maternal and neonatal outcomes [ 35 ] . state that although varying considerably , the reproductive health of immigrant women in europe is fairly unsatisfactory . research indicates that many migrant women find it difficult to access optimal maternity care and have worse maternal outcomes than native born women [ 79 ] . in sweden and norway the reproductive health of immigrant women is similar to that of the native born population , while it is inferior in uk and italy , which may be due to the fact that receiving countries have different attitudes to immigrants and that the health and welfare of immigrant communities vary . . found that migrant women in maternity care in europe were concerned with preserving their integrity in the new country . many struggled to find meaning , which was related to inadequate communication , lack of connection , striving to cope , struggling to ensure a safe pregnancy and childbirth , and maintaining bodily integrity . some felt insecure and not taken seriously during childbirth . organizational barriers to maternity care also existed , as they were not designed for migrants or adjusted to their needs . on the other hand , although migrant women face many challenges , they also encounter opportunities in their new country of residence . appropriate maternity care presupposes recognition of and feedback from immigrant women . according to mccourt and pearce , minority ethnic women share similar fundamental expectations of maternity care services to those of the wider population of which they are a part , but they experience a greater dissonance between expectations and experiences . small et al . found that immigrant women were less positive about maternity care than nonimmigrant women , although few differences were found in terms of their wishes . migrant women focused on the need for respectful care , assistance with communication , and better information about how care is provided in their new country . according to heaman et al . , migrant women are more likely to have inadequate prenatal care and the factors that contribute to their prenatal care utilization are poorly understood . research on migrant women in maternity care has traditionally focused on the reduction of risk during pregnancy and adverse birth outcomes , as well as the difficulties involved in caring for these women . although there has been little interest in the coping strategies of migrant women , some research has been carried out in this area . for example , gagnon et al . demonstrated that migrant women in canada draw on a wide range of coping strategies and resources to respond to maternal - child health and psychosocial concerns . among the 16 participants in the study , social inclusion was identified as an overarching factor for enhancing resiliency , while margioula - siarkou et al . revealed that immigrant women had better obstetric and neonatal outcomes compared to native women in greece . the increasing diversity of the population in many countries presents a challenge for healthcare professionals . correa - velez and ryan discussed the development of the best practice model of refugee maternity care comprising continuity of care , quality interpreter services , educational strategies , and psychosocial support . in sweden , women from the middle east and sweden who were interviewed three months postpartum were found to focus on well - being and ability to care for the baby . however , beliefs about health and illness differ and affect risk awareness as well as self - care practice . the researchers concluded that professionals play a significant role in supporting women and their families during the transition to motherhood , particularly those who are migrants . a critical factor for women 's experiences at all stages of care is the nature of the women 's interactions with caregivers . the number of immigrants living in norway has more than doubled since 2004 to 600,000 persons in 2013 , corresponding to 12% of the population . norwegian immigrants mainly come from pakistan , iraq , vietnam , somalia , bosnia and herzegovina , iran , turkey , serbia , sri lanka , and poland . as a result , norwegian society has become more multicultural than ever before in the sense that different ethnic groups are a natural part of the same society . the growth in the number of immigrants and the fact that most immigrant women are of child - bearing age explain why an increasing number of babies born in norway have an immigrant mother . in 2012 , 23% of newborns , nearly one in four , had an immigrant mother . equal rights are enshrined in norwegian legislation enabling immigrants and asylum seekers to access health care and medical treatment . the patients ' rights act is intended to ensure that all citizens have equal access to good quality health care by granting patients ' rights in their relations with the health service . the provisions of this act are designed to promote a trusting relationship between the patient and the health service while safeguarding respect for life , integrity , and human dignity . in norway refugees and their relatives who joined them under the family reunification scheme have a right and duty to participate in the introductory programme , the purpose of which is to promote their participation in work and social life . adult immigrants with a residence permit who are eligible for a settlement permit also have a right and duty to attend 300 hours of norwegian language training and social knowledge . the norwegian national clinical guideline for antenatal care states that women with a normal pregnancy should be cared for by a midwife or a general medical practitioner ( gp ) . the woman herself should choose whether to be attended by a gp , a midwife , or a gp and midwife working in cooperation . the midwife in the community works in the maternal and child health clinic together with public health nurses . the national professional guideline for postnatal care emphasizes equity in the postnatal health services , implying that healthcare personnel must respect diversity and recognize all individuals as equal members of society with the same right to health services . a study from norway on healthcare professionals ' perspectives revealed that equity in maternity care for migrant woman is related to managing and supporting educational , relational , and cultural diversity , about which public health nurses and midwives require more information and knowledge . the maternal health coping strategies of migrant women should be viewed in the context of where they live . psychologist urie bronfenbrenner stated that human abilities and their realization depend to a significant degree on the larger social and institutional context . he presented an ecological model , which provides the framework from which researchers study an individual 's relationship within communities and society in general . the model was originally used to explain how a child 's development is influenced by different environments , but is now considered a valuable perspective in health promotion [ 28 , 29 ] . in the present paper a description of the systems in the ecological model thurston and vissandje argue that the health of immigrant women is best understood in a framework that takes gender and the migratory experience into consideration , caring for the individual by focusing on social factors at meso- and macrolevels . the salutogenic perspective means focusing on what creates health rather than taking a traditional medical approach that explores the roots of illness . the sociologist aron antonovsky posed the question : what are the origins of health ? he explored the idea of health as a continuum from ease to dis - ease . migration as well as pregnancy and childbirth can be considered stressful life events . in a salutogenic approach , stressors are open - ended , initiating an interaction between the individual and her / his immediate environment . the emphasis is on the person 's ability to use both internal and external general resistance resources ( grrs ) to manage stressful situations . grrs are found within people bound to their person and capacity , but also as material and nonmaterial resources in their immediate or distant environment . grrs constitute the prerequisites for the development of a person 's sense of coherence ( soc ) . soc is a coping resource that enables a person to choose different strategies for solving different problems or managing life events . the theory of salutogenesis corresponds closely with the key principles of the who ottawa charter for health promotion and is useful in the making of healthy public policy . the focus of this study was migrant women 's movement on the health / dis - ease continuum and how to strengthen their ability to make the best choices for safeguarding health . the aim is to explore the maternal health coping strategies of migrant women in norway . the research questions were as follows : what are migrant women 's expectations and experiences of the maternal and child health services ? in this study a qualitative exploratory , descriptive design with a hermeneutic approach was employed [ 35 , 36 ] . the study , in which 17 migrant women participated , was conducted at a maternal and child health centre by means of semistructured interviews . the participants had attended the centre during pregnancy and after childbirth during the previous year . they were purposively selected and invited to take part in the study by a midwife at the health centre . the midwife contacted all women with a foreign background who had given birth during the previous year . the inclusion criteria were as follows : women who ( 1 ) were born in another country with two foreign born parents , ( 2 ) attended the maternity and child health centre , and ( 3 ) had given birth in the previous 12 months up to six weeks before the interview . they comprised refugees , labour migrants , students , and women married to norwegian men . most of them spoke norwegian , but somali , amharic , and kurdish interpreters were employed in five of the interviews ( table 1 ) . the interviews were conducted between may and december 2012 at the participants ' usual maternal and child health centre . each interview lasted between 45 and 75 minutes , was audio - taped , and later transcribed verbatim . the interviewers ( berit viken and anne lyberg ) encouraged the participants to narrate their expectations and experiences of norwegian maternity services and their life situation in general in the new country and as mothers in particular . some examples of the questions posed are as follows : what are your experiences of maternity health care ? was the cooperation between you and the midwife , public health nurse , and doctor satisfactory ? how did the health providers show respect for you ? do you have any suggestions for changes in the health services ? who do you ask when you are uncertain about your own or your baby 's health ? the interviewers listened to the women 's narratives and posed probing questions to further illuminate their experiences . the study was approved by the manager of the maternal and child health centre . the midwives and public health nurses working at the centre were also informed about the study , which was carried out according to the ethical guidelines for nursing research in the nordic countries . the participants were provided with written and verbal information about the purpose and design of the study by the midwife who tried to ascertain the women 's ability to understand it . the other women who needed a translation spoke different languages and received a verbal translation of the information from an interpreter . the women were advised about the voluntary nature of participation that their name and identity would not be disclosed as well as their right to withdraw at any time without any consequences in terms of maternal and child health care . they were also informed that the audio - tapes and transcripts would be destroyed at the end of the project . as the researchers are responsible for the safety of the participants , the latter were invited to contact a named midwife at the maternity and child health centre if they in any way felt upset after the interview . it started with the authors reading the transcribed text to gain an overview of the data as a whole as well as an understanding of each interview . thereafter , the two research questions what are migrant women 's expectations and experiences of the maternal and child health services ? and what are the maternal health coping strategies of migrant women ? were focused on . the codes were compared , combined , reduced , and sorted into condensed meaning , subthemes , themes , and a main theme ( table 2 ) . to avoid misunderstanding the meaning of the text and ensure validity , the analysis moved back and forth between the emerging categories and the data . when interpreting the interview texts , the three authors discussed and shared the meanings arrived at , thus expanding and adjusting their individual understandings of the participants ' expectations and experiences . thereafter the results of the analysis were interpreted in the light of bronfenbrenner 's ecological model . bronfenbrenner identified four environmental systems with which an individual interacts : the micro- , meso- , exo- , and macrosystem . the microsystem refers to the institutions and groups that most directly influence the child 's development ( or a person 's adaptation , author 's note ) , including family , school , religious institutions , neighbourhood , and peers . the exosystem comprises links between a social setting in which the individual does not play an active role and her / his immediate context . the two interviewers are both public health nurses . this could have affected the participants ' answers in that they might have wanted to be positive and not criticize the norwegian health services . on the other hand , all participants had a residence permit in norway and did not have a reason to fear that their answers could have negative consequences for them , as described by liamputtong . it became clear when the interviews started that some of the participants had not understood all the information provided beforehand . informed consent presupposes that the individual understands the information and can make a voluntary decision to enrol and participate in the research . this can be a challenging task in cross - cultural research because issues of culture come into play . when the participants afterwards were asked how it was to talk with the interviewer , they all had a positive attitude towards giving their opinions about subjects that were important to them . the interview data revealed the participants ' strong will to integrate into the new society while at the same time maintaining traditions from their country of origin . the analysis revealed one main theme : keeping original traditions and at the same time being willing to integrate into norwegian society , based on four themes , balancing their sense of belongingness , seeking information and support from healthcare professionals , being open to new opportunities , and focusing on feeling safe in the new country . the results will be presented and explained in the light of the four systems in the ecological model ( table 3 ) . the migrant women 's life in a family and connections to other relatives and friends are part of the microsystem . several mothers stated that having a child gave them a feeling of belonging and that they followed the child 's rhythm . for example , one of the women could not understand why she should pump breast milk and keep it in a bottle because as she expressed : passes so quickly , i breastfeed at least three times every night and sleep when the baby sleeps . i do not have much time to do other things than taking care of her . ( p15 ) the day passes so quickly , i breastfeed at least three times every night and sleep when the baby sleeps . i do not have much time to do other things than taking care of her . ( p15 ) faith in god was important for several of the migrant women and gave them confidence in daily life.i am comforted when i read the koran , as you do not get any negative thoughts , just positive ones and trust . ( p6 ) i am comforted when i read the koran , as you do not get any negative thoughts , just positive ones and trust . ( p6 ) other women stated that their belief in god is not strong , but that it is important to follow some religious traditions such as the celebration of eid al - fitr after ramadan . this is a very common activity among norwegian families with children and everybody has a pram , but for migrants it can be a new experience.at weekends we normally take a walk in the forest . many took cod liver oil to prevent lack of vitamin d , which is a tradition in norway and recommended for both mothers and children . at the same time many of the women said that they made use of advice from their country of origin , for example , herbal tea for the baby 's stomach ache . an asian woman struggled with contradictory advice from healthcare professionals in norway and from her mother and healthcare professionals in her native country . she says that her gp had advised her to continue exercising as there was no need to change her lifestyle . she believed that exercise was the reason for her miscarriage because in her native country it is usual for women to be very careful during the first three months of pregnancy . therefore it was confusing to receive different advice in norway both from her norwegian husband and the gp . when this woman became pregnant again , she travelled back to her country of origin and followed the advice not to exercise . she also brought some medicine back to norway to prevent a miscarriage and this made her feel safer . ( p14 ) the participants appreciated contact with other migrants but also longed for friendships with norwegians . some women found it difficult to get to know people in their new surroundings.i feel a bit lonely in this town . my midwife introduced me to a woman from poland in the same situation as myself and she has become my friend now . it is important to be open and speak norwegian . in another town , where i used to live , the health centre organised a group for women who had given birth , in which i felt very welcome . my midwife introduced me to a woman from poland in the same situation as myself and she has become my friend now . it is important to be open and speak norwegian . in another town , where i used to live , the health centre organised a group for women who had given birth , in which i felt very welcome . ( p12 ) it was of major importance for all the migrant women to keep in contact with family and relatives in their country of origin , which they achieved by means of telephone and the internet . they also travelled there on holidays or to a neighbouring country if they were unable to go back to their own country , especially in the winter , to experience sun and hot weather . ( p11 ) many women highlighted family unity as the most important aspect of life.unity in the family is the most important thing in life . having gone through what we experienced , we never ever want to be separated again . ( p5 ) unity in the family is the most important thing in life . having gone through what we experienced , we never ever want to be separated again . ( p5 ) a woman who had no family from her native country in norway said that she received a great deal of help from her husband 's family . the women wanted to strengthen their sense of belonging to norwegian society while at the same time maintaining their sense of belonging to their native country . one woman who came from a country during a war twenty years ago commented that she could not imagine living anywhere other than norway now . however , her dream was to move back to her native country and be buried there . migrant women 's interactions with persons and institutions in the local community , such as the health services , make up the mesosystem . with the exception of one woman , all the participants had undergone several check - ups with the midwife during pregnancy and found the prenatal care very satisfactory . some women compared the health services in norway with the poor health services in their native country . a woman from an african country plagued by war said : i think norway is best when you are pregnant . the culture is very different to that of my home country and i miss my family , but i had very good discussions with the midwife , who made me relax and not think too much about the delivery . we have midwives in my country too , but we do not have a big health centre . sometimes the mother or the child can die . but here in norway it is the best you can get . the child check - ups , the hospital and the midwife are all very good . the culture is very different to that of my home country and i miss my family , but i had very good discussions with the midwife , who made me relax and not think too much about the delivery . we have midwives in my country too , but we do not have a big health centre . sometimes the mother or the child can die . but here in norway it is the best you can get . the child check - ups , the hospital and the midwife are all very good . ( p4 ) the participants required knowledge about pregnancy to gain confidence and feel secure about their own and their baby 's health . they were well informed and prepared for the birth , while many had visited the maternity ward before delivery . some said they were lucky to have had the same midwife at the pregnancy check - ups and at childbirth . the women who had experienced pregnancies or deliveries that required extra care were very grateful to be in norway.i had long contractions and they helped me so that everything went well . i am thankful to the midwives who stood beside me the whole day and ensured the baby came out . if my child had been born in my home country he would not have survived . ( p16 ) i had long contractions and they helped me so that everything went well . i am thankful to the midwives who stood beside me the whole day and ensured the baby came out . if my child had been born in my home country he would not have survived . ( p16 ) some of the women were circumcised and needed surgery to open the vagina before delivery.it was my first child and i had just arrived in norway . she sent the information to the hospital and i got the help i needed to open the vagina before the baby came out . ( p6 ) it was my first child and i had just arrived in norway . she sent the information to the hospital and i got the help i needed to open the vagina before the baby came out . ( p6 ) there were both good and bad experiences of childbirth in norway , sometimes due to poor communication . when this woman was in labour she described being treated with indifference and lack of respect , which she thought was because she was new in norway and did not speak the language . ( p10 ) the women considered it important to accept what was offered by the health services , also after childbirth.i am satisfied with the health centre . i got very good follow - up when i had difficulty with breast - feeding . i got very good follow - up when i had difficulty with breast - feeding . ( p12 ) the women appreciated the information they received from the midwife during pregnancy and from the public health nurse in early motherhood . the information was provided during regular check - ups as well as open hours at the maternal and child health centre when there was no need to make an appointment . some women need more follow - up from the maternal and child health centre because certain aspects of knowledge are less familiar to them compared with many ethnic norwegian women . this can concern relatively minor issues such as different cultural traditions when the child is ready to eat solid food , for example , what sort of porridge they should start with . another more serious matter is lack of knowledge about norwegian legislation that prohibits hitting children . she explained that she was new in norway , did not speak the language , and did not know anything about norwegian culture and parenting . she did not understand what to do and the child welfare service took her children into care . she has become very preoccupied with obtaining new knowledge and wants new immigrants to be informed about the right thing to do . this system consists of decision makers ( such as municipal officials and politicians ) with whom the women have no direct contact but who still affect their lives . many saw life in norway as a new opportunity . living in a welfare state allowed them an opportunity to learn norwegian and study culture and society whilst receiving financial benefit . all the participants wanted to learn more norwegian and obtain a higher educational qualification and at a later stage paid employment . they wanted their children to attend kindergarten and were impressed by the opportunities it presented to their children and themselves . learning norwegian as a part of the introductory programme was very important to all the participants . they wished to improve their language skills and have an opportunity to practise the language.i have a positive opinion of the municipality . i also had the opportunity to learn norwegian and earn money by attending the introductory programme . i also had the opportunity to learn norwegian and earn money by attending the introductory programme . ( p6 ) one woman appreciated not needing social security payment because her husband was employed and she was in receipt of introduction money . the migrant women wanted to work outside the home and were prepared to send their children to kindergarten . nevertheless , not all of them could find suitable work . a woman from africa had a job for several years in another european country , but she found it difficult to obtain a permanent position in norway . she had been in trial placements but when they finished she did not obtain any further work.if you try you can manage anything , i just need a chance . if we are given a chance we will communicate and learn the language . i will go to high school next year and then my child will attend kindergarten . then i will learn to drive and maybe start studying . ( p8 ) if you try you can manage anything , i just need a chance . if we are given a chance we will communicate and learn the language . many came from countries in the throes of war and conflict with no public schools and social security . when they longed for their family and native country , the thought of their own and their children 's safe future in norway helped them to manage their feelings.sometimes when i think of moving back , i remind myself of how safe our future is in norway . i think it is a good environment for children , with fresh air and clean , safe food . ( p14 ) sometimes when i think of moving back , i remind myself of how safe our future is in norway . i think it is a good environment for children , with fresh air and clean , safe food . ( p14 ) one woman who had lived for many years in norway expressed her need to process memories from the war but also to move on . a woman from asia explained how her opinion changed about the use of medications for the common cold.when i first came i was very irritated because i could not buy medication without a prescription . after a while i understood that we do not need medications if we just have a cold . ( p2 ) when i first came i was very irritated because i could not buy medication without a prescription . after a while i understood that we do not need medications if we just have a cold . ( p2 ) other women wanted more examinations by specialists , for example , by a gynaecologist instead of a gp when pregnant and by a paediatrician to check their children in the maternal and child health centre . several women wished for more ultrasound scans during pregnancy.i would have liked more ultrasound scans . ( p7 ) this woman paid for extra check - ups with a private doctor to obtain the ultrasound examinations she wanted and felt safer after those examinations . the aim of this study was to explore the maternal health coping strategies of migrant women in norway . the main theme that emerged was keeping original traditions while at the same time being willing to integrate into norwegian society . they had mostly positive experiences of the maternal health services , which were reflected in their willingness to integrate into society . in situations where they had negative experiences , the women used a wider range of strategies that involved keeping their original traditions and travelling to their native country to seek advice . the discussion will focus on migrant women 's coping strategies in accordance with a salutogenic approach , including their expectations and experiences , both within and outside the maternal health services . sense of coherence ( soc ) appears to influence the women 's actions and coping strategies when they become pregnant and after childbirth . balancing their sense of belongingness to family members and institutions in norway with that to their relatives in their country of origin affected the women 's sense of meaningfulness , which is the motivational component of the soc . the most meaningful task for the women is taking care of their own and the baby 's health during pregnancy and childbirth . this is consistent with other studies which underline childbirth as a positive life event [ 41 , 42 ] . another important strategy is being open to new opportunities , which involves the willingness and efforts of migrant women to learn norwegian by taking part in the introductory programme . despite the fact that this programme is mandatory for all new refugees , the migrant women in our study were all very interested in it , especially learning norwegian . the programme is provided in the community and is thus a part of migrant women 's mesosystem . in the mesosystem can increase the women 's capabilities ; for example language skills can help them to comprehend health information and social structures in norwegian society . it also strengthens their social networks and makes their daily life more manageable by creating independency through not having to rely on translation by family members and interpreters . ability to communicate in norwegian also provides a stronger sense of belonging to society as it enables women to strengthen their social network and start new activities such as entering into paid employment . nussbaum contends that economic independence enhances women 's self - respect and capacity for choice . balancing their sense of belongingness can also be seen from a global perspective in accordance with haith - cooper and bradshaw and the model the pregnant woman in the global context , which emphasizes the environment of migrant women prior to , during , and after migration . another coping strategy is seeking information and support from family and healthcare professionals , where the women interact both within their microsystem and with the maternal and child health services , which are a part of their mesosystem . this strategy can be seen in connection with personal agency , the notion that the person has the capacity to make her ( or his ) own choices and exercise autonomy over personal life events and circumstances . support during childbirth was also significant for their capacity building . however , some of the women felt exposed when meeting healthcare professionals as they experienced a lack of communication and social interaction . the women also searched for information from the media both in norway and other countries as well as from healthcare professionals and family in their native country . similar findings are described in a study where somali women said they received health information from , among other sources , an english television channel . most of the women in the present study maintained close links with family members in their country of origin through the internet , telephone , and/or international travel . this can be considered part of the macrosystem and is supported by other studies that have a transnational perspective on migration . the identity and social practices of migrants transcend national borders [ 48 , 49 ] . for example , a traditional medical perspective on pregnancy and childbirth was maintained by several of the participants , which contrasts with the concept of normal childbirth held by many norwegian midwives and described in the international literature [ 50 , 51 ] . the maternal health coping strategies of migrant women are also influenced by their health literacy , which is a composite term used to describe the capacities of persons to meet the complex demands related to health information in modern society . it is a resource that equips people to navigate healthcare systems , critically assess information , and take greater control of their health . two different concepts reflect health literacy , a clinical risk and a personal asset . boerleider et al . found that suboptimal health literacy was one of the difficulties midwives encountered when caring for nonwestern women in netherlands , which can be said to represent the risk approach . in the present study we used the asset approach , in which health literacy applies to the development of skills and capacities that enable migrant women to exert greater control over their health and the factors that shape it . consequently , health literacy affects women 's maternal health coping strategies . the introductory programme , maternal and child health services , and social media interact and contribute to the women 's health literacy and thus their comprehensibility and manageability . aasen highlighted the importance of language skills for refugee women to cope with their daily life . some of the participants in this study had poor education , which affected their health literacy . the women who could not read and write were largely dependent on verbal information from healthcare professionals to increase their comprehensibility . consistent with these findings is the need for more innovative forms of communication that go beyond the written word [ 10 , 47 ] . in their encounters with healthcare professionals the women who did not speak norwegian were highly dependent on interpreters to achieve understanding . an important coping strategy is focusing on feeling safe in the new country , which is connected to structures in norwegian society and thus part of the macrosystem . when the women longed for their home country , they focused on the safety of themselves and their children . while this is related to norway being a relatively peaceful country , it can also be seen as confidence in the welfare system . experiencing comfort and social security in a safe society constituted a major part of the women 's meaningfulness . norway 's welfare system , migrant , and refugee policy affect the social and economic conditions as well as the health services offered to migrant women . this is in accordance with the results of a group of researchers from germany , canada , and the united kingdom , who compared migration and maternity care in the three countries . they found that while the design and delivery of maternal health services play a crucial role in ensuring positive experiences and good maternity outcomes for migrant women , these services must be understood within a much wider framework . the main resources of migrant women in this study were their own capacity to readjust their lives , support from family and friends both in norway and abroad , and information and support from healthcare professionals as well as financial support and educational opportunities provided by the municipality . thus migrant women 's maternal health coping strategies are affected by participation in the micro- and mesosystems as well as being influenced by the exo- and macrosystems . reorienting health services is an important strategy in the ottawa charter for health promotion and implies that the health sector should move in a health promotion direction , beyond its responsibility for providing clinical and curative services . it is vital that this leads to a change of attitude towards the organization of health services , where the focus is on the total needs of the individual as a whole person . the results of this study reveal that migrant women 's maternal health coping strategies involve multisetting participation and can be understood by means of a system approach . we advocate a salutogenic approach to maternity care research in order to strengthen the capabilities of migrant women to take care of their own and their baby 's health . this will encourage healthcare professionals , policy makers , and maternity service users to consider maternity care from a health oriented and person centred perspective as opposed to one based on morbidity and mortality . maternal healthcare services should therefore be adjusted to the needs of migrant women . in order to provide culturally competent care , healthcare professionals must be culturally sensitive and aware of their own cultural background . cooperation with other sectors and multidisciplinary work can be part of an ecological approach to strengthen migrant women 's capabilities and wellbeing . this study had a salutogenic approach with focus on migrant women 's resources and capabilities . it was concluded that migrant women were concerned with keeping their own traditions in pregnancy and childbirth while at the same time showing a willingness to integrate into norwegian society . they used different coping strategies connected to family , healthcare professionals , and the education sector . in relation to ecological systems they were also affected by the guidelines and decisions taken in the exo- and macrosystems . to provide quality care , healthcare professionals should use a system approach and focus on the development of migrant women 's capabilities . adaptation of maternal health services for culturally diverse migrant women also requires a culturally sensitive approach on the part of healthcare professionals .

the aim of the study was to explore the maternal health coping strategies of migrant women in norway . the ethnic and cultural background of the norwegian population have become increasingly diverse . a challenge in practice is to adjust maternal health services to migrant women 's specific needs . previous studies have revealed that migrant women have difficulty achieving safe pregnancies and childbirths . data were obtained by means of 17 semistructured interviews with women from south america , europe , the middle east , asia , and africa . qualitative content analysis was employed . one overall theme is as follows : keeping original traditions while at the same time being willing to integrate into norwegian society , and four themes emerged as follows : balancing their sense of belongingness ; seeking information and support from healthcare professionals ; being open to new opportunities and focusing on feeling safe in the new country . the results were interpreted in the light of bronfenbrenner 's ecological model . to provide quality care , healthcare professionals should focus on the development of migrant women 's capabilities . adaptation of maternal health services for culturally diverse migrant women also requires a culturally sensitive approach on the part of healthcare professionals .

1. Introduction 2. Methods 3. Results and Interpretation 4. Discussion 5. Recommendations 6. Conclusions

the healthcare needs of migrant women have been an area of concern both nationally and internationally , with particular focus on issues of sexual and maternal health including access to maternity care as well as maternal and neonatal outcomes [ 35 ] . state that although varying considerably , the reproductive health of immigrant women in europe is fairly unsatisfactory . in sweden and norway the reproductive health of immigrant women is similar to that of the native born population , while it is inferior in uk and italy , which may be due to the fact that receiving countries have different attitudes to immigrants and that the health and welfare of immigrant communities vary . found that migrant women in maternity care in europe were concerned with preserving their integrity in the new country . on the other hand , although migrant women face many challenges , they also encounter opportunities in their new country of residence . according to mccourt and pearce , minority ethnic women share similar fundamental expectations of maternity care services to those of the wider population of which they are a part , but they experience a greater dissonance between expectations and experiences . migrant women focused on the need for respectful care , assistance with communication , and better information about how care is provided in their new country . research on migrant women in maternity care has traditionally focused on the reduction of risk during pregnancy and adverse birth outcomes , as well as the difficulties involved in caring for these women . although there has been little interest in the coping strategies of migrant women , some research has been carried out in this area . demonstrated that migrant women in canada draw on a wide range of coping strategies and resources to respond to maternal - child health and psychosocial concerns . among the 16 participants in the study , social inclusion was identified as an overarching factor for enhancing resiliency , while margioula - siarkou et al . revealed that immigrant women had better obstetric and neonatal outcomes compared to native women in greece . the increasing diversity of the population in many countries presents a challenge for healthcare professionals . correa - velez and ryan discussed the development of the best practice model of refugee maternity care comprising continuity of care , quality interpreter services , educational strategies , and psychosocial support . in sweden , women from the middle east and sweden who were interviewed three months postpartum were found to focus on well - being and ability to care for the baby . a critical factor for women 's experiences at all stages of care is the nature of the women 's interactions with caregivers . the number of immigrants living in norway has more than doubled since 2004 to 600,000 persons in 2013 , corresponding to 12% of the population . as a result , norwegian society has become more multicultural than ever before in the sense that different ethnic groups are a natural part of the same society . the growth in the number of immigrants and the fact that most immigrant women are of child - bearing age explain why an increasing number of babies born in norway have an immigrant mother . the provisions of this act are designed to promote a trusting relationship between the patient and the health service while safeguarding respect for life , integrity , and human dignity . in norway refugees and their relatives who joined them under the family reunification scheme have a right and duty to participate in the introductory programme , the purpose of which is to promote their participation in work and social life . the midwife in the community works in the maternal and child health clinic together with public health nurses . the national professional guideline for postnatal care emphasizes equity in the postnatal health services , implying that healthcare personnel must respect diversity and recognize all individuals as equal members of society with the same right to health services . a study from norway on healthcare professionals ' perspectives revealed that equity in maternity care for migrant woman is related to managing and supporting educational , relational , and cultural diversity , about which public health nurses and midwives require more information and knowledge . the maternal health coping strategies of migrant women should be viewed in the context of where they live . he presented an ecological model , which provides the framework from which researchers study an individual 's relationship within communities and society in general . in the present paper a description of the systems in the ecological model thurston and vissandje argue that the health of immigrant women is best understood in a framework that takes gender and the migratory experience into consideration , caring for the individual by focusing on social factors at meso- and macrolevels . the emphasis is on the person 's ability to use both internal and external general resistance resources ( grrs ) to manage stressful situations . grrs constitute the prerequisites for the development of a person 's sense of coherence ( soc ) . the theory of salutogenesis corresponds closely with the key principles of the who ottawa charter for health promotion and is useful in the making of healthy public policy . the focus of this study was migrant women 's movement on the health / dis - ease continuum and how to strengthen their ability to make the best choices for safeguarding health . the aim is to explore the maternal health coping strategies of migrant women in norway . the research questions were as follows : what are migrant women 's expectations and experiences of the maternal and child health services ? the study , in which 17 migrant women participated , was conducted at a maternal and child health centre by means of semistructured interviews . they were purposively selected and invited to take part in the study by a midwife at the health centre . the inclusion criteria were as follows : women who ( 1 ) were born in another country with two foreign born parents , ( 2 ) attended the maternity and child health centre , and ( 3 ) had given birth in the previous 12 months up to six weeks before the interview . they comprised refugees , labour migrants , students , and women married to norwegian men . most of them spoke norwegian , but somali , amharic , and kurdish interpreters were employed in five of the interviews ( table 1 ) . the interviews were conducted between may and december 2012 at the participants ' usual maternal and child health centre . each interview lasted between 45 and 75 minutes , was audio - taped , and later transcribed verbatim . the interviewers ( berit viken and anne lyberg ) encouraged the participants to narrate their expectations and experiences of norwegian maternity services and their life situation in general in the new country and as mothers in particular . some examples of the questions posed are as follows : what are your experiences of maternity health care ? was the cooperation between you and the midwife , public health nurse , and doctor satisfactory ? do you have any suggestions for changes in the health services ? the study was approved by the manager of the maternal and child health centre . the midwives and public health nurses working at the centre were also informed about the study , which was carried out according to the ethical guidelines for nursing research in the nordic countries . the participants were provided with written and verbal information about the purpose and design of the study by the midwife who tried to ascertain the women 's ability to understand it . they were also informed that the audio - tapes and transcripts would be destroyed at the end of the project . as the researchers are responsible for the safety of the participants , the latter were invited to contact a named midwife at the maternity and child health centre if they in any way felt upset after the interview . it started with the authors reading the transcribed text to gain an overview of the data as a whole as well as an understanding of each interview . thereafter , the two research questions what are migrant women 's expectations and experiences of the maternal and child health services ? and what are the maternal health coping strategies of migrant women ? to avoid misunderstanding the meaning of the text and ensure validity , the analysis moved back and forth between the emerging categories and the data . when interpreting the interview texts , the three authors discussed and shared the meanings arrived at , thus expanding and adjusting their individual understandings of the participants ' expectations and experiences . thereafter the results of the analysis were interpreted in the light of bronfenbrenner 's ecological model . this could have affected the participants ' answers in that they might have wanted to be positive and not criticize the norwegian health services . on the other hand , all participants had a residence permit in norway and did not have a reason to fear that their answers could have negative consequences for them , as described by liamputtong . it became clear when the interviews started that some of the participants had not understood all the information provided beforehand . informed consent presupposes that the individual understands the information and can make a voluntary decision to enrol and participate in the research . the interview data revealed the participants ' strong will to integrate into the new society while at the same time maintaining traditions from their country of origin . the analysis revealed one main theme : keeping original traditions and at the same time being willing to integrate into norwegian society , based on four themes , balancing their sense of belongingness , seeking information and support from healthcare professionals , being open to new opportunities , and focusing on feeling safe in the new country . the results will be presented and explained in the light of the four systems in the ecological model ( table 3 ) . the migrant women 's life in a family and connections to other relatives and friends are part of the microsystem . ( p15 ) faith in god was important for several of the migrant women and gave them confidence in daily life.i am comforted when i read the koran , as you do not get any negative thoughts , just positive ones and trust . many took cod liver oil to prevent lack of vitamin d , which is a tradition in norway and recommended for both mothers and children . at the same time many of the women said that they made use of advice from their country of origin , for example , herbal tea for the baby 's stomach ache . an asian woman struggled with contradictory advice from healthcare professionals in norway and from her mother and healthcare professionals in her native country . my midwife introduced me to a woman from poland in the same situation as myself and she has become my friend now . in another town , where i used to live , the health centre organised a group for women who had given birth , in which i felt very welcome . my midwife introduced me to a woman from poland in the same situation as myself and she has become my friend now . in another town , where i used to live , the health centre organised a group for women who had given birth , in which i felt very welcome . ( p12 ) it was of major importance for all the migrant women to keep in contact with family and relatives in their country of origin , which they achieved by means of telephone and the internet . ( p5 ) unity in the family is the most important thing in life . ( p5 ) a woman who had no family from her native country in norway said that she received a great deal of help from her husband 's family . the women wanted to strengthen their sense of belonging to norwegian society while at the same time maintaining their sense of belonging to their native country . migrant women 's interactions with persons and institutions in the local community , such as the health services , make up the mesosystem . some women compared the health services in norway with the poor health services in their native country . but here in norway it is the best you can get . the child check - ups , the hospital and the midwife are all very good . some said they were lucky to have had the same midwife at the pregnancy check - ups and at childbirth . the women who had experienced pregnancies or deliveries that required extra care were very grateful to be in norway.i had long contractions and they helped me so that everything went well . ( p16 ) some of the women were circumcised and needed surgery to open the vagina before delivery.it was my first child and i had just arrived in norway . ( p6 ) it was my first child and i had just arrived in norway . ( p6 ) there were both good and bad experiences of childbirth in norway , sometimes due to poor communication . the information was provided during regular check - ups as well as open hours at the maternal and child health centre when there was no need to make an appointment . she explained that she was new in norway , did not speak the language , and did not know anything about norwegian culture and parenting . learning norwegian as a part of the introductory programme was very important to all the participants . when they longed for their family and native country , the thought of their own and their children 's safe future in norway helped them to manage their feelings.sometimes when i think of moving back , i remind myself of how safe our future is in norway . ( p14 ) sometimes when i think of moving back , i remind myself of how safe our future is in norway . ( p2 ) other women wanted more examinations by specialists , for example , by a gynaecologist instead of a gp when pregnant and by a paediatrician to check their children in the maternal and child health centre . the aim of this study was to explore the maternal health coping strategies of migrant women in norway . the main theme that emerged was keeping original traditions while at the same time being willing to integrate into norwegian society . they had mostly positive experiences of the maternal health services , which were reflected in their willingness to integrate into society . in situations where they had negative experiences , the women used a wider range of strategies that involved keeping their original traditions and travelling to their native country to seek advice . the discussion will focus on migrant women 's coping strategies in accordance with a salutogenic approach , including their expectations and experiences , both within and outside the maternal health services . sense of coherence ( soc ) appears to influence the women 's actions and coping strategies when they become pregnant and after childbirth . balancing their sense of belongingness to family members and institutions in norway with that to their relatives in their country of origin affected the women 's sense of meaningfulness , which is the motivational component of the soc . another important strategy is being open to new opportunities , which involves the willingness and efforts of migrant women to learn norwegian by taking part in the introductory programme . despite the fact that this programme is mandatory for all new refugees , the migrant women in our study were all very interested in it , especially learning norwegian . the programme is provided in the community and is thus a part of migrant women 's mesosystem . in the mesosystem can increase the women 's capabilities ; for example language skills can help them to comprehend health information and social structures in norwegian society . balancing their sense of belongingness can also be seen from a global perspective in accordance with haith - cooper and bradshaw and the model the pregnant woman in the global context , which emphasizes the environment of migrant women prior to , during , and after migration . another coping strategy is seeking information and support from family and healthcare professionals , where the women interact both within their microsystem and with the maternal and child health services , which are a part of their mesosystem . however , some of the women felt exposed when meeting healthcare professionals as they experienced a lack of communication and social interaction . the women also searched for information from the media both in norway and other countries as well as from healthcare professionals and family in their native country . most of the women in the present study maintained close links with family members in their country of origin through the internet , telephone , and/or international travel . this can be considered part of the macrosystem and is supported by other studies that have a transnational perspective on migration . for example , a traditional medical perspective on pregnancy and childbirth was maintained by several of the participants , which contrasts with the concept of normal childbirth held by many norwegian midwives and described in the international literature [ 50 , 51 ] . the maternal health coping strategies of migrant women are also influenced by their health literacy , which is a composite term used to describe the capacities of persons to meet the complex demands related to health information in modern society . it is a resource that equips people to navigate healthcare systems , critically assess information , and take greater control of their health . found that suboptimal health literacy was one of the difficulties midwives encountered when caring for nonwestern women in netherlands , which can be said to represent the risk approach . in the present study we used the asset approach , in which health literacy applies to the development of skills and capacities that enable migrant women to exert greater control over their health and the factors that shape it . consequently , health literacy affects women 's maternal health coping strategies . the introductory programme , maternal and child health services , and social media interact and contribute to the women 's health literacy and thus their comprehensibility and manageability . some of the participants in this study had poor education , which affected their health literacy . the women who could not read and write were largely dependent on verbal information from healthcare professionals to increase their comprehensibility . an important coping strategy is focusing on feeling safe in the new country , which is connected to structures in norwegian society and thus part of the macrosystem . when the women longed for their home country , they focused on the safety of themselves and their children . experiencing comfort and social security in a safe society constituted a major part of the women 's meaningfulness . norway 's welfare system , migrant , and refugee policy affect the social and economic conditions as well as the health services offered to migrant women . this is in accordance with the results of a group of researchers from germany , canada , and the united kingdom , who compared migration and maternity care in the three countries . they found that while the design and delivery of maternal health services play a crucial role in ensuring positive experiences and good maternity outcomes for migrant women , these services must be understood within a much wider framework . the main resources of migrant women in this study were their own capacity to readjust their lives , support from family and friends both in norway and abroad , and information and support from healthcare professionals as well as financial support and educational opportunities provided by the municipality . thus migrant women 's maternal health coping strategies are affected by participation in the micro- and mesosystems as well as being influenced by the exo- and macrosystems . reorienting health services is an important strategy in the ottawa charter for health promotion and implies that the health sector should move in a health promotion direction , beyond its responsibility for providing clinical and curative services . it is vital that this leads to a change of attitude towards the organization of health services , where the focus is on the total needs of the individual as a whole person . the results of this study reveal that migrant women 's maternal health coping strategies involve multisetting participation and can be understood by means of a system approach . we advocate a salutogenic approach to maternity care research in order to strengthen the capabilities of migrant women to take care of their own and their baby 's health . this will encourage healthcare professionals , policy makers , and maternity service users to consider maternity care from a health oriented and person centred perspective as opposed to one based on morbidity and mortality . maternal healthcare services should therefore be adjusted to the needs of migrant women . in order to provide culturally competent care , healthcare professionals must be culturally sensitive and aware of their own cultural background . cooperation with other sectors and multidisciplinary work can be part of an ecological approach to strengthen migrant women 's capabilities and wellbeing . this study had a salutogenic approach with focus on migrant women 's resources and capabilities . it was concluded that migrant women were concerned with keeping their own traditions in pregnancy and childbirth while at the same time showing a willingness to integrate into norwegian society . they used different coping strategies connected to family , healthcare professionals , and the education sector . to provide quality care , healthcare professionals should use a system approach and focus on the development of migrant women 's capabilities . adaptation of maternal health services for culturally diverse migrant women also requires a culturally sensitive approach on the part of healthcare professionals .

a cohort of 4,069 patients with iga nephropathy ( igan ) from swedish biopsy registers was collected for this study . the positive predictive value ( ppv ) of a biopsy consistent with igan was 95% . igan , which was first described by berger and hinglais in 1968,1 is now recognized as the most frequent type of primary glomerulonephritis worldwide.2,3 since subclinical disease is common and renal biopsy practice varies between countries , true incidence and prevalence data are difficult to establish.3 incidence rates have been reported as 210 per 100,000 person - years38 with male to female ratios ranging from 1.3:1 to 6:1.4,7 igan is histologically characterized by iga deposits in the glomerular mesangium . a renal biopsy with immunofluorescence staining or immunohistochemistry the archetypical clinical presentation of igan is that of a young adult with episodic macroscopic hematuria during an upper respiratory tract infection . other patients are identified through asymptomatic urine abnormalities or through uremic symptoms late in the disease course . some patients with a renal manifestation of igan also have systemic features of vasculitis in the skin ( henoch schnlein purpura [ hsp ] ) , intestine ( gastrointestinal bleeding ) , or joints ( arthritis ) , now classified as iga vasculitis ( igav ) . histopathologically , there is no clear distinction between primary igan and igan related to hsp / igav , although the latter commonly shows more active inflammatory changes in the kidney . an important step in the pathogenesis of igan is the synthesis of high levels of poorly galactosylated iga1 and igg autoantibodies directed against gdiga1 , resulting in immune complex formation and deposition in the glomerular mesangium . furthermore , this leads to mesangial cell proliferation , endocapillary proliferation , and varying degrees of segmental glomerulosclerosis and tubulointerstitial damage.9 identification of mesangial iga deposits by immunofluorescence or immunohistochemistry in renal biopsy is the key feature of the diagnosis . mesangial iga deposits may rarely occur in other conditions , including human immunodeficiency virus ( hiv ) infection , chronic liver disease , and systemic lupus erythematosus ( sle ) . the prognosis is variable , ranging from subclinical disease with isolated microscopic hematuria to severe renal disease progression . up to 25% of affected individuals develop end - stage renal disease within 1020 years from diagnosis.10,11 recurrence in renal transplants occurs in ~30% of patients , with great discrepancy in different studies , mainly because of differences in follow - up times and in the threshold for performing a transplant biopsy.12 all renal biopsy specimens in sweden are assessed in any of four pathology departments at the university hospitals in stockholm , gteborg , linkping , and malm / lund . previously , data have been collected on a cohort consisting of all patients diagnosed with igan in sweden , by performing computerized searches in all these four departments.13 this yielded a cohort of 4,069 unique individuals with a biopsy record of igan , diagnosed between 1974 and 2011 . from all biopsies , data on 1 ) personal identity number , 2 ) date of biopsy , 3 ) topography ( that the biopsy took place in the kidney ) , and 4 ) histologic diagnosis are available . this cohort could be an excellent source for further epidemiological studies . since asymptomatic igan is common and renal biopsy practices vary between countries and regions , this cohort needs a thorough clinical description to increase external validity . as all registers may include imperfections due to ambiguous findings or simple typing errors , the igan diagnosis in this register - based cohort also requires internal validation . the primary aim of this study was to validate the diagnosis of igan in swedish biopsy registers against patient charts . a second aim was to describe the clinical characteristics of patients with a biopsy indicating igan . the current study is a descriptive observational study of biopsy - verified igan in sweden . the patient cohort consists of all individuals with a renal biopsy record of igan from 1974 to 2011 , retrieved from pathology registers in the four units where all renal biopsies in sweden are assessed . from this cohort , a random sample of 150 individuals were selected to validate the igan diagnosis and to characterize the patients . epitools ( epitools.ausvet.com.au ) was used and it was estimated that 139 patients need to be examined to detect a ppv of 90% with a 95% confidence interval ( ci ) of 85%95% . as patient charts in all the patients could not be expected , 150 patient charts were requested . renal biopsy with immunofluorescence staining or immunohistochemistry is required for the diagnosis of igan . in sweden , evaluation of renal biopsies is centralized to four pathology departments ( stockholm , gteborg , malm / lund , and linkping ) . computerized records of biopsy reports are available in all of sweden since the early 1990s ; recording started in the 1980s , with some backward registration of older reports . renal biopsy data were collected through computerized searches for relevant snomed ( systemized nomenclature of medicine clinical terms14 ) codes for pathology diagnosis ( igan , d67300 ) and topography ( kidney , t71000 ) . one region ( malm / lund ) provided renal biopsy data but no snomed codes . biopsy reports from this region were screened manually by a pathologist.13 for each biopsy , the study also obtained data on arrival date of the renal biopsy and personal identity number.15 thus , a cohort of 4,069 unique individuals were identified with a biopsy report of igan . for the aforementioned 150 patients , the pathology departments provided information on the caregiver who had referred the patient for renal biopsy . charts were received from 127 ( 84.7% ) patients and reviewed according to a standardized template . this included data on the cause of consultation , presenting symptoms , age , sex , comorbidities ( including hepatitis and hiv infection ) , and symptoms or laboratory results that may suggest other diagnoses , like antineutrophil cytoplasmic antibody ( anca ) vasculitis or sle . in ambiguous cases also , the clinical course during follow - up was evaluated to distinguish igan from differential diagnoses . glomerular filtration rate ( gfr ) was described with iohexol or cr - ethylenediaminetetraacetic acid clearance data , when available , and otherwise estimated from serum creatinine levels , using the modification of diet in renal disease study ( mdrd ) formula in adults ( 16 years)16 and the schwartz bedside formula in children ( < 16 years).17 isolated igan and igan with extra - renal vascular manifestations ( hsp / igav ) were treated as one common entity . patients were divided into either of three categories ( figure 1 ) : confirmed igan , likely igan , or unlikely igan . a confirmed igan diagnosis required 1 ) a description of mesangial iga deposits in biopsy report , 2 ) igan as primary biopsy diagnosis , 3 ) a clear statement of igan in the patient chart , and 4 ) no clinical or biopsy data contradicting igan . likely igan required 1 ) a description of mesangial iga deposits in biopsy report , 2 ) either igan as primary biopsy diagnosis or a statement in patient chart of igan as the most plausible diagnosis , and 3 ) no clinical or biopsy data contradicting igan . when these criteria were not met , the diagnosis was considered unlikely . the ppv was calculated with 95% ci of having an igan+ biopsy , using confirmed and/or probable igan as the gold standard of diagnosis . calculations were made using microsoft excel program ( microsoft corporation , redmond , wa , usa ) . the current study was approved by the stockholm ethics review board ( jan 22 , 2014 ; approval number : 2013/2095 - 31/2 ) . for this type of study , the current study is a descriptive observational study of biopsy - verified igan in sweden . the patient cohort consists of all individuals with a renal biopsy record of igan from 1974 to 2011 , retrieved from pathology registers in the four units where all renal biopsies in sweden are assessed . from this cohort , a random sample of 150 individuals were selected to validate the igan diagnosis and to characterize the patients . epitools ( epitools.ausvet.com.au ) was used and it was estimated that 139 patients need to be examined to detect a ppv of 90% with a 95% confidence interval ( ci ) of 85%95% . as patient charts in all the patients could not be expected , 150 patient charts were requested . renal biopsy with immunofluorescence staining or immunohistochemistry is required for the diagnosis of igan . in sweden , evaluation of renal biopsies is centralized to four pathology departments ( stockholm , gteborg , malm / lund , and linkping ) . computerized records of biopsy reports are available in all of sweden since the early 1990s ; recording started in the 1980s , with some backward registration of older reports . renal biopsy data were collected through computerized searches for relevant snomed ( systemized nomenclature of medicine clinical terms14 ) codes for pathology diagnosis ( igan , d67300 ) and topography ( kidney , t71000 ) . one region ( malm / lund ) provided renal biopsy data but no snomed codes . biopsy reports from this region were screened manually by a pathologist.13 for each biopsy , the study also obtained data on arrival date of the renal biopsy and personal identity number.15 thus , a cohort of 4,069 unique individuals were identified with a biopsy report of igan . for the aforementioned 150 patients , the pathology departments provided information on the caregiver who had referred the patient for renal biopsy . charts were received from 127 ( 84.7% ) patients and reviewed according to a standardized template . this included data on the cause of consultation , presenting symptoms , age , sex , comorbidities ( including hepatitis and hiv infection ) , and symptoms or laboratory results that may suggest other diagnoses , like antineutrophil cytoplasmic antibody ( anca ) vasculitis or sle . in ambiguous cases also , the clinical course during follow - up was evaluated to distinguish igan from differential diagnoses . glomerular filtration rate ( gfr ) was described with iohexol or cr - ethylenediaminetetraacetic acid clearance data , when available , and otherwise estimated from serum creatinine levels , using the modification of diet in renal disease study ( mdrd ) formula in adults ( 16 years)16 and the schwartz bedside formula in children ( < 16 years).17 isolated igan and igan with extra - renal vascular manifestations ( hsp / igav ) were treated as one common entity . patients were divided into either of three categories ( figure 1 ) : confirmed igan , likely igan , or unlikely igan . a confirmed igan diagnosis required 1 ) a description of mesangial iga deposits in biopsy report , 2 ) igan as primary biopsy diagnosis , 3 ) a clear statement of igan in the patient chart , and 4 ) no clinical or biopsy data contradicting igan . likely igan required 1 ) a description of mesangial iga deposits in biopsy report , 2 ) either igan as primary biopsy diagnosis or a statement in patient chart of igan as the most plausible diagnosis , and 3 ) no clinical or biopsy data contradicting igan . when these criteria were not met , the diagnosis was considered unlikely . the ppv was calculated with 95% ci of having an igan+ biopsy , using confirmed and/or probable igan as the gold standard of diagnosis . calculations were made using microsoft excel program ( microsoft corporation , redmond , wa , usa ) . the current study was approved by the stockholm ethics review board ( jan 22 , 2014 ; approval number : 2013/2095 - 31/2 ) . for this type of study , table 1 presents the characteristics of the 127 patients with patient chart data and 23 patients without such data . two units ( with a total of 10 patients ) did not provide any patient charts . for another 13 patients , charts were either incomplete or could not be located . out of the remaining 127 patients , iga deposits were found to at least some degree in 123 patients ( 97% ) , and igan was confirmed or likely in 121 patients ( ppv = 95% , 95% ci = 92%99% ) , with a confirmed igan diagnosis in 116 patients ( ppv = 91% , 95% ci = 86%96% ) . in three patients with confirmed igan , ancas were demonstrated in low titers ; none of those showed clinical or histopathological features of anca - associated vasculitis , and anca was judged unspecific . in two patients with confirmed igan , there was evidence of a second renal diagnosis ( anca - associated vasculitis and drug - induced interstitial nephritis , respectively ) . one patient had a confirmed chronic hepatitis c infection and demonstrated virus - like endothelial inclusions , indicating that igan might be secondary to hepatitis c. hsp - related igan / igav with extra - renal manifestations was present in 16 patients ( 13% ) . in six patients , igan was ruled out or considered unlikely : in three patients where iga deposits were not mentioned in the biopsy report , igan was ruled out as the primary renal diagnosis ( one patient had sle , and the other two had acute tubular necrosis due to puumala virus ) ; in three patients with non - igan - renal diagnoses , mesangial iga and c3 deposits were considered incidental findings , although a concurrent igan could not be excluded ( one patient with acute steroid responding nephrotic syndrome , where electronic microscopy showed widening of podocyte foot processes as in minimal change glomerulonephritis , one patient with acute renal failure , high titers of antibodies to glomerular basement membrane , and linear igg fluorescence pattern as in goodpasture s disease , and one patient with acute onset flank pain and macroscopic hematuria , where a toxic effect of ethylene glycol was considered a more likely explanation ) . in five patients , igan was considered likely but could not be fully confirmed ( failed immunofluorescence staining , n=1 ; mainly capillary location of iga deposits and incomplete patient chart , n=1 ; chronic changes dominating and igan stated the majority were adults at the time of first renal biopsy ; 6% were children ( < 16 years ; table 1 ) . microscopic or macroscopic hematuria was recorded in 116 patients ( 91% ) , and data of eight patients were missing . / l , or protein excretion of 1.5 g / l , or an urinary albumin to creatinine ratio of 100 g / mole , was present in 56 patients . forty - two patients ( 33% ) had less severe or no proteinuria ( data missing in 29 cases ) . in a majority of patients ( 59% ) , renal function was well preserved at inclusion ( gfr 60 ml / min/1.73 m ) . in 17% , there was a severe reduction in gfr ( < 30 ml / min/1.73 m ) . hypertension was noted in 46% of the patients , and 28% were on angiotensin converting enzyme inhibitor or angiotensin ii receptor antagonist therapy at the time of renal biopsy . approximately 16% had a family history of renal disease : in eight cases ( 6% ) , there was a family history of igan ( table 2 ) . data from these biopsy reports mentioned iga deposits of at least moderate fluorescence or abundance in 84% of the cases ( n=107 ) , mesangial hypercellularity in 76% ( n=96 ) , c3 ( table 3 ) . in 21 patients , a second renal biopsy had been performed : in 13 cases because of ambiguous findings in the first biopsy , and in the remaining cases of renal transplant to assess disease recurrence or transplant rejection . age and sex distribution were similar in patients with and without available patient charts ( table 1 ) . however , in patients without charts , the renal biopsy had been performed on average 10 years earlier than in patients with available clinical data ( median year of biopsy 1991 vs 2001 ) . the earlier year of biopsy in those with a missing patient chart may reflect the fact that patient charts are only stored for a certain period of time in sweden . table 1 presents the characteristics of the 127 patients with patient chart data and 23 patients without such data . two units ( with a total of 10 patients ) did not provide any patient charts . for another 13 patients , charts were either incomplete or could not be located . out of the remaining 127 patients , iga deposits were found to at least some degree in 123 patients ( 97% ) , and igan was confirmed or likely in 121 patients ( ppv = 95% , 95% ci = 92%99% ) , with a confirmed igan diagnosis in 116 patients ( ppv = 91% , 95% ci = 86%96% ) . in three patients with confirmed igan , ancas were demonstrated in low titers ; none of those showed clinical or histopathological features of anca - associated vasculitis , and anca was judged unspecific . in two patients with confirmed igan , there was evidence of a second renal diagnosis ( anca - associated vasculitis and drug - induced interstitial nephritis , respectively ) . one patient had a confirmed chronic hepatitis c infection and demonstrated virus - like endothelial inclusions , indicating that igan might be secondary to hepatitis c. hsp - related igan / igav with extra - renal manifestations was present in 16 patients ( 13% ) . in six patients , igan was ruled out or considered unlikely : in three patients where iga deposits were not mentioned in the biopsy report , igan was ruled out as the primary renal diagnosis ( one patient had sle , and the other two had acute tubular necrosis due to puumala virus ) ; in three patients with non - igan - renal diagnoses , mesangial iga and c3 deposits were considered incidental findings , although a concurrent igan could not be excluded ( one patient with acute steroid responding nephrotic syndrome , where electronic microscopy showed widening of podocyte foot processes as in minimal change glomerulonephritis , one patient with acute renal failure , high titers of antibodies to glomerular basement membrane , and linear igg fluorescence pattern as in goodpasture s disease , and one patient with acute onset flank pain and macroscopic hematuria , where a toxic effect of ethylene glycol was considered a more likely explanation ) . in five patients , igan was considered likely but could not be fully confirmed ( failed immunofluorescence staining , n=1 ; mainly capillary location of iga deposits and incomplete patient chart , n=1 ; chronic changes dominating and igan stated the majority were adults at the time of first renal biopsy ; 6% were children ( < 16 years ; table 1 ) . clinical characteristics according to the 127 reviewed patient charts are presented in table 2 . microscopic or macroscopic hematuria was recorded in 116 patients ( 91% ) , and data of eight patients were missing . g / l , or an urinary albumin to creatinine ratio of 100 g / mole , was present in 56 patients . forty - two patients ( 33% ) had less severe or no proteinuria ( data missing in 29 cases ) . in a majority of patients ( 59% ) , renal function was well preserved at inclusion ( gfr 60 ml / min/1.73 m ) . in 17% , there was a severe reduction in gfr ( < 30 ml / min/1.73 m ) . hypertension was noted in 46% of the patients , and 28% were on angiotensin converting enzyme inhibitor or angiotensin ii receptor antagonist therapy at the time of renal biopsy . approximately 16% had a family history of renal disease : in eight cases ( 6% ) , there was a family history of igan ( table 2 ) . data from these biopsy reports mentioned iga deposits of at least moderate fluorescence or abundance in 84% of the cases ( n=107 ) , mesangial hypercellularity in 76% ( n=96 ) , c3 ( table 3 ) . in 21 patients , a second renal biopsy had been performed : in 13 cases because of ambiguous findings in the first biopsy , and in the remaining cases of renal transplant to assess disease recurrence or transplant rejection . age and sex distribution were similar in patients with and without available patient charts ( table 1 ) . however , in patients without charts , the renal biopsy had been performed on average 10 years earlier than in patients with available clinical data ( median year of biopsy 1991 vs 2001 ) . the earlier year of biopsy in those with a missing patient chart may reflect the fact that patient charts are only stored for a certain period of time in sweden . this paper describes the validation of the igan diagnosis according to renal biopsy reports in sweden . in total , 4,069 unique individuals were identified with a biopsy indicating igan through computerized searches in pathology reports using snomed codes , supplemented by manual screening in one department not providing snomed codes . since a renal biopsy is required for igan diagnosis , and renal biopsy evaluation is centralized to only four departments in sweden , this cohort can be regarded as truly population - based . patient charts for a subset of 127 randomly selected individuals were examined for igan validation including thorough reevaluation of biopsy reports . in all , the results indicate that a biopsy report of igan from swedish pathology departments is highly specific for true igan , with a ppv of 95% for confirmed or likely igan . the current patient chart review found that ~19 out of 20 patients with a biopsy report with igan had clinical igan . among other diagnoses with igan - like biopsies were sle and acute tubular necrosis due to puumala virus infection . sle patients sometimes have sparse immune depositions of iga and c3 and only mild mesangial proliferation early in the disease course before developing the typical picture of full house clinical follow - up in general helps to differentiate between the diseases . on rare occasions , mesangial iga deposits can also be found when capillary damage occurs , for example , in conditions characterized by acute tubular necrosis and gross hematuria . there is a debate of whether or not the presence of c1q in renal biopsy should rule out the diagnosis of igan or is a marker of more severe prognosis in this disease.18,19 sle nephritis and c1q nephropathy are among the differential diagnoses here . the decision to classify these cases as confirmed or likely igan is based on the evaluation of the patient charts where no other clinical or laboratory signs of sle were described . the predominance of iga over c1q staining makes the diagnosis of c1q nephropathy less likely . combining renal biopsy data with data from the national patient register20 in future studies should help rule out some cases with false - positive igan since at least sle is often diagnosed in the swedish national patient register . the male to female ratio in this study ( 2.8:1 ) is consistent with earlier literature,4 and a vast majority of individuals were diagnosed in adulthood . two retrospective japanese studies6,7 based on school urinary dipstick screening programs show a less marked male predominance . since igan is often asymptomatic and spontaneous recovery sometimes occurs , this might be an indication that female igan incidence is underestimated in studies that are not screening - based or could reflect a true difference in sexual predilection between genetically diverse populations . the present study shows relatively few cases of igan diagnosed in childhood , compared to older studies . this can in part be explained by the fact that one of the departments failing to provide patient charts cared for children . dropouts included ( n=150 ) , 9.3% of all individuals were diagnosed in childhood . in sweden , renal biopsy is often postponed in children with suspected mild to moderate igan with normal renal function and minimal proteinuria , especially when nephropathy is associated with cutaneous vasculitis ( hsp ) ; therefore , this study may underestimate the true proportion of igan diagnosed during childhood . the most common causes of referral for renal biopsy were macroscopic hematuria related to infectious disease , urinary abnormalities detected by screening , and purpura of the skin . these findings are comparable to those in other patient cohorts.11 one in six individuals in this validation study had a family history of renal disease , which is in line with one previous swedish study.21 so far , most of the epidemiological studies on renal disease emanating from sweden have used the swedish national patient registry to ascertain the disease . while that register has a high ppv for many chronic disorders,20 95% as seen for renal biopsy data must be regarded as very high . also , the use of biopsy data to ascertain igan has a high sensitivity , since renal biopsy is a condition for diagnosis . renal biopsy data also have the advantage that they enable researchers to examine the risk factors for igan and future complications for the same disease according to biopsy status . renal biopsy registers are an important source of research worldwide.2 in a study by the spanish society of nephrology , renal biopsy data showed a biopsy rate of 4.8/100,000 inhabitants . the most common pathological finding in adults was that of igan.22 renal biopsy data have been used with great success elsewhere not only to examine both geographical and socioeconomic differences in disease but also to improve patient care . investigations based on the scottish renal biopsy register showed that both the incidence of renal biopsy and a diagnosis of igan were overrepresented in socially deprived areas , independent of demographic data , and there were some differences in the clinical picture between patients from different socioeconomic groups.8 in norway , record linkage between the kidney biopsy registry , the norwegian cause of death registry , and the norwegian renal registry unraveled a twofold mortality risk for patients with igan compared to the general population . this increase of mortality was mainly seen after initiation of renal replacement therapy.23 a nationwide kidney biopsy registry , linked to the well - established swedish renal registry , has recently been started up in sweden and will in the near future allow for a wide range of epidemiologic studies on all forms of glomerulonephritis and other renal diagnoses . it is concluded that in sweden , renal biopsies consistent with igan have a high predictive value for the diagnosis of igan confirmed by review of patient charts , and patient characteristics of individuals with biopsy - verified igan are similar to that reported from international literature .

aimsthe aims of this study were to validate the diagnosis of iga nephropathy ( igan ) in swedish biopsy registers against patient charts and to describe the clinical characteristics of patients with a biopsy indicating igan.methodsthis is a population - based cohort study . out of 4,069 individuals with a renal biopsy consistent with igan ( biopsies performed in 19742011 ) , this study reviewed patient charts of a random subset of 127 individuals . clinical and biopsy characteristics at the time of biopsy were evaluated , and positive predictive values ( ppv ) were calculated with 95% confidence intervals ( ci).resultsout of 127 individuals with a renal biopsy consistent with igan , 121 had a likely or confirmed clinical diagnosis of igan , primary or secondary to henoch schnlein purpura , yielding a ppv of 95% ( 95% ci = 92%99% ) . the median age at biopsy was 39 years ( range : 479 years ) ; seven patients ( 6% ) were < 16 years . the male to female ratio was 2.8:1 . the most common causes for consultation were macroscopic hematuria ( n=37 ; 29% ) , screening ( n=33 ; 26% ) , and purpura ( n=14 , 11% ) . in patients with available data , the median creatinine level was 104 mol / l ( range 26986 mol / l , n=110 ) and glomerular filtration rate 75 ml / min/1.73m2 ( range 5173 ml / min/1.73m2 , n=114 ) . hypertension was noted in 59 ( 46% ) individuals . iga deposits were reported in 97% of the biopsy records ( n=123 ) , mesangial hypercellularity in 76% ( n=96 ) , c3 deposits in 89% ( n=113 ) , and c1q deposits in 12% ( n=15).conclusiona histologic diagnosis of igan has a high ppv for a diagnosis of igan confirmed by review of patient charts .

Brief summary Introduction Subjects and methods Study design and primary study population Collection of renal biopsy data Collection of clinical data and validation of IgAN diagnosis Statistics Compliance with ethical standards Results Validation of IgAN diagnosis from biopsy reports Age and sex of individuals with an IgAN+ renal biopsy Clinical characteristics of individuals with IgAN at the time of biopsy Biopsy findings Patients with unavailable patient charts Discussion Conclusion

a cohort of 4,069 patients with iga nephropathy ( igan ) from swedish biopsy registers was collected for this study . the positive predictive value ( ppv ) of a biopsy consistent with igan was 95% . igan , which was first described by berger and hinglais in 1968,1 is now recognized as the most frequent type of primary glomerulonephritis worldwide.2,3 since subclinical disease is common and renal biopsy practice varies between countries , true incidence and prevalence data are difficult to establish.3 incidence rates have been reported as 210 per 100,000 person - years38 with male to female ratios ranging from 1.3:1 to 6:1.4,7 igan is histologically characterized by iga deposits in the glomerular mesangium . a renal biopsy with immunofluorescence staining or immunohistochemistry the archetypical clinical presentation of igan is that of a young adult with episodic macroscopic hematuria during an upper respiratory tract infection . some patients with a renal manifestation of igan also have systemic features of vasculitis in the skin ( henoch schnlein purpura [ hsp ] ) , intestine ( gastrointestinal bleeding ) , or joints ( arthritis ) , now classified as iga vasculitis ( igav ) . furthermore , this leads to mesangial cell proliferation , endocapillary proliferation , and varying degrees of segmental glomerulosclerosis and tubulointerstitial damage.9 identification of mesangial iga deposits by immunofluorescence or immunohistochemistry in renal biopsy is the key feature of the diagnosis . up to 25% of affected individuals develop end - stage renal disease within 1020 years from diagnosis.10,11 recurrence in renal transplants occurs in ~30% of patients , with great discrepancy in different studies , mainly because of differences in follow - up times and in the threshold for performing a transplant biopsy.12 all renal biopsy specimens in sweden are assessed in any of four pathology departments at the university hospitals in stockholm , gteborg , linkping , and malm / lund . previously , data have been collected on a cohort consisting of all patients diagnosed with igan in sweden , by performing computerized searches in all these four departments.13 this yielded a cohort of 4,069 unique individuals with a biopsy record of igan , diagnosed between 1974 and 2011 . from all biopsies , data on 1 ) personal identity number , 2 ) date of biopsy , 3 ) topography ( that the biopsy took place in the kidney ) , and 4 ) histologic diagnosis are available . as all registers may include imperfections due to ambiguous findings or simple typing errors , the igan diagnosis in this register - based cohort also requires internal validation . the primary aim of this study was to validate the diagnosis of igan in swedish biopsy registers against patient charts . a second aim was to describe the clinical characteristics of patients with a biopsy indicating igan . the patient cohort consists of all individuals with a renal biopsy record of igan from 1974 to 2011 , retrieved from pathology registers in the four units where all renal biopsies in sweden are assessed . from this cohort , a random sample of 150 individuals were selected to validate the igan diagnosis and to characterize the patients . epitools ( epitools.ausvet.com.au ) was used and it was estimated that 139 patients need to be examined to detect a ppv of 90% with a 95% confidence interval ( ci ) of 85%95% . renal biopsy with immunofluorescence staining or immunohistochemistry is required for the diagnosis of igan . renal biopsy data were collected through computerized searches for relevant snomed ( systemized nomenclature of medicine clinical terms14 ) codes for pathology diagnosis ( igan , d67300 ) and topography ( kidney , t71000 ) . biopsy reports from this region were screened manually by a pathologist.13 for each biopsy , the study also obtained data on arrival date of the renal biopsy and personal identity number.15 thus , a cohort of 4,069 unique individuals were identified with a biopsy report of igan . glomerular filtration rate ( gfr ) was described with iohexol or cr - ethylenediaminetetraacetic acid clearance data , when available , and otherwise estimated from serum creatinine levels , using the modification of diet in renal disease study ( mdrd ) formula in adults ( 16 years)16 and the schwartz bedside formula in children ( < 16 years).17 isolated igan and igan with extra - renal vascular manifestations ( hsp / igav ) were treated as one common entity . a confirmed igan diagnosis required 1 ) a description of mesangial iga deposits in biopsy report , 2 ) igan as primary biopsy diagnosis , 3 ) a clear statement of igan in the patient chart , and 4 ) no clinical or biopsy data contradicting igan . likely igan required 1 ) a description of mesangial iga deposits in biopsy report , 2 ) either igan as primary biopsy diagnosis or a statement in patient chart of igan as the most plausible diagnosis , and 3 ) no clinical or biopsy data contradicting igan . the ppv was calculated with 95% ci of having an igan+ biopsy , using confirmed and/or probable igan as the gold standard of diagnosis . for this type of study , the current study is a descriptive observational study of biopsy - verified igan in sweden . the patient cohort consists of all individuals with a renal biopsy record of igan from 1974 to 2011 , retrieved from pathology registers in the four units where all renal biopsies in sweden are assessed . from this cohort , a random sample of 150 individuals were selected to validate the igan diagnosis and to characterize the patients . epitools ( epitools.ausvet.com.au ) was used and it was estimated that 139 patients need to be examined to detect a ppv of 90% with a 95% confidence interval ( ci ) of 85%95% . renal biopsy with immunofluorescence staining or immunohistochemistry is required for the diagnosis of igan . renal biopsy data were collected through computerized searches for relevant snomed ( systemized nomenclature of medicine clinical terms14 ) codes for pathology diagnosis ( igan , d67300 ) and topography ( kidney , t71000 ) . biopsy reports from this region were screened manually by a pathologist.13 for each biopsy , the study also obtained data on arrival date of the renal biopsy and personal identity number.15 thus , a cohort of 4,069 unique individuals were identified with a biopsy report of igan . glomerular filtration rate ( gfr ) was described with iohexol or cr - ethylenediaminetetraacetic acid clearance data , when available , and otherwise estimated from serum creatinine levels , using the modification of diet in renal disease study ( mdrd ) formula in adults ( 16 years)16 and the schwartz bedside formula in children ( < 16 years).17 isolated igan and igan with extra - renal vascular manifestations ( hsp / igav ) were treated as one common entity . a confirmed igan diagnosis required 1 ) a description of mesangial iga deposits in biopsy report , 2 ) igan as primary biopsy diagnosis , 3 ) a clear statement of igan in the patient chart , and 4 ) no clinical or biopsy data contradicting igan . likely igan required 1 ) a description of mesangial iga deposits in biopsy report , 2 ) either igan as primary biopsy diagnosis or a statement in patient chart of igan as the most plausible diagnosis , and 3 ) no clinical or biopsy data contradicting igan . the ppv was calculated with 95% ci of having an igan+ biopsy , using confirmed and/or probable igan as the gold standard of diagnosis . for this type of study , table 1 presents the characteristics of the 127 patients with patient chart data and 23 patients without such data . out of the remaining 127 patients , iga deposits were found to at least some degree in 123 patients ( 97% ) , and igan was confirmed or likely in 121 patients ( ppv = 95% , 95% ci = 92%99% ) , with a confirmed igan diagnosis in 116 patients ( ppv = 91% , 95% ci = 86%96% ) . in three patients with confirmed igan , ancas were demonstrated in low titers ; none of those showed clinical or histopathological features of anca - associated vasculitis , and anca was judged unspecific . in two patients with confirmed igan , there was evidence of a second renal diagnosis ( anca - associated vasculitis and drug - induced interstitial nephritis , respectively ) . one patient had a confirmed chronic hepatitis c infection and demonstrated virus - like endothelial inclusions , indicating that igan might be secondary to hepatitis c. hsp - related igan / igav with extra - renal manifestations was present in 16 patients ( 13% ) . in six patients , igan was ruled out or considered unlikely : in three patients where iga deposits were not mentioned in the biopsy report , igan was ruled out as the primary renal diagnosis ( one patient had sle , and the other two had acute tubular necrosis due to puumala virus ) ; in three patients with non - igan - renal diagnoses , mesangial iga and c3 deposits were considered incidental findings , although a concurrent igan could not be excluded ( one patient with acute steroid responding nephrotic syndrome , where electronic microscopy showed widening of podocyte foot processes as in minimal change glomerulonephritis , one patient with acute renal failure , high titers of antibodies to glomerular basement membrane , and linear igg fluorescence pattern as in goodpasture s disease , and one patient with acute onset flank pain and macroscopic hematuria , where a toxic effect of ethylene glycol was considered a more likely explanation ) . in five patients , igan was considered likely but could not be fully confirmed ( failed immunofluorescence staining , n=1 ; mainly capillary location of iga deposits and incomplete patient chart , n=1 ; chronic changes dominating and igan stated the majority were adults at the time of first renal biopsy ; 6% were children ( < 16 years ; table 1 ) . microscopic or macroscopic hematuria was recorded in 116 patients ( 91% ) , and data of eight patients were missing . in a majority of patients ( 59% ) , renal function was well preserved at inclusion ( gfr 60 ml / min/1.73 m ) . hypertension was noted in 46% of the patients , and 28% were on angiotensin converting enzyme inhibitor or angiotensin ii receptor antagonist therapy at the time of renal biopsy . approximately 16% had a family history of renal disease : in eight cases ( 6% ) , there was a family history of igan ( table 2 ) . data from these biopsy reports mentioned iga deposits of at least moderate fluorescence or abundance in 84% of the cases ( n=107 ) , mesangial hypercellularity in 76% ( n=96 ) , c3 ( table 3 ) . age and sex distribution were similar in patients with and without available patient charts ( table 1 ) . however , in patients without charts , the renal biopsy had been performed on average 10 years earlier than in patients with available clinical data ( median year of biopsy 1991 vs 2001 ) . the earlier year of biopsy in those with a missing patient chart may reflect the fact that patient charts are only stored for a certain period of time in sweden . table 1 presents the characteristics of the 127 patients with patient chart data and 23 patients without such data . out of the remaining 127 patients , iga deposits were found to at least some degree in 123 patients ( 97% ) , and igan was confirmed or likely in 121 patients ( ppv = 95% , 95% ci = 92%99% ) , with a confirmed igan diagnosis in 116 patients ( ppv = 91% , 95% ci = 86%96% ) . in three patients with confirmed igan , ancas were demonstrated in low titers ; none of those showed clinical or histopathological features of anca - associated vasculitis , and anca was judged unspecific . in two patients with confirmed igan , there was evidence of a second renal diagnosis ( anca - associated vasculitis and drug - induced interstitial nephritis , respectively ) . one patient had a confirmed chronic hepatitis c infection and demonstrated virus - like endothelial inclusions , indicating that igan might be secondary to hepatitis c. hsp - related igan / igav with extra - renal manifestations was present in 16 patients ( 13% ) . in six patients , igan was ruled out or considered unlikely : in three patients where iga deposits were not mentioned in the biopsy report , igan was ruled out as the primary renal diagnosis ( one patient had sle , and the other two had acute tubular necrosis due to puumala virus ) ; in three patients with non - igan - renal diagnoses , mesangial iga and c3 deposits were considered incidental findings , although a concurrent igan could not be excluded ( one patient with acute steroid responding nephrotic syndrome , where electronic microscopy showed widening of podocyte foot processes as in minimal change glomerulonephritis , one patient with acute renal failure , high titers of antibodies to glomerular basement membrane , and linear igg fluorescence pattern as in goodpasture s disease , and one patient with acute onset flank pain and macroscopic hematuria , where a toxic effect of ethylene glycol was considered a more likely explanation ) . in five patients , igan was considered likely but could not be fully confirmed ( failed immunofluorescence staining , n=1 ; mainly capillary location of iga deposits and incomplete patient chart , n=1 ; chronic changes dominating and igan stated the majority were adults at the time of first renal biopsy ; 6% were children ( < 16 years ; table 1 ) . clinical characteristics according to the 127 reviewed patient charts are presented in table 2 . microscopic or macroscopic hematuria was recorded in 116 patients ( 91% ) , and data of eight patients were missing . in a majority of patients ( 59% ) , renal function was well preserved at inclusion ( gfr 60 ml / min/1.73 m ) . in 17% , there was a severe reduction in gfr ( < 30 ml / min/1.73 m ) . hypertension was noted in 46% of the patients , and 28% were on angiotensin converting enzyme inhibitor or angiotensin ii receptor antagonist therapy at the time of renal biopsy . approximately 16% had a family history of renal disease : in eight cases ( 6% ) , there was a family history of igan ( table 2 ) . data from these biopsy reports mentioned iga deposits of at least moderate fluorescence or abundance in 84% of the cases ( n=107 ) , mesangial hypercellularity in 76% ( n=96 ) , c3 ( table 3 ) . age and sex distribution were similar in patients with and without available patient charts ( table 1 ) . however , in patients without charts , the renal biopsy had been performed on average 10 years earlier than in patients with available clinical data ( median year of biopsy 1991 vs 2001 ) . the earlier year of biopsy in those with a missing patient chart may reflect the fact that patient charts are only stored for a certain period of time in sweden . in total , 4,069 unique individuals were identified with a biopsy indicating igan through computerized searches in pathology reports using snomed codes , supplemented by manual screening in one department not providing snomed codes . since a renal biopsy is required for igan diagnosis , and renal biopsy evaluation is centralized to only four departments in sweden , this cohort can be regarded as truly population - based . patient charts for a subset of 127 randomly selected individuals were examined for igan validation including thorough reevaluation of biopsy reports . in all , the results indicate that a biopsy report of igan from swedish pathology departments is highly specific for true igan , with a ppv of 95% for confirmed or likely igan . the current patient chart review found that ~19 out of 20 patients with a biopsy report with igan had clinical igan . there is a debate of whether or not the presence of c1q in renal biopsy should rule out the diagnosis of igan or is a marker of more severe prognosis in this disease.18,19 sle nephritis and c1q nephropathy are among the differential diagnoses here . the decision to classify these cases as confirmed or likely igan is based on the evaluation of the patient charts where no other clinical or laboratory signs of sle were described . the predominance of iga over c1q staining makes the diagnosis of c1q nephropathy less likely . the male to female ratio in this study ( 2.8:1 ) is consistent with earlier literature,4 and a vast majority of individuals were diagnosed in adulthood . since igan is often asymptomatic and spontaneous recovery sometimes occurs , this might be an indication that female igan incidence is underestimated in studies that are not screening - based or could reflect a true difference in sexual predilection between genetically diverse populations . in sweden , renal biopsy is often postponed in children with suspected mild to moderate igan with normal renal function and minimal proteinuria , especially when nephropathy is associated with cutaneous vasculitis ( hsp ) ; therefore , this study may underestimate the true proportion of igan diagnosed during childhood . the most common causes of referral for renal biopsy were macroscopic hematuria related to infectious disease , urinary abnormalities detected by screening , and purpura of the skin . while that register has a high ppv for many chronic disorders,20 95% as seen for renal biopsy data must be regarded as very high . also , the use of biopsy data to ascertain igan has a high sensitivity , since renal biopsy is a condition for diagnosis . renal biopsy registers are an important source of research worldwide.2 in a study by the spanish society of nephrology , renal biopsy data showed a biopsy rate of 4.8/100,000 inhabitants . the most common pathological finding in adults was that of igan.22 renal biopsy data have been used with great success elsewhere not only to examine both geographical and socioeconomic differences in disease but also to improve patient care . investigations based on the scottish renal biopsy register showed that both the incidence of renal biopsy and a diagnosis of igan were overrepresented in socially deprived areas , independent of demographic data , and there were some differences in the clinical picture between patients from different socioeconomic groups.8 in norway , record linkage between the kidney biopsy registry , the norwegian cause of death registry , and the norwegian renal registry unraveled a twofold mortality risk for patients with igan compared to the general population . it is concluded that in sweden , renal biopsies consistent with igan have a high predictive value for the diagnosis of igan confirmed by review of patient charts , and patient characteristics of individuals with biopsy - verified igan are similar to that reported from international literature .

patients with neuroendocrine tumors ( nets ) often suffer from neuroendocrine liver metastases ( nlms ) causing significant morbidity and mortality . the excess hormone production , the multitude of hepatic lesions , and ultimate liver disease lend to the poorer prognosis . in fact , 46%93% of patients with nets will find nlms involved at the time of diagnosis . patients with liver metastases have a significantly worse prognosis than those without liver involvement . the 5-year survival of patients with nlms on supportive care is 0%20% [ 13 ] . this dismal prognosis paints a much more stark reality for a pathological process often described as indolent . surgical interventions for nlms have consistently been shown to have superior outcomes to nonoperative therapies . resection alone is supported by favorable long - term outcomes in large retrospective trials ; however , complete surgical extirpation is an option for a very small percentage of the neuroendocrine cancer patient population . due to excessive metastatic tumor burden in difficult locations , surgical resections treatment options for patients that are not surgical candidates have evolved over the last several years . use of ablative techniques , as well as development of new medical therapies , has expanded the treatment options for the majority of patients with nlms . surgery remains the only potential for cure in patients with nlms . even in the setting of incurable disease , surgery offers the best chance for prolonged survival . in patients treated with resection , the five - year survival has been shown to be greater than 60% [ 4 , 5 ] and even approaches 80% in some studies , with minimal mortality ( < 5% ) and morbidity ( < 30% ) . a precise review of the literature available on patients who undergo liver resection for neuroendocrine tumors is difficult due to the small number of patients who are candidates and the varied approaches to surgical treatment . historically , patients were selected to undergo palliative resection if greater that 90% of the tumor burden could be excised . one of the earlier prospective studies concluded from their study of 47 patients that hepatic resection is indicated only when all gross disease can be removed safely . in this study , they determined that number , size , and location of primary tumor were less important than the completeness of resection . patients that underwent a complete resection had a 5-year survival of 80% . however , the patients that underwent an r1 resection had a 5-year survival of 70% and r2 resection still had a 5-year survival of 60% . although patients in this study were included only if it appeared that they could be completely resected , patients that had incomplete resection still did well . several other series have reported similar results [ 4 , 6 ] . a more recent retrospective review of 74 cases demonstrated a greater than 60% 5-year survival in all patients that underwent resection . although an aggressive surgical approach is considered to prolong survival and contribute to better symptom control , the criteria for patient selection are ill defined . in an effort to identify variables that have prognostic relevance to patients who undergo hepatic resection , a prospective review of 70 patients ' outcomes was performed based on tumor grade . the tumors were categorized as low grade ( < 2 mitotic figures/50 hpf and no necrosis ) , intermediate grade ( 250 mitotic figures/50 hpf and/or focal necrosis ) , and high grade ( > 50 mitotic figures/50 hpf and/or extensive necrosis ) . the majority of the neoplasms were considered low grade ( 37 ) or intermediate grade ( 26 ) . none of the patients with high - grade malignancy survived 5 years with a median survival of only 6 months . the importance of tumor grade to patient 's outcomes after resection has been confirmed by several investigations [ 10 , 11 ] . tumor size , number , and location have also been shown to influence postresection survival [ 4 , 12 ] . in 2008 , the enets proposed guideline for surgical resection based on the 3 distinct patterns of liver involvement : ( 1 ) simple pattern of metastasis located in one or two contiguous lobes ( 2025% ) , ( 2 ) complex pattern where there is one major focus and other lesions are contained in the contralateral lobe ( 1015% ) , and ( 3 ) diffuse disease in both lobes ( 6070% ) . the type of surgical resection is based on the patient 's overall medical condition , size , number , and location of lesions , and adequacy of remnant liver size / function . in patients with the simple pattern of disease patients with the complex pattern of disease can be treated with several different methods . an anatomic lobectomy can be performed for the majority of the disease and either a wedge resection or locally ablative therapy can treat the remainder of the tumors . staged , multiple surgical procedures have also been shown to be beneficial with little increase in morbidity and mortality . the majority of patients with diffuse disease are not candidates for resection . it is usually recommended only in cases where > 90% of the tumor volume can be excised or in very young patients . in patients that are symptomatic , cytoreductive surgery has been shown to improve or alleviate their symptoms for a prolonged period of time . it has been estimated that less than 20% of patients with metastatic neuroendocrine tumors are candidates for hepatic resection . resection is not a viable option for the majority of patients with diffuse hepatic disease . based on their slow growth and good response to resection , liver transplantation has been tried in an attempt to cure , prolong survival or control symptoms . although many centers are reluctant to allocate liver allografts to patients with metastatic disease , liver transplantation for neuroendocrine tumors is one of the only accepted indications for transplant in the setting of metastatic disease . in 1998 , lehnert analyzed a total of 103 patients transplanted for metastatic neuroendocrine carcinoma . the overall 5-year survival was 47% , and disease - free survival was 24% . tumor histology or location of primary did not appear to effect survival in this study . however , extent of surgery at the time of transplantation and age of recipient were significant prognostic factors for survival . the overall 5-year survival was comparable at 47% and disease - free survival of 20% at 5 years . in this study , primary tumor location in the duodenum or pancreas was noted to be an indicator of poor prognosis . this finding was not supported in several other investigations [ 2 , 17 ] . one of the larger single center studies attempted to analyze tumor biology in relation to postliver transplant outcomes . based on evaluation of 19 cases , they demonstrated that patients with a low ki-67 ( < 5% ) and normal e - cadherin staining did significantly better than patients with high ki-67 or abnormal e - cadherin expression . analysis of the united network for organ sharing database reveals that between november 1988 and march 2011 , only 185 liver transplants were performed for metastatic neuroendocrine tumors in the united states . although the long - term survival is not comparable to other patients with benign disease , most liver transplant programs will consider evaluating patients with nlms . many liver transplant programs will consider a patient with metastatic neuroendocrine for liver transplantation if the following criteria are met : not a resection candidate , identification and complete resection of primary malignancy at least one year prior to evaluation , no evidence of extrahepatic disease demonstrated on cross - sectional imaging or nuclear medicine scan , evidence of stability of disease for at least one year , failure of nonoperative treatments . this radical treatment occasionally provides a cure , but the long - term survival is still significantly less than in patients transplanted for other diseases . it can prolong survival and provide symptomatic relief in a very small subset of patients . patients that are younger than 50 years in the setting of low ki-67 and e - cadherin expression with symptoms that are difficult to control appear to benefit the most from liver transplantation . nets are predisposed to form highly vascular metastatic lesions in the liver and derive more than 90% of their oxygenation and nutrition from the hepatic artery . thus , the hepatic artery offers a viable mode of introducing directed chemotherapy and/or creating an ischemic environment . this effectively starves the tumors of their nutrient and oxygen supply while sparing healthy hepatic cells , which derive the majority of their nutrient and oxygen supply from the portal venous system . several ablative techniques have been developed that exploit the dual blood supply of the liver in an effort to control the disease process . defining the treatment best suited for the tumor load is dependent on number and location of the lesions , invasiveness and size of the tumor , physiology and effects of hormone secretion , and extent of metastatic disease within the patient . this is in conjunction with the ultimate goals of cure or palliation . considering the rate of recurrence , liver - directed therapies have been considered more as debulking modalities . in a review of the literature , general guidelines for the treatment pathways are : for fewer nodular liver metastatic lesions , local resection or thermal ablation is recommended ; for a higher - tumor load due to unresectable multinodular disease or recurrent disease after resection , hepatic artery embolization , hepatic artery chemoembolization , or radioembolization is warranted . these modalities are also useful as neoadjuvants to decrease the size of previously unresectable metastatic disease . unless 80%90% of the tumor load is debulked , treatment does not serve useful as palliation therapy to prolong survival and improve symptom control . radiofrequency ablation ( rfa ) uses an image - guided technique , percutaneous , laparoscopic , or open , to provide local control with short - term symptomatic relief by subjecting tumors to intense , destructive heat using an alternating electric current . this technique is amenable to patients with fewer liver metastases who are ineligible for hepatic resection . it is used as a single modality , often more than once , or as an adjunct to other nlms therapies for debulking . the largest study to date , with the longest followup , was done at the cleveland clinic , a prospective trial of 89 patients with nlms who underwent 119 laparoscopic rfa sessions in total . ninety - seven percent of the sample immediately felt improvement of symptoms after the procedure , where median disease - free survival was 1.3 years and overall survival at 6 years after rfa . of note , 22% of this sample developed local recurrence , with 63% developing new lesions and 59% developing extrahepatic disease . prior to that study , mazzaglia et al . investigated a series of 63 patients who had a total of 452 treated nlms lesions . symptoms were controlled an average of 11 2.3 months after rfa , with greater than 90% of symptomatic patients experiencing relief immediately after procedure . larger dominant tumor size ( > 3 cm ) and male sex were significant variables negatively correlated with survival . median survival of the group was 53 months from liver diagnosis . of those with radiologic followup , 74% of patients were noted to have tumor load control at a median of 21 months after the procedure . hormonal treatment has also been used as an adjunct to improve symptomatic relief , though not improve survival . adjuvant octreotide has been shown to extend median symptom - free duration from 16 to 60 months . morbidity for radiofrequency ablation of liver metastases has been reported in the larger studies to be approximately 5% to 12% , with 30-day mortality at 0% to 1% . [ 21 , 22 ] the complications can include carcinoid crisis , liver abscesses , biliopleural fistulas , bile leakage , and pleural effusion , as well as postablation syndrome , and liver failure . capitalizing on the dual blood supply of the liver hepatic artery embolization ( hae ) induces ischemia within the tumor , using a variety of agents such as cyanoacrylate , gel foam particles , polyvinyl alcohol , and microspheres . indications for hae or hepatic artery chemoembolization ( hace ) generally include unresectability with symptoms related to tumor bulk , excessive hormone production , and rapid progression of liver disease . hae has been shown to improve biophysical markers , palliate symptoms and reduce tumor burden by radiographic evaluation [ 2 , 23 ] . because of the observation that higher disease regression rate and longer length of regression with systemic chemotherapy after hae was published , chemotherapy has been added to the embolic agents , and hace is now generally favored over hae . hace , also known as transarterial chemoembolization , combines the hepatic artery embolization with the hepatic artery chemoinfusion where the microspheres are bound to chemotherapy agents , which are then injected into the hepatic artery to lodge downstream within capillaries . not only do the emboli block the blood supply causing ischemic necrosis , but the chemotherapy agents are localized within the region of the metastatic lesions , creating a much more concentrated effect ( up to 20 times greater ) than systemic chemotherapy alone as well . despite this theoretical advantage , little evidence has suggested a significant difference in the outcomes of hepatic artery embolization versus hepatic artery chemoembolization . in a review of the literature , hace has shown a 5-year survival between 50% to 65% whereas hae has a 5-year survival between 40% to 67% . in one study of 100 patients with nlms who received hace or hae , the authors found no difference in overall survival , median survival after diagnosis of metastatic disease , or median survival after first embolization . on univariate analysis , the only predictor that significantly improved survival was concurrent resection of the primary tumor , which increased median survival from 28.0 months to 73.1 months . contrary to this study , ho et al . reported results on 46 patients with nlms who received hace or hae , and showed that there was no statistically significant survival benefit in a small subset of population that also had resection of the primary tumor , although mean survival after resection increased by a mean of 558 days . regardless , these therapies have increased versatility as reflected in a study of 48 patients and 123 treatment sessions which revealed hace or hae could even benefit carefully selected patients with a tumor load of greater than 75% liver involvement , so long as the patients did not have additional risk factors . having said this , a number of reports reveal worse outcomes for patients with greater than 50% liver involvement [ 28 , 29 ] . this is tempered by the fact that extent of liver involvement did not serve as an independent prognostic indicator . in order to mediate the complications arising from disease which takes up the bulk of the liver , it is recommended to divvy small portions of the liver for treatment during each session . there are adjuvants to hace or hae in those patients with severely limited therapeutic options to improve otherwise bleak outcomes , and hace or hae can be used as adjuvant therapy to other treatments . adding hepatic artery chemoinfusion ( hai ) to hace offers an increased probability of clinical benefits to those with unresectable , refractory disease , as presented in a study of 77 patients . the response rate was 80% of islet and carcinoid tumors with a median progression - free survival of 19 months . of the different types of neuroendocrine tumors , carcinoid tumors seem to consistently have better outcomes to the combination of hace and hai [ 21 , 23 , 30 ] . although studies on patients with nlms are limited , in one study of 32 patients with hepatocellular carcinoma , the authors found that there was no survival advantage in patients with preoperative hace prior to surgical resection . in fact , the recurrence - free survival rates were statistically higher , and cumulative recurrence rates were statistically lower at 1 , 2 , and 5 years compared between the two groups . one study from iowa on patients with nlms showed that preoperative hace followed by olt can result favorably for the patients with progression - free intervals up to 29 months , but this was a small study , and statistical inferences could not be made due to the inclusion of only four patients . along a different treatment strategy , hao et al . showed that survival improved when patients with hepatocellular carcinoma received combination therapy with hace plus thalidomide versus hace alone . this improvement reached statistically significant improvement , resulting in median overall survival increases of 15 months . similarly , in an experimental model utilizing liver tumors in rabbits , favorable outcomes resulting in significantly decreased vascular endothelial growth factor and microvascular density levels were achieved when hace plus antiangiogenic therapies were used . however , tumor size was not significantly different between these two groups . an important point of hace or hae is that response can be incomplete as the periphery of the tumor is spared from ischemia or chemotherapy . with proximal embolization of arterial branches feeding the tumors , multiple sessions are usually needed . as all other procedures , there are risks involved with liver - directed therapy through the hepatic artery . liver abscesses , transient liver failure with or without encephalopathy , carcinoid crisis , pleural effusions , and postembolization syndrome ( i.e. , fever , abdominal pain , leukocytosis , and transient increases in hepatic enzymes and bilirubin ) are some of the more common and worrisome . relative contraindications for these procedures include coagulopathy , renal failure , portal vein occlusion , and liver failure . interest in one particular liver - directed therapy is under further investigation for this indication : hepatic artery embolization ( har ) , also known as selective internal radiation therapy ( sirt ) . sirt acts by delivering microspheres of glass or resin , labeled by yttrium ( y ) to deliver radiation directly into the hepatic artery . rather than using peptides to localize the lesions , this therapy mechanically targets the metastases and lodges within the nutrient - supplying capillaries , thereby delivering radiation therapy . while this modality has been tested in a limited number of nets patients , the results thus far have shown promise [ 31 , 3638 ] . saxena et al . have been investigating the safety and efficacy of treatment with y radioactive microspheres for patients with unresectable nlms . in this study , 34 such patients were treated with sirt to achieve long - term responses with a mean overall survival of 29.4 3.4 months , and radiological improvement in 50% . biochemical marker levels of chromogranin a fell in nearly 50% of survivors by 30 months . in one multicenter retrospective review by kennedy et al . this study reports favorable results with radiological response in 63.2% of patients , stable disease in 22.7% , and progression of disease in only 4.9% . the authors state that one of the largest benefits of this treatment is the stabilization of extensive disease allowing for longer survival periods . radiographic and serology studies revealed median survival of 35 months with a followup of 41 months , and 55% of patients had complete or partial responses . prognostic factors were assessed , and 6 of significance were found to influence survivorship : complete / partial response , low hepatic tumor burden , female gender , well - differentiated tumors , and absence of extrahepatic metastases . this was important in identifying a subset of the nlms patient population who would be best served by this newer technique . radiation gastritis and duodenal ulcers have been described , and as all liver - directed therapies , the risk of liver failure is present . of note , this promising modality of care , while approved for treatment of colonic cancer metastases to the liver , is still under fda investigation for treatment of nlms . current literature suggests there is significant potential in sirt / har as part of the armamentarium against neuroendocrine tumors and its hepatic metastases . since nlms is a rare disease , large - scale , randomized trials prove difficult , and although these therapies have been used in the treatment of nets , not all have been specifically used in the treatment of nlms . due to the multiple therapies available , the effectiveness of one versus another is difficult to study , and many times nonsurgical , non - liver - directed therapies tend to be lumped together in studies that are available . moreover , there continues to be a lack of consensus on a nonsurgical treatment algorithm ; however , most agree that nonsurgical , non - liver - directed treatments of nets and nlms constitute palliative care . at least , one single - center study in the medical literature has proven that aggressive treatment of nlms with nonsurgical therapy can extend 3- and 5-year survival rates in patients to 76.4% and 63.9% as compared to previously stated survival rates of 39% and 25% , respectively . with these encouraging results and the boom in treatment advancements , the older perspective of peptide receptor radionuclide therapy ( prrt ) is an upcoming option with enticing advantages , most useful in symptomatic patients with somatostatin receptor - positive tumors , who are not surgical candidates . between 80% to 95% of gastroenteropancreatic , nets express somatostatin receptors as demonstrated by in - pentetreotide scans ( octreoscan , covidien - mallinckrodt imaging , hazelwood , mo 63042 ) , so prrt may be useful for a large percentage of nlms , perhaps in up to 25% of patients . prrt utilizes the targeting of a molecule to specific receptors located on the surface of tumor cells . once the molecule interacts with the receptor , it is internalized , thereby delivering specific and localized radiotherapy . this technique allows precise destruction of tumor cells [ 42 , 43 ] , with little interference of nontumor tissue , except for some exposure of renal , bladder , and bone marrow tissues . y , lutetium ( lu ) , or indium ( in ) are radionuclides that are linked with a somatostatin analog : octreotide , octreotate , or lanreotide . the more the tumor expresses somatostatin receptors as compared to the surrounding tissue , the more effective the prrt will be . somatostatin scintigraphy can predict the effectiveness of prrt : low uptake indicates 20% chance of effect on liver metastases , whereas high uptake indicates a 60% chance . lu - dotatyroctreotate seems to be the most effective prrt , with a tumor response rate of 35% and tumor stabilization of 80% to 90% of nets , versus y - dotatyroctreotide with a tumor response of 4% and tumor stabilization of 70% . after therapy with y - dotatyroctreotide or lu - dotatyroctreotate , median duration of results were 30 months and 36 months , respectively . in one study with 310 patients , further , patients experiencing benefit after one round of prrt who develop recurrent or progressive disease may benefit from a second round of prrt . side effects of prrt are rare and usually mild consisting most commonly of nausea and vomiting occurring within 24 hours of administration , and although anemia and transient toxicity grade 1 have been reported , long - lasting adverse side effects are extremely rare . patients that seem to benefit the most from prrt have strong radiotracer uptake on octreoscan , at least as much as the liver . newer positron emission tomography ( pet ) imaging platforms such as gallium - dotatyroctreotide - pet and gallium - dotatyroctreotate - pet are increasingly used to evaluate tumors as they are even more sensitive to radiotracer uptake and may be able to better predict responsiveness to prrt . unfortunately , prrt is not available in the united states until september 2011 , when the first clinical trial will begin ( http://clinicaltrials.gov/ ; identifier : nct01237457 ) . several chemotherapeutic agents have been used in multiple trials , but mainly in the study of nets only , with limited success and restrictions from side effects and toxicities . the usefulness of chemotherapy in the treatment of nets seems to be related to primary tumor location and tumor grade . the greatest efficacy seems to be related to the use of stz with other chemotherapy agents , including 5-fluorouracil and doxorubicin , but still only results in a median response time of 9.3 months . dacarbazine ( dtic ) is another chemotherapy agent with proven effectiveness in pancreatic nets , and in one phase ii trial demonstrated a response rate of 34% . the alkylating agent , temozolomide , has also shown promise in pancreatic nets : a phase ii study using temozolomide and thalidomide showed a response rate of 45% , and a retrospective study of temozolomide and capecitabine showed a response rate of 70% , a median pfs of 18 months , and an overall 2-year survival of 92% . platinum - based chemotherapy regimens may be useful in patients with high - grade , poorly differentiated nets , with response rates of 42% to 80% with the use of cisplatin and etoposide [ 5456 ] , and 78% with use of oxaliplatin - based regimens . even with increased response rates , median survival times are of short duration of 8 to 11 months . therefore , chemotherapy can be used as salvage treatment , but is generally not considered as first - line , nonsurgical treatment . moreover , the presence of nlms may be related to worse response to chemotherapy as compared to nets . somatostatin exerts its affect by integration with one of five somatostatin receptors , ssts1 - 5 , but due to a half - life of only two minutes , somatostatin analogs ( ssa ) have been developed . newer formulations may be even easier to administer to patients due to a longer half - life of approximately two hours . the principle use of ssa is in the symptomatic relief of nets and nlms , although it may be useful for other indications . the use of ssa produces a median biochemical response rate between 0% to 77% ; and biochemical and radiographic tumor stability of 28% and 55% , respectively [ 6171 ] . one review article found symptomatic and tumor response to octreotide , octreotide long - acting repeatable ( lar ) , lanreotide , and lanreotide slow - release depot ( autogel ) in 74.2% , 77.3% , 63.0% , and 67.5% , and in 57.4% , 69.8% , 46.6% , and 64.4% , respectively . another investigation demonstrated relief from flushing and diarrhea in 88% of patients after octreotide administration . interim data from the promid study with metastatic midgut nets , showed a 66% reduction in the risk of disease progression and arrested tumor growth in 69% for a median of 14.3 months . however , over 75% of patients in this study had limited liver involvement of 10% or less , and the response was highest in patients with relatively low tumor burden . the greatest response rates have been witnessed with octreotide doses of 30 mg / day or greater or with lanreotide doses of 5 mg / day or greater ; octreotide doses greater than 60 mg / day likely do not have additive effect due to oversaturation of receptor sites . similar to prrt , the level of uptake on somatostatin scintigraphy may be an indicator of patient 's response to ssa therapy . the newest ssa , pasireotide , is still in clinical development stages , but is promising due to binding of ssts1 , ssts2 , ssts3 , and ssts5 , as compared to octreotide and lanreotide which bind to ssts2 and ssts5 only . preliminary data indicate that pasireotide may be useful in patients with symptoms refractory to octreotide , possibly controlling symptoms in up to 27% of these patients . side effects are infrequent , but nausea , stomach cramping or discomfort , diarrhea , steatorrhea , cardiac abnormalities and arrhythmias , hypothyroidism , and hypoglycemia may occur [ 40 , 41 , 77 ] . cholelithiasis may arise in up to 50% of patients due to inhibition of gallbladder contractility , but only a handful will develop symptoms requiring cholecystectomy . interferons have multiple antitumor effects , and they may upregulate somatostatin receptors in nets , thereby providing a useful combination therapeutic option . interferon- can ameliorate symptoms in 30% to 70% of patients [ 81 , 82 ] , and in some studies has shown promising results with tumor response rate or stabilization in up to 70% of patients . however , the results of three randomized clinical trials involving interferon- and octreotide have mixed results . two demonstrated increased 5-year survival rate and median survival time in the combination group versus the octreotide - only group , 57% versus 37% and 51 months versus 35 months , respectively ; but another trial showed minimal response rates . the side effect profile of interferons may preclude wide utilization . interferon- can cause fevers , chills , myalgias , depression , and myelosuppression , and is considered inferior to ssa . however , in patients with progressive disease , combination therapy may be a viable option . others have examined the role of dopamine receptors and interferon- as other possible targets , but currently , neither of these targets seems promising at this time due to ineffectiveness and short half - life . patients who have exhausted other therapies may find acceptable treatment through the use of newer treatment strategies . these interventions remain in the investigative process , including targeting vascular endothelial growth factors ( vegf ) , mtor pathways , other growth factor receptors , antiproliferative factors , and antiangiogenic factors . monoclonal antibodies against insulin - like growth factor-1 receptor ( igf-1r ) : amg479 , imc - a12 , and mk-0646 , are currently in clinical phase ii studies in patients with metastatic nets ( http://clinicaltrials.gov/ , identifier : nct01024387 , nct00781911 , nct00610129 ) . others are looking at genetic copy number alterations of tumor suppressor genes and the detection and characterization of circulating tumor cells to reduce metastatic burden as other possible avenues to treat nets and nlms . nets and nlms frequently overexpress the vascular endothelial growth factor ( vegf ) ligand and receptor ( vegfr ) . tumor progression of nets has also been associated with circulating levels of vegf , therefore vegf and vegfr are promising targets . in a study where patients on octreotide therapy were randomized into either treatment with bevacizumab , a humanized monoclonal antibody against vegf , or interferon- , 95% of patients receiving bevacizumab were progression - free after 18 weeks , compared to 67% of patients receiving interferon- . bevacizumab is associated with reduction of tumor blood flow and longer progression - free survival ( pfs ) when compared to alternative treatments . currently , multiple clinical trials of bevacizumab are ongoing ( http://clinicaltrials.gov/ , identifiers : nct00569127 , nct00137774 , nct00398320 , nct00227617 , nct00607113 ) . bevacizumab may cause hypertension and proteinuria , so optimal patient selection prior to treatment is mandatory . sunitinib is a tyrosine kinase receptor inhibitor currently approved in the treatment of renal cell carcinoma and gastrointestinal stromal tumors and inhibits vegfr1 , vegfr2 , and vegfr3 . phase iii trials resulted in median pfs of 11.1 months for patients on sunitinib versus 5.5 months for patients receiving placebo ( p < 0.001 ) [ 40 , 90 , 91 ] . in europe , sunitinib is approved for the treatment of unresectable or metastatic , well - differentiated pancreatic nets with disease progression in adults . side effects of sunitinib include fatigue , asthenia , diarrhea , nausea , vomiting , anorexia , bleeding complications , mucosal inflammation , hypertension , anemia , granulocytopenia , thrombocytopenia , and hypothyroidism . the mammalian target of rapamycin ( mtor ) pathway is central to the control of cell growth , protein synthesis , and apoptosis and is activated in nets . two mtor inhibitors have been developed and approved for use in renal cell carcinomas , everolimus , and temsirolimus , and have been studied in nets [ 41 , 9395 ] . everolimus has a potential in conjunction with octreotide lar and as a monotherapeutic agent with a response rate of 20% , a median pfs between 11 and 16 months in three separate phase iii trials [ 41 , 96 ] , and with stabilization of disease in 70% with low- to - intermediate grade nets . side effects of everolimus include stomatitis , rash , diarrhea , fatigue , infections , noninfectious pneumonitis , anemia , lymphopenia , hypercholesterolemia , hyperlipidemia , and hyperglycemia . micrornas are small , noncoding rnas that can function as gene regulators by posttranscriptional processes , such as inducing mrna degradation or repression of translation [ 97100 ] . micrornas are usually downregulated in cancers [ 97100 ] and have been studied for possible therapeutic interventions . one study identified microrna-133a , -145 , -146 , -222 , and -106 to be important in primary nets , whereas microrna-183 , -488 , -19a+b were found to be important in metastatic nets . further , the same group determined that decreasing levels of microrna-133a has an important role in the development , progression , and possible metastasis of midgut carcinoid tumors . a different study identified microrna-142 - 3p , -142 - 5p , -155 , -146a , and -483 as up - regulated in pancreatic nets as compared to normal tissue . this study also found that microrna-210 , -431 , and -424 were up - regulated in metastases as compared with tumors , suggesting that certain micrornas could be used to predict the probability of metastasis . another study showed that anti - microrna-182 targeting had a therapeutic effect against melanoma liver metastasis , which may be extended to other tumors . additional studies are warranted in this area pertaining to microrna - regulated pathways , but already possible therapeutic targets have been identified by researchers including the high - mobility group a proteins , hmga1 , hmga2 , and the microrna family let-7 [ 103 , 104 ] . these targets will be useful as better strategies evolve to care for patients with nlms and extend their survival . the treatment modalities available to a patient diagnosed with liver metastases due to nets are vast . the options range from surgical treatments , to locally liver - directed therapies , to systemic approaches ( figure 1 ) . however , most , if not all clinicians , agree that the treatment must be tailored specifically to the patient . generally , surgical therapies are preferred as they can give the longest disease - free interval . yet , not all patients with nlms are candidates for surgical therapy , and in the case of an elderly asymptomatic patient with a slow - growing nets , the patient may not desire surgical therapy . liver - directed treatment can also produce great results , extending the lifetime of the patient without riskier surgical interventions . moreover , liver - directed therapies may clearly benefit a patient who is symptomatic from their tumor or may even allow that patient to be a candidate for surgical treatment in the future . lastly , nonsurgical , non - liver - directed therapies are considered palliative care in the treatment of nlms . these systemic therapies are not first line , but can still achieve longer lifespans as a salvage therapy . newer technologies , including genetic targets such as microrna subtypes , are fast evolving and will continue to allow patients with nlms several options . even though nets are rare tumors , nlms are even more rare , and this characteristic prevents large , randomized - controlled trials and modalities of treatment for these tumors continue to improve as there is an obvious need . these treatments are expected to maintain this progression well into the future , especially as knowledge of nlms increases with additional studies , so that we may provide patients diagnosed with nlms with every possible chance towards increased survival .

patients diagnosed with neuroendocrine tumors ( net ) often are also diagnosed with neuroendocrine liver metastases ( nlm ) during the course of their disease . nlm can cause significant morbidity and mortality , oftentimes much more than compared to patients with net . treatment options have been limited in the past , focusing on surgical resections , for which only a minority of patients are candidates . however , developments of new treatment modalities have progressed rapidly and patients with nlm now have significantly more options , including surgical - directed therapies ; liver - directed therapies ; and nonsurgical , non - liver - directed therapies . this review provides information about the roles of hepatic resection , orthotopic liver resection , radiofrequency ablation , hepatic artery embolization and hepatic artery chemoembolization , hepatic artery radioembolization and selective internal radiation therapy , peptide receptor radionuclide therapy , systemic chemotherapy , biotherapies including somatostatin analogs and interferon- , vascular endothelial growth factor and mtor targets , and microrna - regulated pathways . given these new options , the clinician can tailor therapy specific to the patient diagnosed with nlm , thereby giving the patient the best possible chance of prolonged survival .

1. Introduction 2. Surgical-Directed Therapies 3. Liver-Directed Therapies 4. Nonsurgical, Non-Liver-Directed Therapies 5. Conclusion

patients with neuroendocrine tumors ( nets ) often suffer from neuroendocrine liver metastases ( nlms ) causing significant morbidity and mortality . the excess hormone production , the multitude of hepatic lesions , and ultimate liver disease lend to the poorer prognosis . in fact , 46%93% of patients with nets will find nlms involved at the time of diagnosis . patients with liver metastases have a significantly worse prognosis than those without liver involvement . the 5-year survival of patients with nlms on supportive care is 0%20% [ 13 ] . this dismal prognosis paints a much more stark reality for a pathological process often described as indolent . due to excessive metastatic tumor burden in difficult locations , surgical resections treatment options for patients that are not surgical candidates have evolved over the last several years . use of ablative techniques , as well as development of new medical therapies , has expanded the treatment options for the majority of patients with nlms . even in the setting of incurable disease , surgery offers the best chance for prolonged survival . in patients treated with resection , the five - year survival has been shown to be greater than 60% [ 4 , 5 ] and even approaches 80% in some studies , with minimal mortality ( < 5% ) and morbidity ( < 30% ) . a precise review of the literature available on patients who undergo liver resection for neuroendocrine tumors is difficult due to the small number of patients who are candidates and the varied approaches to surgical treatment . however , the patients that underwent an r1 resection had a 5-year survival of 70% and r2 resection still had a 5-year survival of 60% . in an effort to identify variables that have prognostic relevance to patients who undergo hepatic resection , a prospective review of 70 patients ' outcomes was performed based on tumor grade . in 2008 , the enets proposed guideline for surgical resection based on the 3 distinct patterns of liver involvement : ( 1 ) simple pattern of metastasis located in one or two contiguous lobes ( 2025% ) , ( 2 ) complex pattern where there is one major focus and other lesions are contained in the contralateral lobe ( 1015% ) , and ( 3 ) diffuse disease in both lobes ( 6070% ) . the type of surgical resection is based on the patient 's overall medical condition , size , number , and location of lesions , and adequacy of remnant liver size / function . staged , multiple surgical procedures have also been shown to be beneficial with little increase in morbidity and mortality . the majority of patients with diffuse disease are not candidates for resection . it has been estimated that less than 20% of patients with metastatic neuroendocrine tumors are candidates for hepatic resection . resection is not a viable option for the majority of patients with diffuse hepatic disease . although many centers are reluctant to allocate liver allografts to patients with metastatic disease , liver transplantation for neuroendocrine tumors is one of the only accepted indications for transplant in the setting of metastatic disease . analysis of the united network for organ sharing database reveals that between november 1988 and march 2011 , only 185 liver transplants were performed for metastatic neuroendocrine tumors in the united states . although the long - term survival is not comparable to other patients with benign disease , most liver transplant programs will consider evaluating patients with nlms . it can prolong survival and provide symptomatic relief in a very small subset of patients . nets are predisposed to form highly vascular metastatic lesions in the liver and derive more than 90% of their oxygenation and nutrition from the hepatic artery . thus , the hepatic artery offers a viable mode of introducing directed chemotherapy and/or creating an ischemic environment . this effectively starves the tumors of their nutrient and oxygen supply while sparing healthy hepatic cells , which derive the majority of their nutrient and oxygen supply from the portal venous system . defining the treatment best suited for the tumor load is dependent on number and location of the lesions , invasiveness and size of the tumor , physiology and effects of hormone secretion , and extent of metastatic disease within the patient . considering the rate of recurrence , liver - directed therapies have been considered more as debulking modalities . in a review of the literature , general guidelines for the treatment pathways are : for fewer nodular liver metastatic lesions , local resection or thermal ablation is recommended ; for a higher - tumor load due to unresectable multinodular disease or recurrent disease after resection , hepatic artery embolization , hepatic artery chemoembolization , or radioembolization is warranted . these modalities are also useful as neoadjuvants to decrease the size of previously unresectable metastatic disease . this technique is amenable to patients with fewer liver metastases who are ineligible for hepatic resection . the largest study to date , with the longest followup , was done at the cleveland clinic , a prospective trial of 89 patients with nlms who underwent 119 laparoscopic rfa sessions in total . morbidity for radiofrequency ablation of liver metastases has been reported in the larger studies to be approximately 5% to 12% , with 30-day mortality at 0% to 1% . [ 21 , 22 ] the complications can include carcinoid crisis , liver abscesses , biliopleural fistulas , bile leakage , and pleural effusion , as well as postablation syndrome , and liver failure . capitalizing on the dual blood supply of the liver hepatic artery embolization ( hae ) induces ischemia within the tumor , using a variety of agents such as cyanoacrylate , gel foam particles , polyvinyl alcohol , and microspheres . indications for hae or hepatic artery chemoembolization ( hace ) generally include unresectability with symptoms related to tumor bulk , excessive hormone production , and rapid progression of liver disease . because of the observation that higher disease regression rate and longer length of regression with systemic chemotherapy after hae was published , chemotherapy has been added to the embolic agents , and hace is now generally favored over hae . hace , also known as transarterial chemoembolization , combines the hepatic artery embolization with the hepatic artery chemoinfusion where the microspheres are bound to chemotherapy agents , which are then injected into the hepatic artery to lodge downstream within capillaries . not only do the emboli block the blood supply causing ischemic necrosis , but the chemotherapy agents are localized within the region of the metastatic lesions , creating a much more concentrated effect ( up to 20 times greater ) than systemic chemotherapy alone as well . despite this theoretical advantage , little evidence has suggested a significant difference in the outcomes of hepatic artery embolization versus hepatic artery chemoembolization . in one study of 100 patients with nlms who received hace or hae , the authors found no difference in overall survival , median survival after diagnosis of metastatic disease , or median survival after first embolization . reported results on 46 patients with nlms who received hace or hae , and showed that there was no statistically significant survival benefit in a small subset of population that also had resection of the primary tumor , although mean survival after resection increased by a mean of 558 days . there are adjuvants to hace or hae in those patients with severely limited therapeutic options to improve otherwise bleak outcomes , and hace or hae can be used as adjuvant therapy to other treatments . adding hepatic artery chemoinfusion ( hai ) to hace offers an increased probability of clinical benefits to those with unresectable , refractory disease , as presented in a study of 77 patients . of the different types of neuroendocrine tumors , carcinoid tumors seem to consistently have better outcomes to the combination of hace and hai [ 21 , 23 , 30 ] . although studies on patients with nlms are limited , in one study of 32 patients with hepatocellular carcinoma , the authors found that there was no survival advantage in patients with preoperative hace prior to surgical resection . in fact , the recurrence - free survival rates were statistically higher , and cumulative recurrence rates were statistically lower at 1 , 2 , and 5 years compared between the two groups . one study from iowa on patients with nlms showed that preoperative hace followed by olt can result favorably for the patients with progression - free intervals up to 29 months , but this was a small study , and statistical inferences could not be made due to the inclusion of only four patients . similarly , in an experimental model utilizing liver tumors in rabbits , favorable outcomes resulting in significantly decreased vascular endothelial growth factor and microvascular density levels were achieved when hace plus antiangiogenic therapies were used . as all other procedures , there are risks involved with liver - directed therapy through the hepatic artery . interest in one particular liver - directed therapy is under further investigation for this indication : hepatic artery embolization ( har ) , also known as selective internal radiation therapy ( sirt ) . rather than using peptides to localize the lesions , this therapy mechanically targets the metastases and lodges within the nutrient - supplying capillaries , thereby delivering radiation therapy . have been investigating the safety and efficacy of treatment with y radioactive microspheres for patients with unresectable nlms . this study reports favorable results with radiological response in 63.2% of patients , stable disease in 22.7% , and progression of disease in only 4.9% . radiographic and serology studies revealed median survival of 35 months with a followup of 41 months , and 55% of patients had complete or partial responses . radiation gastritis and duodenal ulcers have been described , and as all liver - directed therapies , the risk of liver failure is present . since nlms is a rare disease , large - scale , randomized trials prove difficult , and although these therapies have been used in the treatment of nets , not all have been specifically used in the treatment of nlms . due to the multiple therapies available , the effectiveness of one versus another is difficult to study , and many times nonsurgical , non - liver - directed therapies tend to be lumped together in studies that are available . moreover , there continues to be a lack of consensus on a nonsurgical treatment algorithm ; however , most agree that nonsurgical , non - liver - directed treatments of nets and nlms constitute palliative care . at least , one single - center study in the medical literature has proven that aggressive treatment of nlms with nonsurgical therapy can extend 3- and 5-year survival rates in patients to 76.4% and 63.9% as compared to previously stated survival rates of 39% and 25% , respectively . with these encouraging results and the boom in treatment advancements , the older perspective of peptide receptor radionuclide therapy ( prrt ) is an upcoming option with enticing advantages , most useful in symptomatic patients with somatostatin receptor - positive tumors , who are not surgical candidates . the more the tumor expresses somatostatin receptors as compared to the surrounding tissue , the more effective the prrt will be . somatostatin scintigraphy can predict the effectiveness of prrt : low uptake indicates 20% chance of effect on liver metastases , whereas high uptake indicates a 60% chance . side effects of prrt are rare and usually mild consisting most commonly of nausea and vomiting occurring within 24 hours of administration , and although anemia and transient toxicity grade 1 have been reported , long - lasting adverse side effects are extremely rare . several chemotherapeutic agents have been used in multiple trials , but mainly in the study of nets only , with limited success and restrictions from side effects and toxicities . the greatest efficacy seems to be related to the use of stz with other chemotherapy agents , including 5-fluorouracil and doxorubicin , but still only results in a median response time of 9.3 months . platinum - based chemotherapy regimens may be useful in patients with high - grade , poorly differentiated nets , with response rates of 42% to 80% with the use of cisplatin and etoposide [ 5456 ] , and 78% with use of oxaliplatin - based regimens . moreover , the presence of nlms may be related to worse response to chemotherapy as compared to nets . somatostatin exerts its affect by integration with one of five somatostatin receptors , ssts1 - 5 , but due to a half - life of only two minutes , somatostatin analogs ( ssa ) have been developed . the use of ssa produces a median biochemical response rate between 0% to 77% ; and biochemical and radiographic tumor stability of 28% and 55% , respectively [ 6171 ] . one review article found symptomatic and tumor response to octreotide , octreotide long - acting repeatable ( lar ) , lanreotide , and lanreotide slow - release depot ( autogel ) in 74.2% , 77.3% , 63.0% , and 67.5% , and in 57.4% , 69.8% , 46.6% , and 64.4% , respectively . another investigation demonstrated relief from flushing and diarrhea in 88% of patients after octreotide administration . interim data from the promid study with metastatic midgut nets , showed a 66% reduction in the risk of disease progression and arrested tumor growth in 69% for a median of 14.3 months . however , over 75% of patients in this study had limited liver involvement of 10% or less , and the response was highest in patients with relatively low tumor burden . the newest ssa , pasireotide , is still in clinical development stages , but is promising due to binding of ssts1 , ssts2 , ssts3 , and ssts5 , as compared to octreotide and lanreotide which bind to ssts2 and ssts5 only . cholelithiasis may arise in up to 50% of patients due to inhibition of gallbladder contractility , but only a handful will develop symptoms requiring cholecystectomy . interferons have multiple antitumor effects , and they may upregulate somatostatin receptors in nets , thereby providing a useful combination therapeutic option . interferon- can ameliorate symptoms in 30% to 70% of patients [ 81 , 82 ] , and in some studies has shown promising results with tumor response rate or stabilization in up to 70% of patients . however , the results of three randomized clinical trials involving interferon- and octreotide have mixed results . interferon- can cause fevers , chills , myalgias , depression , and myelosuppression , and is considered inferior to ssa . however , in patients with progressive disease , combination therapy may be a viable option . others have examined the role of dopamine receptors and interferon- as other possible targets , but currently , neither of these targets seems promising at this time due to ineffectiveness and short half - life . these interventions remain in the investigative process , including targeting vascular endothelial growth factors ( vegf ) , mtor pathways , other growth factor receptors , antiproliferative factors , and antiangiogenic factors . monoclonal antibodies against insulin - like growth factor-1 receptor ( igf-1r ) : amg479 , imc - a12 , and mk-0646 , are currently in clinical phase ii studies in patients with metastatic nets ( http://clinicaltrials.gov/ , identifier : nct01024387 , nct00781911 , nct00610129 ) . nets and nlms frequently overexpress the vascular endothelial growth factor ( vegf ) ligand and receptor ( vegfr ) . in a study where patients on octreotide therapy were randomized into either treatment with bevacizumab , a humanized monoclonal antibody against vegf , or interferon- , 95% of patients receiving bevacizumab were progression - free after 18 weeks , compared to 67% of patients receiving interferon- . sunitinib is a tyrosine kinase receptor inhibitor currently approved in the treatment of renal cell carcinoma and gastrointestinal stromal tumors and inhibits vegfr1 , vegfr2 , and vegfr3 . side effects of sunitinib include fatigue , asthenia , diarrhea , nausea , vomiting , anorexia , bleeding complications , mucosal inflammation , hypertension , anemia , granulocytopenia , thrombocytopenia , and hypothyroidism . the mammalian target of rapamycin ( mtor ) pathway is central to the control of cell growth , protein synthesis , and apoptosis and is activated in nets . two mtor inhibitors have been developed and approved for use in renal cell carcinomas , everolimus , and temsirolimus , and have been studied in nets [ 41 , 9395 ] . side effects of everolimus include stomatitis , rash , diarrhea , fatigue , infections , noninfectious pneumonitis , anemia , lymphopenia , hypercholesterolemia , hyperlipidemia , and hyperglycemia . further , the same group determined that decreasing levels of microrna-133a has an important role in the development , progression , and possible metastasis of midgut carcinoid tumors . a different study identified microrna-142 - 3p , -142 - 5p , -155 , -146a , and -483 as up - regulated in pancreatic nets as compared to normal tissue . this study also found that microrna-210 , -431 , and -424 were up - regulated in metastases as compared with tumors , suggesting that certain micrornas could be used to predict the probability of metastasis . additional studies are warranted in this area pertaining to microrna - regulated pathways , but already possible therapeutic targets have been identified by researchers including the high - mobility group a proteins , hmga1 , hmga2 , and the microrna family let-7 [ 103 , 104 ] . the treatment modalities available to a patient diagnosed with liver metastases due to nets are vast . the options range from surgical treatments , to locally liver - directed therapies , to systemic approaches ( figure 1 ) . however , most , if not all clinicians , agree that the treatment must be tailored specifically to the patient . yet , not all patients with nlms are candidates for surgical therapy , and in the case of an elderly asymptomatic patient with a slow - growing nets , the patient may not desire surgical therapy . liver - directed treatment can also produce great results , extending the lifetime of the patient without riskier surgical interventions . moreover , liver - directed therapies may clearly benefit a patient who is symptomatic from their tumor or may even allow that patient to be a candidate for surgical treatment in the future . lastly , nonsurgical , non - liver - directed therapies are considered palliative care in the treatment of nlms . newer technologies , including genetic targets such as microrna subtypes , are fast evolving and will continue to allow patients with nlms several options . these treatments are expected to maintain this progression well into the future , especially as knowledge of nlms increases with additional studies , so that we may provide patients diagnosed with nlms with every possible chance towards increased survival .

depression in pd is very common , with prevalence rates ranging from 50 per cent to 75 per cent . pd patients with depression have faster cognitive and motor decline and poorer quality of life and increased mortality compared to non - depressed pd patients [ 7 , 8 ] . the prevalence of anxiety disorders in pd varies from 25 to 45 per cent , and anxiety disorders are frequently comorbid with depression . pd patients with depression and comorbid anxiety have more severe pd symptoms , show poorer response to treatment for depression , and have higher functional impairment . given the evidence of poorer outcomes for pd patients with depression and anxiety , it is imperative they receive treatment . despite this , only 10 to 20 per cent of pd patients receive treatment for depression or anxiety . pharmacotherapy is the first - line treatment . in the most recent meta - analysis that included randomized placebo controlled treatments for depression and anxiety in pd it was reported that the pooled effect of antidepressants for depression in pd was moderate ( d = 0.71 ) but non - significant and the secondary effect of antidepressants on anxiety in pd was large ( d = 1.13 ) but also non - significant . interestingly , in this meta - analysis cbt was shown to have a larger effect size ( d = 1.57 ) than the antidepressant treatments . as the data is still at an early stage and especially with pharmacological studies outnumbering psychological studies it is premature to compare modalities . in the meta - analysis only two single trials of non - antidepressant treatments for depression in pd were found to have significant effects , that were large , omega-3 supplementation ( d = 0.92 ) and cbt ( d = 1.57 ) . while there is recent literature which has highlighted some promise for other non - pharmacological interventions , such as adapted exercise programs , as one case study described exercise as reducing depression and improving cognitive and motor functioning , and another open trial with nine pd patients indicated an exercise program resulted in decreased depression and motor symptoms . despite the high prevalence of depression and anxiety in pd there is still a lack of controlled trials for both pharmacological and non - pharmacological treatments for depression and anxiety in pd which limits conclusions . given the concerns that have been raised regarding the safety and side effects of pharmacological treatments , it is not surprising a recent review has indicated that cbt is a promising treatment option . a range of case studies [ 1720 ] , single case experimental design series [ 21 , 22 ] and open trials [ 23 , 24 ] , have reported cbt results in decreases in depression and anxiety in pd patients . however it is difficult to generalize from these studies with small sample sizes and lack of control groups . there have only been two randomized controlled trials ( rcts ) to date examining the impact of cbt on depression in pd . in the largest study to date 80 participants with pd and depression were randomized to receive 10 sessions of individual cbt plus clinical monitoring ( cm ) compared to clinical monitoring only . cbt participants had significantly greater reductions in ham - d depression scores , and there were significantly more treatment responders in the cbt+cm group compared to the cm group ( 56% versus 8% ) . gains were maintained at one - month follow - up . in a recent rct of group cbt a waitlist - controlled design was used with 18 adults with pd and a diagnosis of depression and/or anxiety . in the second study , participants were randomized to either an 8 week cbt group or 8 week waitlist consisting of clinical monitoring preceding treatment . at post - treatment cbt participants reported greater reductions in depression than waitlist participants of a large effect ( d = 1.12 ) . large secondary effects on anxiety were also observed for cbt participants ( d = 0.89 ) . treatment gains were maintained and further improved during follow - up , where at 6-month follow - up , significant and large effects were observed in the cbt group for both depression ( d = 2.07 ) and anxiety ( d = 2.26 ) . it is particularly noteworthy that these very large effect sizes of greater than 2.0 at 6 month follow - up indicate not only the durability of effects of group cbt on depression and anxiety , but also that treatment gains are furthered over time . these studies are encouraging given the large effect sizes found particularly in reduction of depression in pd , however more research is still required with rcts with adequate sample sizes and longer follow up periods . there is a need to develop a specific cbt conceptualization for depression and anxiety in pd . as yet no pd - specific cbt conceptual framework exists . central to cbt is the concept that an individual s appraisal of their experience determines its impact . in this way appraisal of the meaning of being diagnosed with a degenerating illness such as pd will influence ones response and coping . cbt clinicians seek to understand the individual experience of the diagnosis of pd and the meanings associated with this as it may determine how the person copes with pd . the opinions and views of those around the person with pd are important as sometimes carers inadvertently reinforce a sense of dependence in the person with pd . cbt involving carers of people with pd may be empowering of both partners in the dyad as the problem - solving orientation of cbt . we suggest that partners and carers should be directly involved in treatment , where possible , and if appropriate given the nature of the relationship , for example , if it is someone that the pd patient has a relatively good relationship with . it is suggested that the partner / carer with the patient s consent , attends part of the formulation feedback session ( usually session two ) , where the cbt therapist and patient have already worked out in a collaborative manner in the first half of the session an individualized model of what specific cognitive and behavioral factors are maintaining their depression and anxiety symptoms , and explain this to the partner / carer . the patient and therapist can then directly ask the partner / carer for their assistance in the treatment strategies to address these maintaining factors . for example , a partner may help behavioral experiments regarding independence by doing less for the person with pd , and a carer may help address experiments aimed at reducing isolation by facilitating the patient s attendance at social gatherings . a comprehensive conceptualization should involve conceptual level suggestions for treatment modification including taking account of individual health beliefs , beliefs about disability , cohort beliefs ( which are beliefs shared amongst a peer group born in the same generation ) and core beliefs . a case example is outlined to demonstrate the benefits of developing a case conceptualization based on a guiding model of cbt for anxiety and depression in pd as shown in fig . 1 . bill was a 73 year old retired shopkeeper who presented with symptoms of generalized anxiety disorder and depression . he had watched his best friend die of end stage pd one year prior to his own diagnosis of pd . understandably , he became anxious the same future awaited him when he received his diagnosis . bill became depressed and stopped doing the activities that gave his life meaning and pleasure such as photography . he believed that his pd would prevent him doing photography well and thus he gave away all of his equipment as he thought my life is over because of my pd and i am going to have to stop doing this sooner or later so may as well get the pain over with now . bill also reported isolating himself due to embarrassment over his motor symptoms , for example worrying he would spill food and drinks . he stopped attending a music club with his friends and also reduced his interest in buying music to share . bill also stated that since his wife had died five years ago he had started to go out and meet with friends , but since his diagnosis of pd had withdrawn from his friends , felt more lonely and was missing her more . bill also worried about being depressed , and that he was losing his identity and was weak . he was worried he would get to the point of no return with his pd and as a result avoided doing many things where his symptoms would be noticeable , and became hypervigilant about his symptoms looking for the start of the end . i am incompetent and can do nothing at all and i am completely not able to function , my life is over . the cbt model outlined in fig . 1 is a new model that has not been outlined before , and is based on standard cognitive - behavioral models where core beliefs are defined as rigid and inflexible beliefs which are developed due to early life experiences and then activated later in life due to stressors such as the diagnosis of pd . in the case of bill his core belief i am incompetent lead him to value being able to try and prove his competence through being highly independent and solving problems and he thus had associated beliefs that a man stands on his two feet and if i can not do things by myself , then i am incompetent . there are also illness beliefs which arise from core beliefs that make someone more likely to react with negative emotions to pd , for bill this was watching his best friend die of pd , and was linked with his fear cohort beliefs refer to values and beliefs shared amongst a generational group of individuals born at a similar time period . for a man of bills generation , he possessed a common cohort belief that people who are disabled are weak , needy and dependent . cbt for depression and anxiety disorders in pd focuses on challenging the negative cognitions that maintain distress in pd . negative pd related cognitions are over - generalized in nature , for example in bill s case his cognition was having pd is terrible it means my life is over . as a result of this negativecognition he experienced a number of specific secondary cognitive biases . these tremors are terrible i might as well sell my photography equipment and car now i am going to lose everything eventually . bill overgeneralized the level of impairment he is experiencing with pd as he remains currently able to function independently but is catastrophizing about reduction in future functioning . another negative cognition that maintains depression in pd is comparison to previous function and function of others . this involves an unhelpful process of comparing one s current functioning with functioning prior to pd , for example bill thinks i used to do this [ task ] without thinking now look what i have come to and everyone else can do simple things without thinking about it . bill s rumination centered on why have i got this , it is unfair , i am a burden which maintained his depression . hypervigilance refers to constantly scanning physical symptoms which may signal decline in functioning . in bill s case this involved monitoring himself for tremors , and he interpreted the presence of tremors as evidence for his illness overgeneralizing and catastrophizing beliefs . another behavioral factor is counterproductive behaviors ( also known as safety behaviors ) , and these are often seen in the individual with anxiety about their pd . for example , bill s counterproductive behavior was to tightly hold his hands when in public to avoid his resting tremor being noticeable to others . the problem however was that it made his tremors worse , further reinforcing anxiety regarding the tremors . avoidance is a very common behavioral response and involves avoidance of situations the individual finds distressing due to their pd symptoms . bill avoided eating in front of others due to a concern that he might spill food or drink , even though this had not happened before , which resulted in social isolation . another factor which was important in bill s case was his feelings of loneliness and missing his wife who had died , which interacted with his withdrawal and social isolation as a result of concern over his pd symptoms . this model is new and has not been proposed before , although combines the commonly known important cognitive and behavioral maintaining factors of anxiety and depression including core beliefs , rumination , hypervigilance for physical symptoms , counterproductive ( safety ) behaviors , and avoidance . while these are common factors that are included in general cognitive behavioral maintenance models , they can be specifically adapted to suit how they are maintaining anxiety and depression for the individual with pd as outlined in the example . in addition to these general factors which can be adapted in an individualized case formulation with the patient with pd , the model also considers several factors which are specific to pd such as illness beliefs , cohort beliefs , and comparison to previous function / function of others . given this model is new it would be useful for future research to examine if the model has validity in accounting for the maintenance of anxiety and depression in parkinson s disease . this could be examined in future research by determining the predictive validity of the model by including measures of the factors specified in the model , e.g. , rumination , avoidance , with measures of anxiety and depression in individuals with pd to determine the degree to which the factors can account for symptoms . however this would only be an indirect test of the model , the model could be more directly tested in a large sample of pd patients through the use of structural equation modeling to determine if the pathways between the hypothesized factors and anxiety and depression are found . in terms of which patients it is appropriate to use cbt with , we suggest that this treatment is utilized in pd patients who do not have dementia . in trials we have typically excluded patients who score less than 24 on the mini mental state examination . it is also helpful if the patient expresses some degree of willingness to attend psychotherapy , those who have been referred by a partner / carer but who are reluctant to attend of their own accord may be difficult to engage in therapy . in a recent book on cbt with older people , guidelines for working with clients with chronicity and comorbidity , laidlaw updated previous recommendations that clinicians may find useful when working with clients presenting with depression and anxiety in pd [ 35 , 36 ] . one of the most common reasons that patients with pd do not come to therapy or dropout is practical problems in accessing therapy such as transport to sessions , or concerns over motor symptoms in session . to overcome this in trials of cbt for depression and anxiety in pd we have typically provided vouchers for taxis for patients to attend sessions so they are not reliant on carers or others to transport them to session or needing to catch public transport if they can not or do not wish to . however , in the reality of clinical practice if funds are not available for taxis , then it is useful to see if a carer or friend can help with transport . for example , in bill s case although he used public transport he was concerned about getting lost coming to a new clinic so a friend provided him with transport a to reduce this barrier . barriers to therapy can also be overcome by the use of telemedicine and videoconferencing as a means to deliver therapy in the client s home who is not able to attend the clinic or lives in a rural or remote area . physical barriers can be overcome by ensuring that the patient with pd is offered regular breaks during sessions , and session times scheduled around what is typically a better period for them for motor symptoms during the day . we recommend that clinicians outline these issues in a phone screening session with the client prior to their first appointment to remove barriers and set the scene that it is possible to engage in therapy even with physical limitations that may be present . we also suggest that in session writing is reduced , and formats of handouts are adapted if necessary for the individual , for example with larger font in handouts and thought records.(2)accepting depression as a separate but reversible problem distinct from their ongoing physical problems . one of the most common presentations that are seen in pd patients with depression is confusion over the symptoms of depression and the patient attributing symptoms of depression to parkinson s disease . often patients are surprised when feedback is given regarding a diagnosis of depression , and that it is possible for depression to improve as they believed the way they were feeling was a part of pd . for example , bill reported that he thought the low mood and anxiety he felt was just a part of living with pd and was surprised when the therapist suggested that they could help to remove those symptoms . this is an important point to collaboratively discuss with patients as it can help them particularly at the start of therapy to feel more hopeful about the future and more engaged in the therapeutic process if they realize that it is possible to have a significant improvement in their mood and quality of life that is independent from the process of the disease of pd , that they are not stuck with feeling depressed and anxious due to the pd that it is distinct from pd and can be improved.(3)identifying and limiting excess disability . it is important to identify in a collaborative manner with patients the extra disability that symptoms of depression and anxiety are adding to their life in addition to the symptoms of pd . this can be done in an initial formulation session with the patient , where the impact of symptoms of depression and anxiety on their life are explored . for example , bill had stopped doing many pleasant events such as photography as he perceived he would no longer be able to do them due to his symptoms of pd , but when this was explored with bill it was discovered that in fact he could still do the photography with his current motor symptoms , but was concerned that in the future it would become so bad he would have to give it up . the therapist helped bill to understand that by reengaging in pleasant activities that he could indeed still engage in he would start to feel better , and that even if he did find in the future that an activity was more difficult due to his pd symptoms , that a more helpful approach would be to adjust how he did the event rather than give up doing it , as most activities could be engaged in with some degree of adjustment while still enjoying them.(4)recognizing and limiting losses of roles and autonomy . many patients with pd report low mood associated with a loss of roles on diagnosis of pd , for example , if patients stop working , or doing other valued activities , and also frustration at a loss of autonomy , for example if they rely on others to help with daily self - care activities or for transport . it is useful to help the patient to see that while there may indeed be some loss of roles due to the pd , and to validate these losses in terms of the grief and loss regarding this , that not all roles are necessarily lost . for example , bill still had a wide circle of friends despite having withdrawn from them and his previous roles of organizing events with friends for music and photography were still possible if he reengaged with them . regarding loss of autonomy , in bill s case he was distressed when he sometimes had to ask others for a lift to an appointment and so avoided doing so and often missed medical appointments or suffered inconvenience due to difficulty with public transport . the therapist helped bill to see that it was ok to sometimes ask others for lifts as they liked to help , and while he had lost some autonomy he was still able to function independently in most aspects of his life , and that he could feel better by offering help to friends in return for their favors.(5)challenging self - perception of oneself as a burden to others . it is very common for patients with pd to report feeling concerned that they are a burden to carers . in bill s case he reported feeling like a burden to his friends if he asked them for favors , therefore in a similar manner to the previous point , the therapist helped bill to understand the positive effect of helping others on the friend feeling good for helping , and also for the social interaction that occurred as a result . bill was encouraged by the therapist to think of other ways that he contributed to his friendships and was not a burden , for example he had helped a friend with his tax return and this was commented on by the friend as being a very helpful activity that bill did . one of the most common reasons that patients with pd do not come to therapy or dropout is practical problems in accessing therapy such as transport to sessions , or concerns over motor symptoms in session . to overcome this in trials of cbt for depression and anxiety in pd we have typically provided vouchers for taxis for patients to attend sessions so they are not reliant on carers or others to transport them to session or needing to catch public transport if they can not or do not wish to . however , in the reality of clinical practice if funds are not available for taxis , then it is useful to see if a carer or friend can help with transport . for example , in bill s case although he used public transport he was concerned about getting lost coming to a new clinic so a friend provided him with transport a to reduce this barrier . barriers to therapy can also be overcome by the use of telemedicine and videoconferencing as a means to deliver therapy in the client s home who is not able to attend the clinic or lives in a rural or remote area . physical barriers can be overcome by ensuring that the patient with pd is offered regular breaks during sessions , and session times scheduled around what is typically a better period for them for motor symptoms during the day . we recommend that clinicians outline these issues in a phone screening session with the client prior to their first appointment to remove barriers and set the scene that it is possible to engage in therapy even with physical limitations that may be present . we also suggest that in session writing is reduced , and formats of handouts are adapted if necessary for the individual , for example with larger font in handouts and thought records . accepting depression as a separate but reversible problem distinct from their ongoing physical problems . one of the most common presentations that are seen in pd patients with depression is confusion over the symptoms of depression and the patient attributing symptoms of depression to parkinson s disease . often patients are surprised when feedback is given regarding a diagnosis of depression , and that it is possible for depression to improve as they believed the way they were feeling was a part of pd . for example , bill reported that he thought the low mood and anxiety he felt was just a part of living with pd and was surprised when the therapist suggested that they could help to remove those symptoms . this is an important point to collaboratively discuss with patients as it can help them particularly at the start of therapy to feel more hopeful about the future and more engaged in the therapeutic process if they realize that it is possible to have a significant improvement in their mood and quality of life that is independent from the process of the disease of pd , that they are not stuck with feeling depressed and anxious due to the pd that it is distinct from pd and can be improved . identifying and limiting excess disability . it is important to identify in a collaborative manner with patients the extra disability that symptoms of depression and anxiety are adding to their life in addition to the symptoms of pd . this can be done in an initial formulation session with the patient , where the impact of symptoms of depression and anxiety on their life are explored . for example , bill had stopped doing many pleasant events such as photography as he perceived he would no longer be able to do them due to his symptoms of pd , but when this was explored with bill it was discovered that in fact he could still do the photography with his current motor symptoms , but was concerned that in the future it would become so bad he would have to give it up . the therapist helped bill to understand that by reengaging in pleasant activities that he could indeed still engage in he would start to feel better , and that even if he did find in the future that an activity was more difficult due to his pd symptoms , that a more helpful approach would be to adjust how he did the event rather than give up doing it , as most activities could be engaged in with some degree of adjustment while still enjoying them . recognizing and limiting losses of roles and autonomy . many patients with pd report low mood associated with a loss of roles on diagnosis of pd , for example , if patients stop working , or doing other valued activities , and also frustration at a loss of autonomy , for example if they rely on others to help with daily self - care activities or for transport . it is useful to help the patient to see that while there may indeed be some loss of roles due to the pd , and to validate these losses in terms of the grief and loss regarding this , that not all roles are necessarily lost . for example , bill still had a wide circle of friends despite having withdrawn from them and his previous roles of organizing events with friends for music and photography were still possible if he reengaged with them . regarding loss of autonomy , in bill s case he was distressed when he sometimes had to ask others for a lift to an appointment and so avoided doing so and often missed medical appointments or suffered inconvenience due to difficulty with public transport . the therapist helped bill to see that it was ok to sometimes ask others for lifts as they liked to help , and while he had lost some autonomy he was still able to function independently in most aspects of his life , and that he could feel better by offering help to friends in return for their favors . it is very common for patients with pd to report feeling concerned that they are a burden to carers . in bill s case he reported feeling like a burden to his friends if he asked them for favors , therefore in a similar manner to the previous point , the therapist helped bill to understand the positive effect of helping others on the friend feeling good for helping , and also for the social interaction that occurred as a result . bill was encouraged by the therapist to think of other ways that he contributed to his friendships and was not a burden , for example he had helped a friend with his tax return and this was commented on by the friend as being a very helpful activity that bill did . cbt for pd typically involves 812 one hour sessions that are conducted on a weekly basis . examples of the cbt strategies that can be employed for the maintaining factors proposed in the model are outlined in table 1 . it should be noted that therapy is not intended to be a strict session by session protocol , rather as is commonplace in most modern cbt approaches for various problems , therapy should be tailored to the individual based on an individualized formulation of the person s problems and disease severity . the model should be used as a guide to help develop this individualized formulation , and then therapy and which specific techniques are to be used based on the particular individual formulation . for example , not all maintaining factors that are proposed in the model may be present for a particular person , and therefore only the maintaining factors that are important to the individual should be targeted , and the specific strategies that can be used to address these as shown in table 1 . in the first phase of treatment , emphasis the early sessions address the first three elements outlined of practical barriers , seeing depression as separate but reversible condition and decreasing excess disability through starting behavioral experiments to challenge beliefs regarding disability . clinicians should also establish goals for treatment and engage in psychoeducation regarding anxiety disorders and depression in this early phase . psychoeducation generally occurs around session 2 - 3 and involves the patient understanding what are symptoms of anxiety and the fight or flight response , what symptoms constitute depression and how these are maintained for example by negative cognitions , social withdrawal and lack of engagement in pleasant events . typically psychoeducation can be conducted in the same session as the collaborative formulation where the cbt therapist is addressing with the patient the understanding of why their problem has arisen and what factors are maintaining their depression and anxiety , and can be continued in the next session if required . in the initial phase of treatment the clinician should also engage caregivers in treatment , given caregiver participation , in particular spouses , has been found to positively influence treatment response to cbt for depression in pd . in the mid - phase of treatment , clinicians challenge beliefs regarding limiting losses of roles and autonomy by introducing the experience of diagnosis as being like a grief process . a phase model of grief ( i.e. worden defined stages of mourning ; bargaining and denial , anger , acceptance and moving on ) can help clients come to terms with pd and focus more explicitly upon what aspects of life they appraise to be most keenly felt in terms of loss post - diagnosis . while becoming aware of one s limitations can be painful , it can also promote a realistic appraisal and hence adjustment to living with pd . this is captured in the framework for optimizing function in the face of realistic challenges of ageing . baltes and colleagues developed selective , optimization with compensation to explain how individuals can proactively and intentionally make adjustments in order to optimize functioning in light of age - related challenges to wellbeing . this recognizes goals can be attained through alternative means and assists the client to function at their most optimal level . although people living with pd often have to deal with realistic challenges , individuals nevertheless often endorse unhelpful or erroneous cognitions about these challenges . challenging these unhelpful cognitions a behavioral experiment challenges negative cognitions by testing out a specific negative prediction an individual believes will occur and is a highly effective technique . bill tested out his belief that he would be incompetent if he made a mistake by testing his negative prediction that others would laugh at him if he spilled his food and drink . he first tested out in session if he would spill food or drink and then out for lunch with his friends . the result of the experiment was that bill spilled a small amount of drink but not an entire glass as he had predicted and his friends did not notice or say anything . as a result , he concluded others are unlikely to laugh at him in public and this helped him to go out and decrease his social isolation which resulted in decreased depressive symptoms . cognitive restructuring focuses on challenging unhelpful thoughts about the impact of pd perceived limitations due to pd . thought diaries are useful in challenging negative pd related specific cognitions and cognitive biases . thought diaries are used to challenge any negative cognition the individual with pd is experiencing as it is these negative cognitions which are seen as maintaining depression and anxiety disorders in pd . behavioral activation which has extensive evidence of efficacy in the treatment of depression in older adults involves engaging the person is pleasant and rewarding events is also used to improve mood , along with decreasing avoidance and social isolation . furthermore , behavioral activation has been shown to be effective in reducing depression when delivered to older adults via videoconferencing so is highly suitable for a pd population where barriers to accessing therapy in a clinic are a problem . the aim of cbt is for the person to become their own therapist and to continue to use the cognitive techniques of thought diaries , behavioral experiments , and behavioral activation to challenge negative cognitions and symptoms of depression and anxiety in the future . it is hoped that this model of cbt helps clinicians to further research on efficacy and lead to widespread dissemination of cbt as a viable treatment option for depression and anxiety in pd given the promising evidence of efficacy [ 3 , 12 ] .

abstractevidence is reviewed demonstrating that cognitive behavior therapy ( cbt ) is effective in the treatment of depression and anxiety in parkinson s disease . the aims were to review the extant literature , specify a model of cognitive and behavioral maintenance factors in depression and anxiety in parkinson s disease and provide a guide to treatment . it is argued that treatment should take into account specific cognitive and behavioral maintaining factors . symptoms of depression and anxiety are highly prevalent in parkinson s disease and therapists should consider how to augment the efficacy of cbt for patients with parkinson s disease . cognitive and behavioral interventions can help people overcome some of the challenges in living with pd by maximizing wellbeing and overall quality of life .

PREVALENCE AND IMPACT OF ANXIETY AND DEPRESSION IN PARKINSONS DISEASE TREATMENT OF ANXIETY AND DEPRESSION IN PARKINSONS DISEASE EFFICACY OF CBT FOR DEPRESSION AND ANXIETY IN PARKINSONS DISEASE RATIONALE FOR A COGNITIVE BEHAVIORAL MODEL OF ANXIETY AND DEPRESSION IN PARKINSONS DISEASE A COGNITIVE BEHAVIORAL MODEL GUIDELINES FOR CBT FOR DEPRESSION AND ANXIETY IN PARKINSONS DISEASE SUMMARY DISCLOSURE STATEMENT

pd patients with depression have faster cognitive and motor decline and poorer quality of life and increased mortality compared to non - depressed pd patients [ 7 , 8 ] . the prevalence of anxiety disorders in pd varies from 25 to 45 per cent , and anxiety disorders are frequently comorbid with depression . pd patients with depression and comorbid anxiety have more severe pd symptoms , show poorer response to treatment for depression , and have higher functional impairment . given the evidence of poorer outcomes for pd patients with depression and anxiety , it is imperative they receive treatment . in the most recent meta - analysis that included randomized placebo controlled treatments for depression and anxiety in pd it was reported that the pooled effect of antidepressants for depression in pd was moderate ( d = 0.71 ) but non - significant and the secondary effect of antidepressants on anxiety in pd was large ( d = 1.13 ) but also non - significant . as the data is still at an early stage and especially with pharmacological studies outnumbering psychological studies it is premature to compare modalities . while there is recent literature which has highlighted some promise for other non - pharmacological interventions , such as adapted exercise programs , as one case study described exercise as reducing depression and improving cognitive and motor functioning , and another open trial with nine pd patients indicated an exercise program resulted in decreased depression and motor symptoms . despite the high prevalence of depression and anxiety in pd there is still a lack of controlled trials for both pharmacological and non - pharmacological treatments for depression and anxiety in pd which limits conclusions . given the concerns that have been raised regarding the safety and side effects of pharmacological treatments , it is not surprising a recent review has indicated that cbt is a promising treatment option . a range of case studies [ 1720 ] , single case experimental design series [ 21 , 22 ] and open trials [ 23 , 24 ] , have reported cbt results in decreases in depression and anxiety in pd patients . however it is difficult to generalize from these studies with small sample sizes and lack of control groups . there have only been two randomized controlled trials ( rcts ) to date examining the impact of cbt on depression in pd . in the largest study to date 80 participants with pd and depression were randomized to receive 10 sessions of individual cbt plus clinical monitoring ( cm ) compared to clinical monitoring only . cbt participants had significantly greater reductions in ham - d depression scores , and there were significantly more treatment responders in the cbt+cm group compared to the cm group ( 56% versus 8% ) . in a recent rct of group cbt a waitlist - controlled design was used with 18 adults with pd and a diagnosis of depression and/or anxiety . in the second study , participants were randomized to either an 8 week cbt group or 8 week waitlist consisting of clinical monitoring preceding treatment . treatment gains were maintained and further improved during follow - up , where at 6-month follow - up , significant and large effects were observed in the cbt group for both depression ( d = 2.07 ) and anxiety ( d = 2.26 ) . it is particularly noteworthy that these very large effect sizes of greater than 2.0 at 6 month follow - up indicate not only the durability of effects of group cbt on depression and anxiety , but also that treatment gains are furthered over time . these studies are encouraging given the large effect sizes found particularly in reduction of depression in pd , however more research is still required with rcts with adequate sample sizes and longer follow up periods . there is a need to develop a specific cbt conceptualization for depression and anxiety in pd . in this way appraisal of the meaning of being diagnosed with a degenerating illness such as pd will influence ones response and coping . cbt clinicians seek to understand the individual experience of the diagnosis of pd and the meanings associated with this as it may determine how the person copes with pd . the opinions and views of those around the person with pd are important as sometimes carers inadvertently reinforce a sense of dependence in the person with pd . cbt involving carers of people with pd may be empowering of both partners in the dyad as the problem - solving orientation of cbt . we suggest that partners and carers should be directly involved in treatment , where possible , and if appropriate given the nature of the relationship , for example , if it is someone that the pd patient has a relatively good relationship with . it is suggested that the partner / carer with the patient s consent , attends part of the formulation feedback session ( usually session two ) , where the cbt therapist and patient have already worked out in a collaborative manner in the first half of the session an individualized model of what specific cognitive and behavioral factors are maintaining their depression and anxiety symptoms , and explain this to the partner / carer . the patient and therapist can then directly ask the partner / carer for their assistance in the treatment strategies to address these maintaining factors . a comprehensive conceptualization should involve conceptual level suggestions for treatment modification including taking account of individual health beliefs , beliefs about disability , cohort beliefs ( which are beliefs shared amongst a peer group born in the same generation ) and core beliefs . a case example is outlined to demonstrate the benefits of developing a case conceptualization based on a guiding model of cbt for anxiety and depression in pd as shown in fig . bill was a 73 year old retired shopkeeper who presented with symptoms of generalized anxiety disorder and depression . he was worried he would get to the point of no return with his pd and as a result avoided doing many things where his symptoms would be noticeable , and became hypervigilant about his symptoms looking for the start of the end . in the case of bill his core belief i am incompetent lead him to value being able to try and prove his competence through being highly independent and solving problems and he thus had associated beliefs that a man stands on his two feet and if i can not do things by myself , then i am incompetent . cbt for depression and anxiety disorders in pd focuses on challenging the negative cognitions that maintain distress in pd . bill s rumination centered on why have i got this , it is unfair , i am a burden which maintained his depression . another behavioral factor is counterproductive behaviors ( also known as safety behaviors ) , and these are often seen in the individual with anxiety about their pd . this model is new and has not been proposed before , although combines the commonly known important cognitive and behavioral maintaining factors of anxiety and depression including core beliefs , rumination , hypervigilance for physical symptoms , counterproductive ( safety ) behaviors , and avoidance . while these are common factors that are included in general cognitive behavioral maintenance models , they can be specifically adapted to suit how they are maintaining anxiety and depression for the individual with pd as outlined in the example . in addition to these general factors which can be adapted in an individualized case formulation with the patient with pd , the model also considers several factors which are specific to pd such as illness beliefs , cohort beliefs , and comparison to previous function / function of others . given this model is new it would be useful for future research to examine if the model has validity in accounting for the maintenance of anxiety and depression in parkinson s disease . this could be examined in future research by determining the predictive validity of the model by including measures of the factors specified in the model , e.g. , rumination , avoidance , with measures of anxiety and depression in individuals with pd to determine the degree to which the factors can account for symptoms . however this would only be an indirect test of the model , the model could be more directly tested in a large sample of pd patients through the use of structural equation modeling to determine if the pathways between the hypothesized factors and anxiety and depression are found . in terms of which patients it is appropriate to use cbt with , we suggest that this treatment is utilized in pd patients who do not have dementia . it is also helpful if the patient expresses some degree of willingness to attend psychotherapy , those who have been referred by a partner / carer but who are reluctant to attend of their own accord may be difficult to engage in therapy . in a recent book on cbt with older people , guidelines for working with clients with chronicity and comorbidity , laidlaw updated previous recommendations that clinicians may find useful when working with clients presenting with depression and anxiety in pd [ 35 , 36 ] . one of the most common reasons that patients with pd do not come to therapy or dropout is practical problems in accessing therapy such as transport to sessions , or concerns over motor symptoms in session . to overcome this in trials of cbt for depression and anxiety in pd we have typically provided vouchers for taxis for patients to attend sessions so they are not reliant on carers or others to transport them to session or needing to catch public transport if they can not or do not wish to . however , in the reality of clinical practice if funds are not available for taxis , then it is useful to see if a carer or friend can help with transport . barriers to therapy can also be overcome by the use of telemedicine and videoconferencing as a means to deliver therapy in the client s home who is not able to attend the clinic or lives in a rural or remote area . physical barriers can be overcome by ensuring that the patient with pd is offered regular breaks during sessions , and session times scheduled around what is typically a better period for them for motor symptoms during the day . one of the most common presentations that are seen in pd patients with depression is confusion over the symptoms of depression and the patient attributing symptoms of depression to parkinson s disease . often patients are surprised when feedback is given regarding a diagnosis of depression , and that it is possible for depression to improve as they believed the way they were feeling was a part of pd . for example , bill reported that he thought the low mood and anxiety he felt was just a part of living with pd and was surprised when the therapist suggested that they could help to remove those symptoms . this is an important point to collaboratively discuss with patients as it can help them particularly at the start of therapy to feel more hopeful about the future and more engaged in the therapeutic process if they realize that it is possible to have a significant improvement in their mood and quality of life that is independent from the process of the disease of pd , that they are not stuck with feeling depressed and anxious due to the pd that it is distinct from pd and can be improved. it is important to identify in a collaborative manner with patients the extra disability that symptoms of depression and anxiety are adding to their life in addition to the symptoms of pd . this can be done in an initial formulation session with the patient , where the impact of symptoms of depression and anxiety on their life are explored . for example , bill had stopped doing many pleasant events such as photography as he perceived he would no longer be able to do them due to his symptoms of pd , but when this was explored with bill it was discovered that in fact he could still do the photography with his current motor symptoms , but was concerned that in the future it would become so bad he would have to give it up . the therapist helped bill to understand that by reengaging in pleasant activities that he could indeed still engage in he would start to feel better , and that even if he did find in the future that an activity was more difficult due to his pd symptoms , that a more helpful approach would be to adjust how he did the event rather than give up doing it , as most activities could be engaged in with some degree of adjustment while still enjoying them. many patients with pd report low mood associated with a loss of roles on diagnosis of pd , for example , if patients stop working , or doing other valued activities , and also frustration at a loss of autonomy , for example if they rely on others to help with daily self - care activities or for transport . it is useful to help the patient to see that while there may indeed be some loss of roles due to the pd , and to validate these losses in terms of the grief and loss regarding this , that not all roles are necessarily lost . it is very common for patients with pd to report feeling concerned that they are a burden to carers . one of the most common reasons that patients with pd do not come to therapy or dropout is practical problems in accessing therapy such as transport to sessions , or concerns over motor symptoms in session . to overcome this in trials of cbt for depression and anxiety in pd we have typically provided vouchers for taxis for patients to attend sessions so they are not reliant on carers or others to transport them to session or needing to catch public transport if they can not or do not wish to . however , in the reality of clinical practice if funds are not available for taxis , then it is useful to see if a carer or friend can help with transport . barriers to therapy can also be overcome by the use of telemedicine and videoconferencing as a means to deliver therapy in the client s home who is not able to attend the clinic or lives in a rural or remote area . physical barriers can be overcome by ensuring that the patient with pd is offered regular breaks during sessions , and session times scheduled around what is typically a better period for them for motor symptoms during the day . we recommend that clinicians outline these issues in a phone screening session with the client prior to their first appointment to remove barriers and set the scene that it is possible to engage in therapy even with physical limitations that may be present . one of the most common presentations that are seen in pd patients with depression is confusion over the symptoms of depression and the patient attributing symptoms of depression to parkinson s disease . often patients are surprised when feedback is given regarding a diagnosis of depression , and that it is possible for depression to improve as they believed the way they were feeling was a part of pd . for example , bill reported that he thought the low mood and anxiety he felt was just a part of living with pd and was surprised when the therapist suggested that they could help to remove those symptoms . this is an important point to collaboratively discuss with patients as it can help them particularly at the start of therapy to feel more hopeful about the future and more engaged in the therapeutic process if they realize that it is possible to have a significant improvement in their mood and quality of life that is independent from the process of the disease of pd , that they are not stuck with feeling depressed and anxious due to the pd that it is distinct from pd and can be improved . it is important to identify in a collaborative manner with patients the extra disability that symptoms of depression and anxiety are adding to their life in addition to the symptoms of pd . this can be done in an initial formulation session with the patient , where the impact of symptoms of depression and anxiety on their life are explored . for example , bill had stopped doing many pleasant events such as photography as he perceived he would no longer be able to do them due to his symptoms of pd , but when this was explored with bill it was discovered that in fact he could still do the photography with his current motor symptoms , but was concerned that in the future it would become so bad he would have to give it up . the therapist helped bill to understand that by reengaging in pleasant activities that he could indeed still engage in he would start to feel better , and that even if he did find in the future that an activity was more difficult due to his pd symptoms , that a more helpful approach would be to adjust how he did the event rather than give up doing it , as most activities could be engaged in with some degree of adjustment while still enjoying them . many patients with pd report low mood associated with a loss of roles on diagnosis of pd , for example , if patients stop working , or doing other valued activities , and also frustration at a loss of autonomy , for example if they rely on others to help with daily self - care activities or for transport . it is useful to help the patient to see that while there may indeed be some loss of roles due to the pd , and to validate these losses in terms of the grief and loss regarding this , that not all roles are necessarily lost . it is very common for patients with pd to report feeling concerned that they are a burden to carers . cbt for pd typically involves 812 one hour sessions that are conducted on a weekly basis . examples of the cbt strategies that can be employed for the maintaining factors proposed in the model are outlined in table 1 . the model should be used as a guide to help develop this individualized formulation , and then therapy and which specific techniques are to be used based on the particular individual formulation . for example , not all maintaining factors that are proposed in the model may be present for a particular person , and therefore only the maintaining factors that are important to the individual should be targeted , and the specific strategies that can be used to address these as shown in table 1 . in the first phase of treatment , emphasis the early sessions address the first three elements outlined of practical barriers , seeing depression as separate but reversible condition and decreasing excess disability through starting behavioral experiments to challenge beliefs regarding disability . psychoeducation generally occurs around session 2 - 3 and involves the patient understanding what are symptoms of anxiety and the fight or flight response , what symptoms constitute depression and how these are maintained for example by negative cognitions , social withdrawal and lack of engagement in pleasant events . typically psychoeducation can be conducted in the same session as the collaborative formulation where the cbt therapist is addressing with the patient the understanding of why their problem has arisen and what factors are maintaining their depression and anxiety , and can be continued in the next session if required . in the initial phase of treatment the clinician should also engage caregivers in treatment , given caregiver participation , in particular spouses , has been found to positively influence treatment response to cbt for depression in pd . in the mid - phase of treatment , clinicians challenge beliefs regarding limiting losses of roles and autonomy by introducing the experience of diagnosis as being like a grief process . worden defined stages of mourning ; bargaining and denial , anger , acceptance and moving on ) can help clients come to terms with pd and focus more explicitly upon what aspects of life they appraise to be most keenly felt in terms of loss post - diagnosis . while becoming aware of one s limitations can be painful , it can also promote a realistic appraisal and hence adjustment to living with pd . this is captured in the framework for optimizing function in the face of realistic challenges of ageing . although people living with pd often have to deal with realistic challenges , individuals nevertheless often endorse unhelpful or erroneous cognitions about these challenges . the result of the experiment was that bill spilled a small amount of drink but not an entire glass as he had predicted and his friends did not notice or say anything . thought diaries are used to challenge any negative cognition the individual with pd is experiencing as it is these negative cognitions which are seen as maintaining depression and anxiety disorders in pd . behavioral activation which has extensive evidence of efficacy in the treatment of depression in older adults involves engaging the person is pleasant and rewarding events is also used to improve mood , along with decreasing avoidance and social isolation . furthermore , behavioral activation has been shown to be effective in reducing depression when delivered to older adults via videoconferencing so is highly suitable for a pd population where barriers to accessing therapy in a clinic are a problem . the aim of cbt is for the person to become their own therapist and to continue to use the cognitive techniques of thought diaries , behavioral experiments , and behavioral activation to challenge negative cognitions and symptoms of depression and anxiety in the future . it is hoped that this model of cbt helps clinicians to further research on efficacy and lead to widespread dissemination of cbt as a viable treatment option for depression and anxiety in pd given the promising evidence of efficacy [ 3 , 12 ] .

anticholinergic medicines bind to the muscarinic receptors , and the decreased cholinergic neurotransmission impacts both peripheral and central body systems . peripheral anticholinergic adverse effects mainly include constipation , urinary retention , dry mouth , blurred vision , peristaltic reduction and tachycardia . the central anticholinergic adverse effects of clinical interest are cognitive impairment , confusion and delirium . the risk of anticholinergic adverse events has been found to increase with age , pre - existing cognitive impairment , polypharmacy , comorbidities and cholinergic neuronal degeneration , and it is influenced by individual pharmacokinetic and pharmacodynamic variability . the frequency of adverse drug reactions reported in older adults ( 65 years ) is 7-fold higher than in younger adults . epidemiological studies have reported that 20 - 50% of the older population are routinely exposed to medicines that possess anticholinergic properties . recent literature has highlighted that up to 40% of all institutionalised older people take medicines with known anticholinergic activity . similarly , it has been reported that among hospitalised older people , the prevalence of those taking anticholinergic medicines increased significantly ( 7.2 3.5 vs. 5.7 3.1 ; p < 0.001 ) during their hospital stay . the cumulative exposure to multiple medicines with anticholinergic properties numerous studies have reported ach burden to be an important predictor of cognitive and physical impairment in older populations . a higher ach burden is associated with greater risks of morbidity and mortality , a longer length of hospital stay , institutionalisation and functional and cognitive decline in older people . reducing the ach burden can cause significant improvements in short - term memory , delirium , behaviour , daily - living and quality of life . our recent systematic review identified two cohort studies that demonstrated an improvement of cognitive performance after the discontinuation of anticholinergic medicines . a retrospective study by nishtala et al . found that a significant interaction exists between age 65 years and the exposure to medicines with anticholinergic properties . in their study , hilmer et al . found a relationship between higher anticholinergic exposure and impaired physical function after adjusting for age , sex , race and comorbidities . the current model assumes that the antimuscarinic response contributed by each drug is linear and additive . however , there is a need to further explore the effect of anticholinergic events using nonlinear models which may reflect receptor binding more closely . studies involving population - level data examining the exposure to anticholinergic drugs and adverse events are limited . furthermore , clinical evidence implicating anticholinergic adverse events and the influence of associated patient characteristics in a nonlinear framework is lacking . to provide additional insights into the relationship between anticholinergic drugs and the influence of patient characteristics , we proposed a model - based approach to predict anticholinergic - type adverse events ( central and peripheral adverse events ) in this study . the main objective of this study was to explore the relationship between effects of important patient characteristics and the occurrence of anticholinergic - type events . this retrospective population - level study included hospitalised individuals in 2010 identified from the national health data collections maintained by the ministry of health of new zealand . pharmaceutical collections are used by the pharmaceutical management agency ( pharmac ) and the ministry of health of new zealand to administer payment to pharmacists for dispensing medicines that are publicly funded under the national medicines budget managed by pharmac . demographic characteristics such as sex , date of birth , ethnicity and district health board of domicile were also extracted from the pharmaceutical collections . patient information from the pharmaceutical collections was matched with hospital event data from the national minimum dataset using unique encrypted national health index numbers . we captured all medicines dispensed 14 days prior to the date of hospital admission to identify possible associations with outcomes of interest . diagnoses in the national minimum dataset are coded using the international classification of diseases and related health problems , 10th revision , australian modification ( icd-10-am ) . we identified icd-10 codes for delirium ( f05 ) , constipation ( k59 ) and urinary retention ( r32 , r39 ) from the coded diagnosis to capture anticholinergic events . overall , all hospitalised individuals aged 65 years living in new zealand who were receiving at least one prescription medicine during the period starting on january 1 , 2010 to december 31 , 2010 were included in this study . individuals with missing data on one or a combination of the following variables were excluded from the dataset : age , sex , medicine and medicine strength / dose . in this study , delirium , constipation and urinary retention were examined for anticholinergic adverse effects . delirium is the most common cause of acute cognitive dysfunction in hospitalised older people , and it is mediated by the antagonism of centrally acting muscarinic receptors . these adverse effects were chosen because they reflect typical adverse pharmacological effects linked to central or peripheral muscarinic receptors associated with drug properties . several studies have highlighted constipation and urinary retention as frequently occurring adverse anticholinergic effects in older people . a validated weighted comorbidity index [ charlson comorbidity index ( cci ) ] was used for the measurement of comorbidity burden . it provides a weighted score of a subject 's comorbidities which can be used to predict short- and long - term outcomes . compared to other comorbidity indices , the cci score can be assigned to the majority of patients and is readily available . studies have shown that the cci score predicts hospitalisation , adverse outcomes as well as mortality . the extracted data were rearranged into nonmem version 7.2 with the g95 fortran compiler - readable format using matlab version r2013b . approval to undertake this study was provided by the human ethics committee of the university of otago , new zealand ( approval no . numerous medicines possess anticholinergic activity , but many of them are unlikely to cause significant clinical symptoms when given as monotherapy . our study focused on the use of medicines with relevant central and peripheral anticholinergic effects . hence , the study considered medicines that have been identified as carrying a mild , moderate or severe risk of anticholinergic properties . anticholinergic exposure was defined based on a list of medicines with known anticholinergic activity from the expert - opinion rating scales . from this exposure , ach burden was defined in this study as the sum of all anticholinergic medicine(s ) taken by an individual . the dataset comprised various patient characteristics such as medicines with anticholinergic properties ( ach burden ) , age , sex , non - anticholinergic medicines ( non - acm ) , cci , ethnicity and number of hospital admissions during the study period . in this study , the total of all medicines is a continuous variable equivalent to the sum of all non - acms and the number of all anticholinergic medicine(s ) ( ach burden ) . we were interested in ach burden , and hence defined the other part of the total number of medicines as non - acm . the linear models were constructed based on a stepwise forward addition of patient characteristics from the structural model to define the maximum number of important characteristics . the nonlinear model building was performed by adding patient characteristics into three different locations to predict various effects . in all models , additional exploration was possible with the nonlinear model , which resembles a pharmacological binding model ( which consider drug(s ) binding to the receptor ) , and patient characteristics could be incorporated to test whether they ( 1 ) increased the risk of events independent of ach burden ( i.e. they pose a risk even in the absence of ach burden ) by adding patient characteristics to the baseline ( 0 ) , ( 2 ) increased the maximal anticholinergic effect of ach burden ( i.e. an overall greater effect is seen with ach burden ) by adding an interaction of patient characteristics on emax or ( 3 ) increased the apparent potency of the ach burden ( i.e. greater effects were seen for a given ach burden value ) by adding a patient characteristics on ach50 that provides 50% of the maximal effect . the following logistic models where used to describe the linear and nonlinear predictors : ( 1)loge{1}=0+1x1+ ... +nxn , where , ( 2)loge{1 } represents the log odds ( logit ) for reporting adverse anticholinergic effects , xn represents the presence or absence of the n - th variable , n represents the parameter estimate and 0 represents the intercept ( base probability ) . , emax is the maximal effect that can be achieved , and ach50 is the ach burden that provides 50% of the maximal effect . in this modelling framework , we can consider the influence of patient characteristics on 0 ( the intercept ) , emax ( the maximal effect ) and ach50 ( the apparent ach burden potency ) . all logit expressions were transformed using the expit function ( e/(1 + e ) ) for a standard logistic regression . model building for the linear and nonlinear frameworks was carried out from the base model ( a model that considered intercept only ) . the data were analysed using logistic regression ( in this case expanded to generalised nonlinear models ) for all three anticholinergic events ( delirium , constipation and urinary retention ) using the software nonmem ( version 7.2 ) . the nonmem ( nonlinear mixed effects modelling ) software is a regression program that can accommodate repeated measurement data , where each individual may contribute multiple observations . since some individuals contributed to more than one occasion ( number of hospital admissions ) , the subject - specific random effects were also considered . in this framework , a second - order conditional estimation method ( the laplacian option ) was used in nonmem . patient characteristics with a statistically significant effect were incorporated in the model , which significantly reduced the unexplained between - subject variability ( in circumstances where this was estimated ) , and the akaike information criterion was used in this study for model selection . continuous data from the patient characteristics ( such as age ) were normalised in the model by the mean population value to derive a stable conversion of data with less variance . the analysis continued with the stepwise forward addition of patient characteristics from the structural base model to define the maximum number of important patient characteristics . the structural model was then revised by considering interaction terms to provide the final model . the 95% confidence interval for the adjusted odds ratio ( aor ) values was estimated using a parametric bootstrap in which 10,000 parameter values were simulated in matlab under the posterior distribution from the nonmem parameter estimates . the confidence interval was then taken as the 2.5th and 97.5th percentiles of the simulations . this retrospective population - level study included hospitalised individuals in 2010 identified from the national health data collections maintained by the ministry of health of new zealand . pharmaceutical collections are used by the pharmaceutical management agency ( pharmac ) and the ministry of health of new zealand to administer payment to pharmacists for dispensing medicines that are publicly funded under the national medicines budget managed by pharmac . demographic characteristics such as sex , date of birth , ethnicity and district health board of domicile were also extracted from the pharmaceutical collections . patient information from the pharmaceutical collections was matched with hospital event data from the national minimum dataset using unique encrypted national health index numbers . we captured all medicines dispensed 14 days prior to the date of hospital admission to identify possible associations with outcomes of interest . diagnoses in the national minimum dataset are coded using the international classification of diseases and related health problems , 10th revision , australian modification ( icd-10-am ) . we identified icd-10 codes for delirium ( f05 ) , constipation ( k59 ) and urinary retention ( r32 , r39 ) from the coded diagnosis to capture anticholinergic events . overall , all hospitalised individuals aged 65 years living in new zealand who were receiving at least one prescription medicine during the period starting on january 1 , 2010 to december 31 , 2010 were included in this study . individuals with missing data on one or a combination of the following variables were excluded from the dataset : age , sex , medicine and medicine strength / dose . in this study , delirium , constipation and urinary retention were examined for anticholinergic adverse effects . delirium is the most common cause of acute cognitive dysfunction in hospitalised older people , and it is mediated by the antagonism of centrally acting muscarinic receptors . these adverse effects were chosen because they reflect typical adverse pharmacological effects linked to central or peripheral muscarinic receptors associated with drug properties . several studies have highlighted constipation and urinary retention as frequently occurring adverse anticholinergic effects in older people . a validated weighted comorbidity index [ charlson comorbidity index ( cci ) ] was used for the measurement of comorbidity burden . it provides a weighted score of a subject 's comorbidities which can be used to predict short- and long - term outcomes . compared to other comorbidity indices , the cci score can be assigned to the majority of patients and is readily available . studies have shown that the cci score predicts hospitalisation , adverse outcomes as well as mortality . the extracted data were rearranged into nonmem version 7.2 with the g95 fortran compiler - readable format using matlab version r2013b . approval to undertake this study was provided by the human ethics committee of the university of otago , new zealand ( approval no . numerous medicines possess anticholinergic activity , but many of them are unlikely to cause significant clinical symptoms when given as monotherapy . our study focused on the use of medicines with relevant central and peripheral anticholinergic effects . hence , the study considered medicines that have been identified as carrying a mild , moderate or severe risk of anticholinergic properties . anticholinergic exposure was defined based on a list of medicines with known anticholinergic activity from the expert - opinion rating scales . from this exposure , ach burden was defined in this study as the sum of all anticholinergic medicine(s ) taken by an individual . the dataset comprised various patient characteristics such as medicines with anticholinergic properties ( ach burden ) , age , sex , non - anticholinergic medicines ( non - acm ) , cci , ethnicity and number of hospital admissions during the study period . in this study , the total of all medicines is a continuous variable equivalent to the sum of all non - acms and the number of all anticholinergic medicine(s ) ( ach burden ) . we were interested in ach burden , and hence defined the other part of the total number of medicines as non - acm . the linear models were constructed based on a stepwise forward addition of patient characteristics from the structural model to define the maximum number of important characteristics . the nonlinear model building was performed by adding patient characteristics into three different locations to predict various effects . in all models , additional exploration was possible with the nonlinear model , which resembles a pharmacological binding model ( which consider drug(s ) binding to the receptor ) , and patient characteristics could be incorporated to test whether they ( 1 ) increased the risk of events independent of ach burden ( i.e. they pose a risk even in the absence of ach burden ) by adding patient characteristics to the baseline ( 0 ) , ( 2 ) increased the maximal anticholinergic effect of ach burden ( i.e. an overall greater effect is seen with ach burden ) by adding an interaction of patient characteristics on emax or ( 3 ) increased the apparent potency of the ach burden ( i.e. greater effects were seen for a given ach burden value ) by adding a patient characteristics on ach50 that provides 50% of the maximal effect . the following logistic models where used to describe the linear and nonlinear predictors : ( 1)loge{1}=0+1x1+ ... +nxn , where , ( 2)loge{1 } represents the log odds ( logit ) for reporting adverse anticholinergic effects , xn represents the presence or absence of the n - th variable , n represents the parameter estimate and 0 represents the intercept ( base probability ) . , emax is the maximal effect that can be achieved , and ach50 is the ach burden that provides 50% of the maximal effect . in this modelling framework , we can consider the influence of patient characteristics on 0 ( the intercept ) , emax ( the maximal effect ) and ach50 ( the apparent ach burden potency ) . all logit expressions were transformed using the expit function ( e/(1 + e ) ) for a standard logistic regression . model building for the linear and nonlinear frameworks was carried out from the base model ( a model that considered intercept only ) . ( 1)loge{1}=0+1x1+ ... +nxn , where , ( 2)loge{1 } represents the log odds ( logit ) for reporting adverse anticholinergic effects , xn represents the presence or absence of the n - th variable , n represents the parameter estimate and 0 represents the intercept ( base probability ) . , emax is the maximal effect that can be achieved , and ach50 is the ach burden that provides 50% of the maximal effect . in this modelling framework , we can consider the influence of patient characteristics on 0 ( the intercept ) , emax ( the maximal effect ) and ach50 ( the apparent ach burden potency ) . all logit expressions were transformed using the expit function ( e/(1 + e ) ) for a standard logistic regression . model building for the linear and nonlinear frameworks was carried out from the base model ( a model that considered intercept only ) . the data were analysed using logistic regression ( in this case expanded to generalised nonlinear models ) for all three anticholinergic events ( delirium , constipation and urinary retention ) using the software nonmem ( version 7.2 ) . the nonmem ( nonlinear mixed effects modelling ) software is a regression program that can accommodate repeated measurement data , where each individual may contribute multiple observations . since some individuals contributed to more than one occasion ( number of hospital admissions ) , the subject - specific random effects were also considered . in this framework , a second - order conditional estimation method ( the laplacian option ) was used in nonmem . patient characteristics with a statistically significant effect were incorporated in the model , which significantly reduced the unexplained between - subject variability ( in circumstances where this was estimated ) , and the akaike information criterion was used in this study for model selection . continuous data from the patient characteristics ( such as age ) were normalised in the model by the mean population value to derive a stable conversion of data with less variance . the analysis continued with the stepwise forward addition of patient characteristics from the structural base model to define the maximum number of important patient characteristics . the structural model was then revised by considering interaction terms to provide the final model . the 95% confidence interval for the adjusted odds ratio ( aor ) values was estimated using a parametric bootstrap in which 10,000 parameter values were simulated in matlab under the posterior distribution from the nonmem parameter estimates . the confidence interval was then taken as the 2.5th and 97.5th percentiles of the simulations . the incidence rates of anticholinergic - type adverse events in the dataset for delirium , constipation and urinary retention were 2.1 , 3.6 and 0.9% , respectively . the mean age ( sd ) of the study population was 79 8 years . the overall incidence rate of all events in the dataset was 6.6% , and the baseline estimate from logistic regression estimated an average incidence rate of the three events of 2.2% . however , considering the akaike information criterion , the complexity of the multivariate model , the biological plausibility and the pharmacological mechanism , the nonlinear model was preferred over the linear model . a detailed summary of both the linear and nonlinear model buildings is depicted in online supplementary table s1 ( see www.karger.com/doi/10.1159/000441718 . table 2 summarises the frequencies of the most commonly prescribed medicines with anticholinergic activity identified from the study population . the anticholinergic activity of medicines is rated based on a published reference scale inferring high ( h ) , moderate ( m ) and low ( l ) activity . the most frequently used anticholinergic medicines are those for the treatment of cardiovascular and neurological disorders ( table 2 ) . the majority of the anticholinergic medicines identified had l anticholinergic activity ( 80% ) , including furosemide , metoprolol , citalopram , metformin , warfarin , digoxin , isosorbide and diltiazem . m to h activity anticholinergic medicines included amitriptyline , quetiapine , nortriptyline , prochlorperazine , haloperidol and paroxetine . l , m and h activity anticholinergic medicines were used for calculating the burden scores shown in table 2 . several anticholinergic activity medicines available in the literature were not applicable to the study population because either they are not available in new zealand or they are funded by pharmac the final model was a nonlinear model for predicting the probability of anticholinergic - type events . those patient characteristics that were found to be influential were ach burden , non - acm , age and cci ( table 3 ) . the main patient characteristics was ach burden ( aor of an anticholinergic event was 3.21 , 95% ci : 1.23 - 5.81 , for those taking an average of 5 anticholinergic medicines compared to those taking 1 ; p < 0.05 ) . other patient characteristics that were found to be significant included : non - acm ( aor 1.41 , 95% ci : 1.31 - 1.51 ) and age ( aor 1.08 , 95% ci : 1.05 - 1.10 ) . the cci score was negatively associated with anticholinergic events ( aor 0.84 , 95% ci : 0.78 - 0.95 ) . the influence of ach burden was nonlinear within the logit domain , and the estimated maximum probability ( emax ) of an adverse anticholinergic event was 0.33 , i.e. with an infinite number of ach burden medicines . the patient characteristics non - acm , age and cci were found to influence the baseline risk of an anticholinergic event . in addition , cci and non - acm were found to influence the maximum risk of an anticholinergic event associated with ach burden ( i.e. they affected emax ) . none of the patient characteristics appeared to modify the effect of risk posed by ach burden drugs . this study examined and explored the relationship between patient characteristics associated with anticholinergic drugs and anticholinergic - type adverse events in older people . the evidence from the current study clearly supports the existing literature that higher ach burden , polypharmacy , age and multimorbidity are independent risk factors for adverse anticholinergic events in older people . findings from a recent cross - sectional study conducted in finland involving 621 older individuals showed a strong association between ach burden and anticholinergic events . in a cohort study conducted by rudolph et al . , higher anticholinergic risk scores were associated with an increased risk of both peripheral and central anticholinergic adverse effects in older people . the study compared two cohorts , one retrospective ( 132 patients ) and one prospective ( 117 patients ) ; in both cohorts , the risk of anticholinergic adverse events increased significantly with higher anticholinergic risk scores . compared to the retrospective cohort , the prospective primary - care cohort reported dry mouth and constipation more frequently with an evidence of a dose - response relationship . a prospective study involving 532 older veterans reported that the prevalence of anticholinergic - type adverse events , such as dry mouth and constipation , was significantly higher in individuals using anticholinergic drugs . the study highlighted that anticholinergic - type adverse events are rare events ( 0.8% ) , which mirrors the incidence rate of anticholinergic - type events in our study population ( 6.6% ) . a study conducted in the usa involving 833 older home - care patients found that polypharmacy , a common occurrence in nursing - home residents , increases the risk of adverse drug events and hospitalisation . similarly , in our study , non - acms were associated with anticholinergic - type events both as an interaction with cci and as an independent predictor . in this study , we anticipated a positive association between comorbidities and anticholinergic events , adjusted for the total medicine burden ; and the finding of a negative association is counterintuitive . we speculate that the burden of disease may take precedence over reporting of anticholinergic events . a new - user design excluding prevalent users ( first - time users ) may provide a clearer picture of the relationships between anticholinergic exposure and anticholinergic events , and this could be an area for future research . the implications of this research could help to identify important patient characteristics that influence ach burden . a reduction in ach burden may result in improved short - term memory , confusion , behaviour and delirium together with an enhanced quality of life and daily functioning of older people . in the context of quantification of ach burden , there is no globally accepted tool to quantify ach burden for commonly prescribed medicines with known anticholinergic properties . the model - based approaches presented in this study illustrate a potential direction for a nonlinear binding model to estimate the relative ach burden of medicines . one of the major strengths of this study was the use of population - level data and allowing the characteristics to be associated with the outcome of interest in a nonlinear model framework . the patient characteristics were derived from national health collections in real settings ; hence , selection bias was negligible and makes our findings relevant to clinical practice . the findings reported in this study are generalisable as it represents the population of older people in new zealand captured by the nationwide prescription database ( pharms ) . however , our findings need replication in other samples of diverse clinical settings for longer periods . the most significant concern is the potential of confounding by indication , something which is inherent to all pharmacoepidemiological studies . the study did not account for all anticholinergic events contained in the national minimum dataset . accurate documentation of anticholinergic - type adverse events relies on rigid clinical coding guidelines . among the 2,248 individuals analysed in this study , only 2.1% had a coded diagnosis of delirium , which is considerably less compared to other database studies . this may have underestimated the risk posed by ach burden . since causality was not assessed , we were unsure whether any of the events were caused by anticholinergics the possibility of protopathic bias could not be entirely ruled out , as many older individuals receive anticholinergic medicines for the management of urinary symptoms . however , the choice of 3 diverse events , delirium , constipation and urinary retention , that arise commonly from anticholinergic activity medicines but less frequently from a single pathology was an attempt to decrease the risk of confounding factors associated with adverse events . this may cause an overestimate of the true association ( positive confounding ) or an underestimate of the association ( negative confounding ) . to our knowledge , this is the first study that examined population - level data in a nonlinear model framework to predict anticholinergic - type adverse events . this study evaluated the relationship between important patient characteristics and the occurrence of anticholinergic - type events . ach burden was found to be an independent risk factor for predicting the probability of the anticholinergic - type events in older people . this study lends support to findings obtained from observational and experimental studies and reinforces the clinical significance of reviewing anticholinergic medicines in older people .

aimsto examine patient characteristics that predict adverse anticholinergic - type events in older people.methodsthis retrospective population - level study included 2,248 hospitalised patients . individual data on medicines that are commonly associated with anticholinergic events ( delirium , constipation and urinary retention ) were identified . patient characteristics examined were medicines with anticholinergic effects ( ach burden ) , age , sex , non - anticholinergic medicines ( non - acm ) , charlson comorbidity index scores and ethnicity . the akaike information criterion was used for model selection . the data were analysed using logistic regression models for anticholinergic events using the software nonmem.resultsach burden was found to be a significant independent predictor for developing an anticholinergic event [ adjusted odds ratio ( aor ) : 3.21 , 95% ci : 1.23 - 5.81 ] for those taking an average of 5 anticholinergic medicines compared to those taking 1 . both non - acm and age were also independent risk factors ( aor : 1.41 , 95% ci : 1.31 - 1.51 and aor : 1.08 , 95% ci : 1.05 - 1.10 , respectively).conclusionto our knowledge , this is the first study that has examined population - level data in a nonlinear model framework to predict anticholinergic - type adverse events . this study evaluated the relationship between important patient characteristics and the occurrence of anticholinergic - type events . these findings reinforce the clinical significance of reviewing anticholinergic medicines in older people .

Introduction Methods Data Source and Study Population Exposure to Anticholinergic Medicines Patient Characteristics and Modelling Linear Framework Nonlinear Framework Statistical Analysis Results Discussion Conclusion Disclosure Statement Supplementary Material

anticholinergic medicines bind to the muscarinic receptors , and the decreased cholinergic neurotransmission impacts both peripheral and central body systems . the risk of anticholinergic adverse events has been found to increase with age , pre - existing cognitive impairment , polypharmacy , comorbidities and cholinergic neuronal degeneration , and it is influenced by individual pharmacokinetic and pharmacodynamic variability . recent literature has highlighted that up to 40% of all institutionalised older people take medicines with known anticholinergic activity . similarly , it has been reported that among hospitalised older people , the prevalence of those taking anticholinergic medicines increased significantly ( 7.2 3.5 vs. 5.7 3.1 ; p < 0.001 ) during their hospital stay . the cumulative exposure to multiple medicines with anticholinergic properties numerous studies have reported ach burden to be an important predictor of cognitive and physical impairment in older populations . a higher ach burden is associated with greater risks of morbidity and mortality , a longer length of hospital stay , institutionalisation and functional and cognitive decline in older people . reducing the ach burden can cause significant improvements in short - term memory , delirium , behaviour , daily - living and quality of life . our recent systematic review identified two cohort studies that demonstrated an improvement of cognitive performance after the discontinuation of anticholinergic medicines . found that a significant interaction exists between age 65 years and the exposure to medicines with anticholinergic properties . found a relationship between higher anticholinergic exposure and impaired physical function after adjusting for age , sex , race and comorbidities . however , there is a need to further explore the effect of anticholinergic events using nonlinear models which may reflect receptor binding more closely . studies involving population - level data examining the exposure to anticholinergic drugs and adverse events are limited . furthermore , clinical evidence implicating anticholinergic adverse events and the influence of associated patient characteristics in a nonlinear framework is lacking . to provide additional insights into the relationship between anticholinergic drugs and the influence of patient characteristics , we proposed a model - based approach to predict anticholinergic - type adverse events ( central and peripheral adverse events ) in this study . the main objective of this study was to explore the relationship between effects of important patient characteristics and the occurrence of anticholinergic - type events . this retrospective population - level study included hospitalised individuals in 2010 identified from the national health data collections maintained by the ministry of health of new zealand . pharmaceutical collections are used by the pharmaceutical management agency ( pharmac ) and the ministry of health of new zealand to administer payment to pharmacists for dispensing medicines that are publicly funded under the national medicines budget managed by pharmac . demographic characteristics such as sex , date of birth , ethnicity and district health board of domicile were also extracted from the pharmaceutical collections . we identified icd-10 codes for delirium ( f05 ) , constipation ( k59 ) and urinary retention ( r32 , r39 ) from the coded diagnosis to capture anticholinergic events . individuals with missing data on one or a combination of the following variables were excluded from the dataset : age , sex , medicine and medicine strength / dose . in this study , delirium , constipation and urinary retention were examined for anticholinergic adverse effects . delirium is the most common cause of acute cognitive dysfunction in hospitalised older people , and it is mediated by the antagonism of centrally acting muscarinic receptors . several studies have highlighted constipation and urinary retention as frequently occurring adverse anticholinergic effects in older people . a validated weighted comorbidity index [ charlson comorbidity index ( cci ) ] was used for the measurement of comorbidity burden . our study focused on the use of medicines with relevant central and peripheral anticholinergic effects . hence , the study considered medicines that have been identified as carrying a mild , moderate or severe risk of anticholinergic properties . from this exposure , ach burden was defined in this study as the sum of all anticholinergic medicine(s ) taken by an individual . the dataset comprised various patient characteristics such as medicines with anticholinergic properties ( ach burden ) , age , sex , non - anticholinergic medicines ( non - acm ) , cci , ethnicity and number of hospital admissions during the study period . in this study , the total of all medicines is a continuous variable equivalent to the sum of all non - acms and the number of all anticholinergic medicine(s ) ( ach burden ) . we were interested in ach burden , and hence defined the other part of the total number of medicines as non - acm . the nonlinear model building was performed by adding patient characteristics into three different locations to predict various effects . in all models , additional exploration was possible with the nonlinear model , which resembles a pharmacological binding model ( which consider drug(s ) binding to the receptor ) , and patient characteristics could be incorporated to test whether they ( 1 ) increased the risk of events independent of ach burden ( i.e. they pose a risk even in the absence of ach burden ) by adding patient characteristics to the baseline ( 0 ) , ( 2 ) increased the maximal anticholinergic effect of ach burden ( i.e. an overall greater effect is seen with ach burden ) by adding an interaction of patient characteristics on emax or ( 3 ) increased the apparent potency of the ach burden ( i.e. greater effects were seen for a given ach burden value ) by adding a patient characteristics on ach50 that provides 50% of the maximal effect . the following logistic models where used to describe the linear and nonlinear predictors : ( 1)loge{1}=0+1x1+ ... +nxn , where , ( 2)loge{1 } represents the log odds ( logit ) for reporting adverse anticholinergic effects , xn represents the presence or absence of the n - th variable , n represents the parameter estimate and 0 represents the intercept ( base probability ) . , emax is the maximal effect that can be achieved , and ach50 is the ach burden that provides 50% of the maximal effect . in this modelling framework , we can consider the influence of patient characteristics on 0 ( the intercept ) , emax ( the maximal effect ) and ach50 ( the apparent ach burden potency ) . all logit expressions were transformed using the expit function ( e/(1 + e ) ) for a standard logistic regression . the data were analysed using logistic regression ( in this case expanded to generalised nonlinear models ) for all three anticholinergic events ( delirium , constipation and urinary retention ) using the software nonmem ( version 7.2 ) . since some individuals contributed to more than one occasion ( number of hospital admissions ) , the subject - specific random effects were also considered . patient characteristics with a statistically significant effect were incorporated in the model , which significantly reduced the unexplained between - subject variability ( in circumstances where this was estimated ) , and the akaike information criterion was used in this study for model selection . continuous data from the patient characteristics ( such as age ) were normalised in the model by the mean population value to derive a stable conversion of data with less variance . the analysis continued with the stepwise forward addition of patient characteristics from the structural base model to define the maximum number of important patient characteristics . the 95% confidence interval for the adjusted odds ratio ( aor ) values was estimated using a parametric bootstrap in which 10,000 parameter values were simulated in matlab under the posterior distribution from the nonmem parameter estimates . this retrospective population - level study included hospitalised individuals in 2010 identified from the national health data collections maintained by the ministry of health of new zealand . pharmaceutical collections are used by the pharmaceutical management agency ( pharmac ) and the ministry of health of new zealand to administer payment to pharmacists for dispensing medicines that are publicly funded under the national medicines budget managed by pharmac . demographic characteristics such as sex , date of birth , ethnicity and district health board of domicile were also extracted from the pharmaceutical collections . we identified icd-10 codes for delirium ( f05 ) , constipation ( k59 ) and urinary retention ( r32 , r39 ) from the coded diagnosis to capture anticholinergic events . individuals with missing data on one or a combination of the following variables were excluded from the dataset : age , sex , medicine and medicine strength / dose . in this study , delirium , constipation and urinary retention were examined for anticholinergic adverse effects . delirium is the most common cause of acute cognitive dysfunction in hospitalised older people , and it is mediated by the antagonism of centrally acting muscarinic receptors . several studies have highlighted constipation and urinary retention as frequently occurring adverse anticholinergic effects in older people . a validated weighted comorbidity index [ charlson comorbidity index ( cci ) ] was used for the measurement of comorbidity burden . our study focused on the use of medicines with relevant central and peripheral anticholinergic effects . from this exposure , ach burden was defined in this study as the sum of all anticholinergic medicine(s ) taken by an individual . the dataset comprised various patient characteristics such as medicines with anticholinergic properties ( ach burden ) , age , sex , non - anticholinergic medicines ( non - acm ) , cci , ethnicity and number of hospital admissions during the study period . in this study , the total of all medicines is a continuous variable equivalent to the sum of all non - acms and the number of all anticholinergic medicine(s ) ( ach burden ) . we were interested in ach burden , and hence defined the other part of the total number of medicines as non - acm . the nonlinear model building was performed by adding patient characteristics into three different locations to predict various effects . in all models , additional exploration was possible with the nonlinear model , which resembles a pharmacological binding model ( which consider drug(s ) binding to the receptor ) , and patient characteristics could be incorporated to test whether they ( 1 ) increased the risk of events independent of ach burden ( i.e. they pose a risk even in the absence of ach burden ) by adding patient characteristics to the baseline ( 0 ) , ( 2 ) increased the maximal anticholinergic effect of ach burden ( i.e. an overall greater effect is seen with ach burden ) by adding an interaction of patient characteristics on emax or ( 3 ) increased the apparent potency of the ach burden ( i.e. greater effects were seen for a given ach burden value ) by adding a patient characteristics on ach50 that provides 50% of the maximal effect . the following logistic models where used to describe the linear and nonlinear predictors : ( 1)loge{1}=0+1x1+ ... +nxn , where , ( 2)loge{1 } represents the log odds ( logit ) for reporting adverse anticholinergic effects , xn represents the presence or absence of the n - th variable , n represents the parameter estimate and 0 represents the intercept ( base probability ) . , emax is the maximal effect that can be achieved , and ach50 is the ach burden that provides 50% of the maximal effect . in this modelling framework , we can consider the influence of patient characteristics on 0 ( the intercept ) , emax ( the maximal effect ) and ach50 ( the apparent ach burden potency ) . ( 1)loge{1}=0+1x1+ ... +nxn , where , ( 2)loge{1 } represents the log odds ( logit ) for reporting adverse anticholinergic effects , xn represents the presence or absence of the n - th variable , n represents the parameter estimate and 0 represents the intercept ( base probability ) . , emax is the maximal effect that can be achieved , and ach50 is the ach burden that provides 50% of the maximal effect . in this modelling framework , we can consider the influence of patient characteristics on 0 ( the intercept ) , emax ( the maximal effect ) and ach50 ( the apparent ach burden potency ) . the data were analysed using logistic regression ( in this case expanded to generalised nonlinear models ) for all three anticholinergic events ( delirium , constipation and urinary retention ) using the software nonmem ( version 7.2 ) . since some individuals contributed to more than one occasion ( number of hospital admissions ) , the subject - specific random effects were also considered . patient characteristics with a statistically significant effect were incorporated in the model , which significantly reduced the unexplained between - subject variability ( in circumstances where this was estimated ) , and the akaike information criterion was used in this study for model selection . the analysis continued with the stepwise forward addition of patient characteristics from the structural base model to define the maximum number of important patient characteristics . the 95% confidence interval for the adjusted odds ratio ( aor ) values was estimated using a parametric bootstrap in which 10,000 parameter values were simulated in matlab under the posterior distribution from the nonmem parameter estimates . the incidence rates of anticholinergic - type adverse events in the dataset for delirium , constipation and urinary retention were 2.1 , 3.6 and 0.9% , respectively . the overall incidence rate of all events in the dataset was 6.6% , and the baseline estimate from logistic regression estimated an average incidence rate of the three events of 2.2% . however , considering the akaike information criterion , the complexity of the multivariate model , the biological plausibility and the pharmacological mechanism , the nonlinear model was preferred over the linear model . table 2 summarises the frequencies of the most commonly prescribed medicines with anticholinergic activity identified from the study population . the majority of the anticholinergic medicines identified had l anticholinergic activity ( 80% ) , including furosemide , metoprolol , citalopram , metformin , warfarin , digoxin , isosorbide and diltiazem . l , m and h activity anticholinergic medicines were used for calculating the burden scores shown in table 2 . several anticholinergic activity medicines available in the literature were not applicable to the study population because either they are not available in new zealand or they are funded by pharmac the final model was a nonlinear model for predicting the probability of anticholinergic - type events . those patient characteristics that were found to be influential were ach burden , non - acm , age and cci ( table 3 ) . the main patient characteristics was ach burden ( aor of an anticholinergic event was 3.21 , 95% ci : 1.23 - 5.81 , for those taking an average of 5 anticholinergic medicines compared to those taking 1 ; p < 0.05 ) . other patient characteristics that were found to be significant included : non - acm ( aor 1.41 , 95% ci : 1.31 - 1.51 ) and age ( aor 1.08 , 95% ci : 1.05 - 1.10 ) . the cci score was negatively associated with anticholinergic events ( aor 0.84 , 95% ci : 0.78 - 0.95 ) . the influence of ach burden was nonlinear within the logit domain , and the estimated maximum probability ( emax ) of an adverse anticholinergic event was 0.33 , i.e. the patient characteristics non - acm , age and cci were found to influence the baseline risk of an anticholinergic event . in addition , cci and non - acm were found to influence the maximum risk of an anticholinergic event associated with ach burden ( i.e. none of the patient characteristics appeared to modify the effect of risk posed by ach burden drugs . this study examined and explored the relationship between patient characteristics associated with anticholinergic drugs and anticholinergic - type adverse events in older people . the evidence from the current study clearly supports the existing literature that higher ach burden , polypharmacy , age and multimorbidity are independent risk factors for adverse anticholinergic events in older people . findings from a recent cross - sectional study conducted in finland involving 621 older individuals showed a strong association between ach burden and anticholinergic events . , higher anticholinergic risk scores were associated with an increased risk of both peripheral and central anticholinergic adverse effects in older people . the study compared two cohorts , one retrospective ( 132 patients ) and one prospective ( 117 patients ) ; in both cohorts , the risk of anticholinergic adverse events increased significantly with higher anticholinergic risk scores . a prospective study involving 532 older veterans reported that the prevalence of anticholinergic - type adverse events , such as dry mouth and constipation , was significantly higher in individuals using anticholinergic drugs . the study highlighted that anticholinergic - type adverse events are rare events ( 0.8% ) , which mirrors the incidence rate of anticholinergic - type events in our study population ( 6.6% ) . similarly , in our study , non - acms were associated with anticholinergic - type events both as an interaction with cci and as an independent predictor . in this study , we anticipated a positive association between comorbidities and anticholinergic events , adjusted for the total medicine burden ; and the finding of a negative association is counterintuitive . we speculate that the burden of disease may take precedence over reporting of anticholinergic events . the implications of this research could help to identify important patient characteristics that influence ach burden . in the context of quantification of ach burden , there is no globally accepted tool to quantify ach burden for commonly prescribed medicines with known anticholinergic properties . the model - based approaches presented in this study illustrate a potential direction for a nonlinear binding model to estimate the relative ach burden of medicines . one of the major strengths of this study was the use of population - level data and allowing the characteristics to be associated with the outcome of interest in a nonlinear model framework . the findings reported in this study are generalisable as it represents the population of older people in new zealand captured by the nationwide prescription database ( pharms ) . accurate documentation of anticholinergic - type adverse events relies on rigid clinical coding guidelines . among the 2,248 individuals analysed in this study , only 2.1% had a coded diagnosis of delirium , which is considerably less compared to other database studies . however , the choice of 3 diverse events , delirium , constipation and urinary retention , that arise commonly from anticholinergic activity medicines but less frequently from a single pathology was an attempt to decrease the risk of confounding factors associated with adverse events . to our knowledge , this is the first study that examined population - level data in a nonlinear model framework to predict anticholinergic - type adverse events . this study evaluated the relationship between important patient characteristics and the occurrence of anticholinergic - type events . ach burden was found to be an independent risk factor for predicting the probability of the anticholinergic - type events in older people . this study lends support to findings obtained from observational and experimental studies and reinforces the clinical significance of reviewing anticholinergic medicines in older people .

inquiry - based approaches should be transforming the way science faculty members teach and undergraduate students learn ( national research council [ nrc ] , 2003a ) . since 1985 , the national science foundation ( nsf ) has promoted the enhancement of undergraduate science instruction , especially in laboratory courses ( tuss et al . , 1998 ) . a push to increase active teaching and learning within undergraduate biology courses occurred in 2003 , with the publication of bio2010 ( nrc , 2003a ) . in the same year , the nrc called for an increase in the scientific assessment of teaching to improve student learning ( nrc , 2003b ) . a year later , handelsman and colleagues seminal paper in science echoed this push for more active learning in science courses , providing examples of scientific teaching in both lecture and laboratory classrooms ( handelsman et al . , 2004 ) . more recently , the vision and change report has once again emphasized the need to incorporate inquiry throughout the undergraduate biology curriculum and to use scientific approaches to assess faculty teaching and student learning ( american association for the advancement of science [ aaas ] , 2011 ) . however , a recent review of curricular innovations in undergraduate science courses ( both lecture and laboratory ) suggests that teaching practices in undergraduate science and engineering courses are changing in the direction of inquiry but , unfortunately , are still not necessarily being assessed rigorously ( ruiz - primo et al . , 2011 ) . the infusion of inquiry - based approaches into the undergraduate biology curriculum is especially important in laboratory courses or within the laboratory component of a course , because students are applying the process of science in these courses ( aaas , 2011 ) and are learning to write in an inquiry - based way ( tuss et al . , 1998 ) . surveys that took place more than 20 yr ago suggest that inquiry - based and open - ended laboratory exercises were used infrequently ( 10% of courses ; sundberg and armstrong , 1993 ) . a more recent survey reporting results from 65 colleges and universities in the united states found close to 80% of institutions are using inquiry - based approaches in their laboratory courses ( sundberg et al . , 2005 ) , indicating a very promising trend . yet ( 2005 ) study did not determine the degree to which these approaches had been implemented or the effectiveness of these inquiry - based teaching methods on student performance . more than 7 yr on , information about inquiry - based approaches is still lacking , with the recent national research council report on discipline - based education research rating the evidence in support of inquiry - based learning in laboratory courses in biology as limited ( nrc , 2012 ) . given the repeated calls for increased inquiry - based teaching and learning in the undergraduate biology curriculum and the important role of laboratory courses in the curriculum , we conducted a meta - analysis of peer - reviewed , published , undergraduate inquiry - based laboratory exercises produced since the publication of the bio2010 report ( nrc , 2003a ) and handelsman et al . 's ( 2004 ) seminal paper on scientific teaching . specifically , we addressed two main questions : to what degree have inquiry - based laboratory exercises been developed and published in different subdisciplines in biology , at what level ( i.e. , nonmajors , introductory , upper - level majors ) , and what type of inquiry ( i.e. , guided inquiry , open - ended inquiry , and research)?to what degree have these exercises been assessed and are they in line with the recommendations of the nrc ( 2003b ) and handelsman et al . have inquiry - based laboratory exercises been developed and published in different subdisciplines in biology , at what level ( i.e. , nonmajors , introductory , upper - level majors ) , and what type of inquiry ( i.e. , guided inquiry , open - ended inquiry , and research ) ? to what degree have these exercises been assessed and are they in line with the recommendations of the nrc ( 2003b ) and handelsman et al . the first question is important , as it tells us about where future efforts need to be made in curriculum development and publication / dissemination . the second question is important , as it tells us whether laboratory exercises are being assessed , how they are being assessed , and what the assessment tells us about the effectiveness of the exercises . the data suggest where future efforts need to be made in assessment and provide a snapshot of the evidence for the value of inquiry - based laboratory exercises in undergraduate biology courses . we searched on inquir * lab * ( where * is a wildcard ) in the topic field of web of science and then limited the results to the years from 2005 to 2012 and to the subject areas of education , educational research , and all areas of biology . the same search was carried out in the education resources information center ( eric ; www.eric.ed.gov ) using proquest , which returned some of the same references as those from the web of science search . as papers on research in laboratory courses inquiry , we also searched on research lab * in web of science and eric and limited the results as described above . finally , we searched research lab * in the tables of contents of journals that were found in our previous searches . we combined the results of all searches and then manually excluded all studies that were not explicitly about an inquiry - based laboratory exercise in biology at the undergraduate level . after the exclusion process , 142 studies remained , which were scored for the current study ( see the supplemental material ) . admittedly , our search did not retrieve all publications of inquiry - based laboratory exercises in biology , as these exercises might not be published in journals catalogued by web of science or eric . however , those studies that include assessment data are perhaps more likely to be published in catalogued journals . because we were interested in changes in the publication of inquiry - based laboratory exercises and the assessment of those exercises following the publication of the bio2010 report ( nrc , 2003a ) , the nrc 's report on assessment of teaching and learning ( nrc , 2003b ) , and handelsman and colleagues paper on scientific teaching ( handelsman et al . , 2004 ) , we only considered published papers from the united states , as these reports likely had the greatest effect in their country of origin . in setting the time windows for our analysis , we allowed for 2 yr after the reports and 1 yr after handelsman et al . 's paper to allow the community to incorporate the recommendations into their work and for new studies to be published . as a result , we considered studies from 2005 to 2012 as those that might reflect the impact of these publications . we coded each paper in the following categories : area of biology : biochemistry , cell biology , developmental biology , genetics , and molecular biology ( bcdgmb ) ; ecology and evolutionary biology ( eeb ) ; plant and animal anatomy and physiology ( organismal biology).inquiry type : guided inquiry , open - ended inquiry , research ( including teacher - collaborative and structured research experiences ) , and other ( e.g. , case studies , problem - based learning , model building ) . faculty members provide students with the research question of interest and guide students to an appropriate experimental design . open - ended inquiry is less structured , with students posing the research question and developing the appropriate methods to address the question . for studies classified as research , the degree of student independence in defining the question and methodology varied . unlike guided inquiry , the outcomes of the experiments are not known to either the students or faculty . unlike open - ended inquiry , in which the emphasis is placed on students being in control of the process , students and faculty work collaboratively in research experiences.course level : nonmajors , introductory majors , upper - level majors , and mixed nonmajors and majors.assessment type : qualitative ( i.e. , student comments , interviews ) , student self - assessment ( self - efficacy and student assessment of learning gains ) , disciplinary content , and other ( e.g. , scientific reasoning , experimental design , information literacy , statistical literacy).whether the assessment was based on a published instrument.whether the paper presented data for a control group.the number of students studied.the results of the study . area of biology : biochemistry , cell biology , developmental biology , genetics , and molecular biology ( bcdgmb ) ; ecology and evolutionary biology ( eeb ) ; plant and animal anatomy and physiology ( organismal biology ) . inquiry type : guided inquiry , open - ended inquiry , research ( including teacher - collaborative and structured research experiences ) , and other ( e.g. , case studies , problem - based learning , model building ) . faculty members provide students with the research question of interest and guide students to an appropriate experimental design . open - ended inquiry is less structured , with students posing the research question and developing the appropriate methods to address the question . for studies classified as research , the degree of student independence in defining the question and methodology varied . unlike guided inquiry , the outcomes of the experiments are not known to either the students or faculty . unlike open - ended inquiry , in which the emphasis is placed on students being in control of the process , students and faculty work collaboratively in research experiences course level : nonmajors , introductory majors , upper - level majors , and mixed nonmajors and majors . assessment type : qualitative ( i.e. , student comments , interviews ) , student self - assessment ( self - efficacy and student assessment of learning gains ) , disciplinary content , and other ( e.g. , scientific reasoning , experimental design , information literacy , statistical literacy ) . whether the assessment was based on a published instrument . whether the paper presented data for a control group . the results of the study . for studies in which the laboratory experiments were in more than one area of biology , the study was included for each area of biology covered . studies that reported multiple types of assessment data were included once for each type of assessment for appropriate analyses . as a result of these multiple codings , the sample sizes for subdiscipline in biology and assessment are greater than the total number of studies in the data set . in most cases , did not provide the required information , we coded them based on the information provided or coded them as not provided . as a result , the sample sizes vary among analyses . all of our data are frequency data ( i.e. , counts of the number of studies that fall into particular categories ) . as a result , we used chi - square tests of independence ( cross - tabulation ) for all analyses . the results of chi - square tests can be biased if the expected count in a particular cell is low . randomization tests have been proposed as an alternative approach for estimating p values in these cases ( sokal and rohlf , 2011 ) . therefore , we used randomization tests based on 10,000 iterations and = 0.05 to estimate p values for the chi - square tests . for analyses that included course level , studies on a mixed majors courses ( six studies ) and studies for which course level was not reported ( eight studies ) were excluded . for analyses that included assessment type or inquiry type , we excluded studies that were coded as other because the number of studies was small , and the assessment types or inquiry types included in the other categories were quite variable . calculation of the effect size ( i.e. , the relative impact ) was possible for the controlled studies . for these studies , we estimated the effect size by standardizing the difference in means between control and treatment groups by dividing the sd of the control group ( see the supplemental materials in ruiz - primo et al . for a discussion of estimation of effect size ) . because of the limited number of studies for which we were able to calculate effect size ( n = 10 ) , we were unable to do any further statistical analysis on whether the effects of inquiry - based learning were greatest at particular course levels , from using particular types of inquiry , or for particular types of assessment . all of our data are frequency data ( i.e. , counts of the number of studies that fall into particular categories ) . as a result , we used chi - square tests of independence ( cross - tabulation ) for all analyses . the results of chi - square tests can be biased if the expected count in a particular cell is low . randomization tests have been proposed as an alternative approach for estimating p values in these cases ( sokal and rohlf , 2011 ) . therefore , we used randomization tests based on 10,000 iterations and = 0.05 to estimate p values for the chi - square tests . for analyses that included course level , studies on a mixed majors courses ( six studies ) and studies for which course level was not reported ( eight studies ) were excluded . for analyses that included assessment type or inquiry type , we excluded studies that were coded as other because the number of studies was small , and the assessment types or inquiry types included in the other categories were quite variable . calculation of the effect size ( i.e. , the relative impact ) was possible for the controlled studies . for these studies , we estimated the effect size by standardizing the difference in means between control and treatment groups by dividing the sd of the control group ( see the supplemental materials in ruiz - primo et al . for a discussion of estimation of effect size ) . because of the limited number of studies for which we were able to calculate effect size ( n = 10 ) , we were unable to do any further statistical analysis on whether the effects of inquiry - based learning were greatest at particular course levels , from using particular types of inquiry , or for particular types of assessment . we found 142 papers that described inquiry - based laboratory exercises that had been implemented in undergraduate biology courses between 2005 and 2012 ( see the supplemental material ) . on average , more than half of the exercises ( 58% ) were in the areas of biochemistry , cell biology , developmental biology , genetics , and molecular biology ( bcdgmb ; figure 1a ) , and the majority were used in courses for upper - level majors ( figure 1b ) . overall , most were guided - inquiry exercises rather than open - ended exercises or research experiences imbedded in a course ( figure 1c ) . the approach to inquiry did not vary significantly based on course level ( table 1 and figure 2a ) or discipline ( table 1 and figure 2b ) . however , bcdgmb exercises were used more often in upper - level courses and ecology and evolutionary biology ( eeb ) experiments were used proportionally more often in nonmajors courses ( table 1 and figure 2c ) . number of published inquiry - based laboratory studies in biology based on ( a ) subdiscipline in biology : bcdgmb , biochemistry , cell biology , developmental biology , genetics , and molecular biology ; eeb , ecology and evolutionary biology ; organismal , organismal biology , including plant and animal anatomy and physiology ( n = 173 ) ; ( b ) course level ( n = 135 ) ; and ( c ) inquiry type ( n = 143 ) . number of studies based on ( a ) inquiry type and level at which experiment was taught ( n = 144 ) ; ( b ) subdiscipline in biology and inquiry type ( n = 185 ) ; and ( c ) subdiscipline in biology and level at which experiment was taught ( n = 159 ) . summary of results of cross - tabulated tests of independence rows marked with * represent significant interactions between the two factors on the number of studies in our data set . for the studies included in our sample , 73% presented assessment data ( see the supplemental material ) . of those studies that presented assessment data , almost two - thirds included more than one type of assessment data . qualitative assessments ( such as interviews and observations ) were the most frequently reported ( 57% ) , with at least one qualitative approach presented in 59 of the 104 articles that contained assessment data ( see the supplemental material ) . this was followed by student self - assessment and self - efficacy measures ( 42% of articles ) and disciplinary content - type assessments like weekly quizzes or final exams ( 40% of the articles ) . the inclusion of assessment data varied significantly based on discipline and type of inquiry ( table 1 ) . proportionally more bcdgmb and research exercises included assessment data , whereas proportionally fewer eeb and guided - inquiry exercises included assessment data ( figure 3 , a and c ) . course levels did not vary significantly in the frequency of assessment data ( table 1 and figure 3b ) . for the studies with assessment data , qualitative assessment was more common than expected , assuming that the four different assessment types would occur with equal frequency ( p = 0.04 ; figure 3 ) . biology subdiscipline , course level , and inquiry type did not influence the type of assessment ( table 1 and figure 3 ) . number of studies based on assessment type and ( a ) subdiscipline in biology ( n = 261 ) ; ( b ) course level ( n = 211 ) ; and ( c ) inquiry - type ( n = 231 ) . although a large portion of the studies included assessment data , of those that did , only 15% were controlled studies and only 12% were based on published instruments . both controlled studies and those based on published instruments were distributed based on course level and subdiscipline in biology in frequencies similar to all studies in our data set ( figures 1 and 4 ) . although guided - inquiry studies were more common ( figure 1c ) , control studies and those based on published instruments were most common in studies in which research inquiry was used ( figure 4c ) . published or validated instruments that were reported include the attitude and intentions survey ( based on silverstein , 1999 , and astrazeneca science teaching trust , 2002 ) , student assessment of their learning gains ( salg ; www.salgsite.org ) , science attitude survey ( moore , 1996 ) , and the classroom undergraduate research experience and summer undergraduate research experience surveys ( www.grinnell.edu/node/25703 ) . in other cases , studies used instruments that had been developed in the context of similar laboratory courses ( marshall , 2007 ; simmons et al . , 2008 ; dean and wilder , 2011 ) . for example , dean and wilder ( 2011 ) assessed their students who carried out a recombinant dna exercise using a survey developed by sleister ( 2007 ) to assess her students who isolated yeast mutants . the most commonly used published instrument , and the only instrument used in more than one study ( n = 3 ) , was the salg . the majority of studies that used published instruments ( 11 out of 17 studies ) were using student self - assessment . of the remainder of the studies , one assessed disciplinary content , one used qualitative assessment , and four were classified as other ( e.g. , critical - thinking skills , scientific reasoning skills ) . percentage of studies with assessment based on published instrument ( n = 17 ) or controlled ( n = 21 ) divided by ( a ) subdiscipline in biology : bcdgmb , biochemistry , cell biology , developmental biology , genetics , and molecular biology ; eeb , ecology and evolutionary biology ; organismal , organismal biology , including plant and animal anatomy and physiology ; ( b ) course level ; and ( c ) inquiry type . most frequently , assessments covered the entire semester ( 61% of studies ) , rather than individual exercises . for papers that reported the number of students studied in their assessment , sample sizes ranged from 3 to 1018 with a median sample size of 54 . although many studies presented assessment data for multiple semesters , relatively few presented assessment data from more than one institution ( campbell et al . generally , data from multiple institutions came from national projects related biology laboratory education like the genome consortium for active teaching ( campbell et al . , 2007 ) and the genomics education partnership ( gep ; shaffer et al . , 2010 ) , or multi - institutional genomics projects ( baumler et al . , 2012 ) . the curricula associated with these projects are predominantly used in upper - level microbiology , genetics , molecular biology , or genomics courses . belanger ( 2009 ) used the same laboratory exercise at two different institutions where he taught . all but one of the studies that included assessment data reported a positive effect of inquiry - based learning for at least one assessment measure , as would be expected for published experiments . in all controlled studies that measured learning gains , students in inquiry - based laboratory courses had significantly higher learning gains than students in non - inquiry courses . in addition , students in inquiry - based laboratory courses enjoyed them more and had more significant positive shifts in science attitudes as compared with students in non - inquiry laboratory courses . the one exception to the trend for a positive effect of inquiry - based learning was a study by basey and francis ( 2011 ) . they found that students had a significantly greater preference for scripted versions of laboratory exercises as compared with inquiry - based versions of the same exercises for one of three exercises used in a first - semester introductory biology laboratory course . students did not differ in their preference for a particular pedagogical approach for the other two exercises . basey and francis ( 2011 ) suggest that students might prefer scripted versions of laboratory exercises that they perceive to be more difficult . of the 16 controlled studies in our sample , for all assessment in those studies , the mean effect size was 0.73 ( median = 0.61 , sd = 0.71 ) . when we took the average effect size for each study first , in order to avoid pseudoreplication , the mean effect size decreased to 0.62 ( median = 0.40 , sd = 0.76 ) , due to several studies with multiple assessments and high effect sizes . the effect sizes seem to be greatest in introductory courses and courses that incorporate research ( table 2 ) . in addition , gains appear to be greatest in student understanding of disciplinary content ( table 2 ) . effect size by assessment type , inquiry type , and course level some studies included more than one assessment for which an effect size could be calculated . we averaged all assessments for each study before calculating sample statistics for the by study comparisons . total sample size by study differs for the assessment - type analysis , because two studies included more than one assessment type for which we could calculate an effect size . we found 142 papers that described inquiry - based laboratory exercises that had been implemented in undergraduate biology courses between 2005 and 2012 ( see the supplemental material ) . on average , more than half of the exercises ( 58% ) were in the areas of biochemistry , cell biology , developmental biology , genetics , and molecular biology ( bcdgmb ; figure 1a ) , and the majority were used in courses for upper - level majors ( figure 1b ) . overall , most were guided - inquiry exercises rather than open - ended exercises or research experiences imbedded in a course ( figure 1c ) . the approach to inquiry did not vary significantly based on course level ( table 1 and figure 2a ) or discipline ( table 1 and figure 2b ) . however , bcdgmb exercises were used more often in upper - level courses and ecology and evolutionary biology ( eeb ) experiments were used proportionally more often in nonmajors courses ( table 1 and figure 2c ) . number of published inquiry - based laboratory studies in biology based on ( a ) subdiscipline in biology : bcdgmb , biochemistry , cell biology , developmental biology , genetics , and molecular biology ; eeb , ecology and evolutionary biology ; organismal , organismal biology , including plant and animal anatomy and physiology ( n = 173 ) ; ( b ) course level ( n = 135 ) ; and ( c ) inquiry type ( n = 143 ) . number of studies based on ( a ) inquiry type and level at which experiment was taught ( n = 144 ) ; ( b ) subdiscipline in biology and inquiry type ( n = 185 ) ; and ( c ) subdiscipline in biology and level at which experiment was taught ( n = 159 ) . summary of results of cross - tabulated tests of independence rows marked with * represent significant interactions between the two factors on the number of studies in our data set . for the studies included in our sample , 73% presented assessment data ( see the supplemental material ) . of those studies that presented assessment data , qualitative assessments ( such as interviews and observations ) were the most frequently reported ( 57% ) , with at least one qualitative approach presented in 59 of the 104 articles that contained assessment data ( see the supplemental material ) . this was followed by student self - assessment and self - efficacy measures ( 42% of articles ) and disciplinary content - type assessments like weekly quizzes or final exams ( 40% of the articles ) . the inclusion of assessment data varied significantly based on discipline and type of inquiry ( table 1 ) . proportionally more bcdgmb and research exercises included assessment data , whereas proportionally fewer eeb and guided - inquiry exercises included assessment data ( figure 3 , a and c ) . course levels did not vary significantly in the frequency of assessment data ( table 1 and figure 3b ) . for the studies with assessment data , qualitative assessment was more common than expected , assuming that the four different assessment types would occur with equal frequency ( p = 0.04 ; figure 3 ) . biology subdiscipline , course level , and inquiry type did not influence the type of assessment ( table 1 and figure 3 ) . number of studies based on assessment type and ( a ) subdiscipline in biology ( n = 261 ) ; ( b ) course level ( n = 211 ) ; and ( c ) inquiry - type ( n = 231 ) . although a large portion of the studies included assessment data , of those that did , only 15% were controlled studies and only 12% were based on published instruments . both controlled studies and those based on published instruments were distributed based on course level and subdiscipline in biology in frequencies similar to all studies in our data set ( figures 1 and 4 ) . although guided - inquiry studies were more common ( figure 1c ) , control studies and those based on published instruments were most common in studies in which research inquiry was used ( figure 4c ) . published or validated instruments that were reported include the attitude and intentions survey ( based on silverstein , 1999 , and astrazeneca science teaching trust , 2002 ) , student assessment of their learning gains ( salg ; www.salgsite.org ) , science attitude survey ( moore , 1996 ) , and the classroom undergraduate research experience and summer undergraduate research experience surveys ( www.grinnell.edu/node/25703 ) . in other cases , studies used instruments that had been developed in the context of similar laboratory courses ( marshall , 2007 ; simmons et al . , 2008 ; dean and wilder , 2011 ) . for example , dean and wilder ( 2011 ) assessed their students who carried out a recombinant dna exercise using a survey developed by sleister ( 2007 ) to assess her students who isolated yeast mutants . the most commonly used published instrument , and the only instrument used in more than one study ( n = 3 ) , was the salg . the majority of studies that used published instruments ( 11 out of 17 studies ) were using student self - assessment . of the remainder of the studies , one assessed disciplinary content , one used qualitative assessment , and four were classified as other ( e.g. , critical - thinking skills , scientific reasoning skills ) . percentage of studies with assessment based on published instrument ( n = 17 ) or controlled ( n = 21 ) divided by ( a ) subdiscipline in biology : bcdgmb , biochemistry , cell biology , developmental biology , genetics , and molecular biology ; eeb , ecology and evolutionary biology ; organismal , organismal biology , including plant and animal anatomy and physiology ; ( b ) course level ; and ( c ) inquiry type . most frequently , assessments covered the entire semester ( 61% of studies ) , rather than individual exercises . for papers that reported the number of students studied in their assessment , sample sizes ranged from 3 to 1018 with a median sample size of 54 . although many studies presented assessment data for multiple semesters , relatively few presented assessment data from more than one institution ( campbell et al . generally , data from multiple institutions came from national projects related biology laboratory education like the genome consortium for active teaching ( campbell et al . , 2007 ) and the genomics education partnership ( gep ; shaffer et al . , 2010 ) , or multi - institutional genomics projects ( baumler et al . , 2012 ) . the curricula associated with these projects are predominantly used in upper - level microbiology , genetics , molecular biology , or genomics courses . belanger ( 2009 ) used the same laboratory exercise at two different institutions where he taught . all but one of the studies that included assessment data reported a positive effect of inquiry - based learning for at least one assessment measure , as would be expected for published experiments . in all controlled studies that measured learning gains , students in inquiry - based laboratory courses had significantly higher learning gains than students in non - inquiry courses . in addition , students in inquiry - based laboratory courses enjoyed them more and had more significant positive shifts in science attitudes as compared with students in non - inquiry laboratory courses . the one exception to the trend for a positive effect of inquiry - based learning was a study by basey and francis ( 2011 ) . they found that students had a significantly greater preference for scripted versions of laboratory exercises as compared with inquiry - based versions of the same exercises for one of three exercises used in a first - semester introductory biology laboratory course . students did not differ in their preference for a particular pedagogical approach for the other two exercises . basey and francis ( 2011 ) suggest that students might prefer scripted versions of laboratory exercises that they perceive to be more difficult . of the 16 controlled studies in our sample , for all assessment in those studies , the mean effect size was 0.73 ( median = 0.61 , sd = 0.71 ) . when we took the average effect size for each study first , in order to avoid pseudoreplication , the mean effect size decreased to 0.62 ( median = 0.40 , sd = 0.76 ) , due to several studies with multiple assessments and high effect sizes . the effect sizes seem to be greatest in introductory courses and courses that incorporate research ( table 2 ) . in addition , gains appear to be greatest in student understanding of disciplinary content ( table 2 ) . effect size by assessment type , inquiry type , and course level some studies included more than one assessment for which an effect size could be calculated . we averaged all assessments for each study before calculating sample statistics for the by study comparisons . total sample size by study differs for the assessment - type analysis , because two studies included more than one assessment type for which we could calculate an effect size . since the publication of bio2010 ( nrc , 2003a ) and handelsman et al . ( 2004 ) , a large number of inquiry - based exercises for biology laboratory courses have been published in peer - reviewed , catalogued journals . our meta - analysis revealed several interesting patterns in the development , implementation , and dissemination of inquiry - based exercises in undergraduate biology laboratory courses . first , published exercises in our data set were more likely to be in bcdgmb than eeb or organismal biology . the greater number of published exercises in bcdgmb could be due to a variety of reasons , including a greater number of laboratory courses , the ease of developing inquiry - based exercises , higher rates of curriculum development , and greater dissemination of inquiry - based labs in the peer - reviewed literature in bcdgmb . whatever the explanation , our results suggest the need for greater emphasis on development and dissemination of inquiry - based laboratory exercises in ecology , evolutionary biology , and organismal biology . second , we found a greater emphasis on guided - inquiry exercises than open - ended inquiry and research - based approaches . moving from guided - inquiry toward research - based laboratory pedagogies in undergraduate science has been shown to help retain students in science majors and to prepare them for the workplace ( weaver et al . , 2008 ) . producing students who not only have developed skills through inquiry , but who know how to carry out research , will aid in the production of more qualified postgraduate students and also provide those not going on to undertake further research with the knowledge of how science is done this is empowering and potentially beneficial to society as a whole . several national projects are working to advance course - based research experiences focusing on particular research projects ( e.g. , howard hughes medical institute [ hhmi ] phage hunters [ hanauer et al . , 2006 ] ; gep [ shaffer et al . , 2010 ] ) or particular experimental approaches ( e.g. , ecological research as education network [ bowne et al . , 2011 ] ; the genome consortium for active teaching [ campbell et al . , 2006 , 2007 ] ; integrated microbial genomes annotation collaboration toolkit [ ditty et al . , 2010 ] ) . in addition , the course - based undergraduate research network ( curenet ) is facilitating collaborations among faculty members interested in incorporating research in their courses ( auchincloss et al . , 2014 ) . finally , more exercises were aimed at upper - level courses for majors as compared with introductory courses for majors and nonmajors , with few introductory courses with research embedded included in this study . this bias toward upper - level courses could be due to the fact that more upper - level laboratory courses are taught than introductory laboratory courses or that development of inquiry - based exercises for upper - level courses might be more straightforward . however , all colleges and universities teach introductory biology laboratory courses , whereas upper - level laboratory course offerings in particular subdisciplines of biology will vary from institution to institution and may be limited . our data suggest the need to continue the development and dissemination of inquiry - based laboratory curricula for introductory biology laboratory courses . while we understand the constraints that faculty members face when moving toward teaching methods that require greater mentorship of students , in order to fulfill the goal of the vision and change report ( aaas , 2011 ) of involving all students in research , we will need to envision how to incorporate open - ended and research - based approaches in courses to a greater degree , especially in nonmajors and introductory majors courses . although there are clearly more impediments to teaching in this way with large introductory or nonmajors courses , the value in terms of learning outcomes is worth the money and the effort . for example , in a large first - year biology course , burke da silva ( 2012 ) found that not only can first - year students undertake research projects and produce quality outcomes , but even nonmajors find value in research projects . ( 2011 ) showed that introductory biology students who engaged in course - based research improved their understanding of how research is done and changed their views on potential career choices . the nrc report on assessing teaching and learning ( nrc , 2003b ) and handelsman et al . ( 2004 ) specifically indicated the need for an increase in the assessment of teaching methods to allow more thorough determinations of their role in student learning . the vast majority of studies in our data set included some type of assessment data , and most studies with assessment data included multiple forms of assessment data . in contrast , this high level of assessment might be more of an artifact of the requirements for publication . although most studies included assessment data , few used published , validated instruments for assessing their students learning . furthermore , the majority of studies using published instruments were using student self - assessment . however , self - reported learning gains are not necessarily correlated with actual learning gains ( falchikov and boud , 1989 ) . given the availability of published , validated instruments for assessing scientific reasoning ( lawson , 1978 ) , experimental design ( sirum and humberg , 2011 ) , and understanding of the nature of science ( reviewed by lederman , 2007 ) , and the recent development of concept inventories and diagnostic question clusters in biology ( davanzo , 2008 ; smith and tanner , 2010 ) , biology faculty members could be using published instruments to assess their students to a greater degree . the use of published , validated instruments will improve our ability to compare the effectiveness of inquiry - based learning in the laboratory across courses and institutions . most studies in our data set were limited to individual courses at a single institution . if the results of these studies can be generalized across courses and institutions remains unclear due to differences in student demographics and faculty implementation of the curricula ( nrc , 2012 ) . in the three studies that assessed students across different institutions ( campbell et al . , 2007 ; , we were unable to determine whether course or institutional contexts had an effect on student learning gains . however , a recently published study by shaffer et al . ( 2014 ) suggests that differences across institutions do not appear to significantly affect learning gains . although many inquiry - based laboratory exercises will not be implemented in different courses at different institutions , comparisons across studies can be facilitated by the reporting of either effect sizes ( e.g. , simmons et al . , 2008 ) or complete summary statistics ( ruiz - primo et al . , 2011 ) . in the controlled studies for which we were unable to estimate effect sizes , studies did not include estimates of variation ( sd or sem ) , did not indicate whether error bars represented sds or sems , or did not provide sample sizes that are necessary to calculate sds from sems . both means and sds are needed to estimate effect sizes , and sample sizes are also needed to estimate weighted effect sizes . clearly , controlled studies allow us to draw stronger inferences about the impact of inquiry - based laboratory courses than studies that do not include a control ( handelsman et al . , 2004 ; ruiz - primo et al . , set , only 15% of the studies included a control group ( i.e. , a group of students who did not conduct the inquiry - based laboratory exercises ) . however , we caution that if a priori we think that a pedagogical change will improve student learning , withholding that innovation from a group of students would be unethical . in these cases , collecting data on students in semesters before a change in teaching approach and then on students in semesters after the change would balance ethical concerns with the need for experimental rigor , particularly if the assessment is done using published instruments . also , this approach is the only tenable one for faculty members who teach at smaller institutions where only a single section of a course is taught at any particular time . not surprisingly , the majority of studies in our data set supports the idea that students in inquiry - based laboratory courses make significantly greater learning gains than students in non - inquiry courses . in the few studies in our data set that have sufficient information to calculate effect size , the mean effect size was 0.73 . in other words , the outcome of the average student in an inquiry - based laboratory course was 0.73 sds better than the outcome of the average student in a non - inquiry course . this effect size is greater than the effect size for a wide variety of course innovations in biology courses ( ruiz - primo et al . , 2011 ) and is well above the threshold of 0.25 that is used as the criterion for an educational innovation to be substantively important ( institute of education sciences , 2013 ) . however , we would caution that this large effect size also might be indicative of a file - drawer effect in which studies that show no effect or negative effects of inquiry - based learning are not submitted for publication . the greatest effects seem to be found in introductory courses and courses that incorporate research ( table 2 ) . students also appear to show the largest gains in disciplinary content knowledge ( table 2 ) . however , the number of studies in each category for which we were able to estimate effect sizes is very small . many more data are needed before we can draw any conclusions as to which students are benefited most by inquiry - based learning in laboratory courses , which approaches to laboratory instruction are most beneficial , and where student learning gains are most pronounced . overall , our meta - analysis of the literature suggests that inquiry - based learning in biology laboratory courses can increase student learning gains . however , whether the impact of individual innovations applies across institution types and course levels is unclear . with so much evidence supporting inquiry - based laboratory teaching and its enhancement of student learning , the question remains as to why inquiry and research - based pedagogies are not found more often in university science courses . the reform is moving slowly , and this is likely due to time for faculty to develop new laboratory exercises ( spell et al . , 2014 ) or potentially due to the conflict between teaching and research in terms of professional identity for faculty ( brownell and tanner , 2012 ) . with the move toward teaching and education focused faculty members who carry out more pedagogical research and who spread their knowledge to more research - focused faculty ( bush et al . , 2013)perhaps the speed of implementation may soon increase . in summary , the biology education community has made strides in developing , implementing , and disseminating new inquiry - based laboratory curricula for undergraduate courses . in addition , the community has taken beginning steps in assessing the effectiveness of these curricular changes . however , substantial gaps still exist in curriculum development and assessment that will need to be filled to meet the grade . we need to redouble our efforts in curriculum development and dissemination , especially with respect to research - based curriculum at the introductory level . moreover , systematic assessment of current and future inquiry - based laboratory curricula across institutions using validated instruments is highly recommended .

the authors reviewed the current literature on inquiry - based learning in laboratory courses and found that most exercises were guided inquiry . the majority of studies included assessment data showing learning gains . few exercises were assessed in multiple courses or at multiple institutions . therefore , whether results can be generalized is unclear .

INTRODUCTION METHODS Data Analysis RESULTS Inquiry-Based Biology Laboratory Exercises Assessment of Inquiry-Based Biology Laboratory Exercises DISCUSSION Supplementary Material

inquiry - based approaches should be transforming the way science faculty members teach and undergraduate students learn ( national research council [ nrc ] , 2003a ) . since 1985 , the national science foundation ( nsf ) has promoted the enhancement of undergraduate science instruction , especially in laboratory courses ( tuss et al . a year later , handelsman and colleagues seminal paper in science echoed this push for more active learning in science courses , providing examples of scientific teaching in both lecture and laboratory classrooms ( handelsman et al . the infusion of inquiry - based approaches into the undergraduate biology curriculum is especially important in laboratory courses or within the laboratory component of a course , because students are applying the process of science in these courses ( aaas , 2011 ) and are learning to write in an inquiry - based way ( tuss et al . surveys that took place more than 20 yr ago suggest that inquiry - based and open - ended laboratory exercises were used infrequently ( 10% of courses ; sundberg and armstrong , 1993 ) . a more recent survey reporting results from 65 colleges and universities in the united states found close to 80% of institutions are using inquiry - based approaches in their laboratory courses ( sundberg et al . yet ( 2005 ) study did not determine the degree to which these approaches had been implemented or the effectiveness of these inquiry - based teaching methods on student performance . more than 7 yr on , information about inquiry - based approaches is still lacking , with the recent national research council report on discipline - based education research rating the evidence in support of inquiry - based learning in laboratory courses in biology as limited ( nrc , 2012 ) . given the repeated calls for increased inquiry - based teaching and learning in the undergraduate biology curriculum and the important role of laboratory courses in the curriculum , we conducted a meta - analysis of peer - reviewed , published , undergraduate inquiry - based laboratory exercises produced since the publication of the bio2010 report ( nrc , 2003a ) and handelsman et al . specifically , we addressed two main questions : to what degree have inquiry - based laboratory exercises been developed and published in different subdisciplines in biology , at what level ( i.e. have inquiry - based laboratory exercises been developed and published in different subdisciplines in biology , at what level ( i.e. the data suggest where future efforts need to be made in assessment and provide a snapshot of the evidence for the value of inquiry - based laboratory exercises in undergraduate biology courses . as papers on research in laboratory courses inquiry , we also searched on research lab * in web of science and eric and limited the results as described above . we combined the results of all searches and then manually excluded all studies that were not explicitly about an inquiry - based laboratory exercise in biology at the undergraduate level . after the exclusion process , 142 studies remained , which were scored for the current study ( see the supplemental material ) . admittedly , our search did not retrieve all publications of inquiry - based laboratory exercises in biology , as these exercises might not be published in journals catalogued by web of science or eric . however , those studies that include assessment data are perhaps more likely to be published in catalogued journals . because we were interested in changes in the publication of inquiry - based laboratory exercises and the assessment of those exercises following the publication of the bio2010 report ( nrc , 2003a ) , the nrc 's report on assessment of teaching and learning ( nrc , 2003b ) , and handelsman and colleagues paper on scientific teaching ( handelsman et al . we coded each paper in the following categories : area of biology : biochemistry , cell biology , developmental biology , genetics , and molecular biology ( bcdgmb ) ; ecology and evolutionary biology ( eeb ) ; plant and animal anatomy and physiology ( organismal biology).inquiry type : guided inquiry , open - ended inquiry , research ( including teacher - collaborative and structured research experiences ) , and other ( e.g. , case studies , problem - based learning , model building ) . unlike guided inquiry , the outcomes of the experiments are not known to either the students or faculty . inquiry type : guided inquiry , open - ended inquiry , research ( including teacher - collaborative and structured research experiences ) , and other ( e.g. , case studies , problem - based learning , model building ) . unlike guided inquiry , the outcomes of the experiments are not known to either the students or faculty . , student comments , interviews ) , student self - assessment ( self - efficacy and student assessment of learning gains ) , disciplinary content , and other ( e.g. studies that reported multiple types of assessment data were included once for each type of assessment for appropriate analyses . as a result of these multiple codings , the sample sizes for subdiscipline in biology and assessment are greater than the total number of studies in the data set . , counts of the number of studies that fall into particular categories ) . for analyses that included assessment type or inquiry type , we excluded studies that were coded as other because the number of studies was small , and the assessment types or inquiry types included in the other categories were quite variable . because of the limited number of studies for which we were able to calculate effect size ( n = 10 ) , we were unable to do any further statistical analysis on whether the effects of inquiry - based learning were greatest at particular course levels , from using particular types of inquiry , or for particular types of assessment . , counts of the number of studies that fall into particular categories ) . the results of chi - square tests can be biased if the expected count in a particular cell is low . for analyses that included assessment type or inquiry type , we excluded studies that were coded as other because the number of studies was small , and the assessment types or inquiry types included in the other categories were quite variable . because of the limited number of studies for which we were able to calculate effect size ( n = 10 ) , we were unable to do any further statistical analysis on whether the effects of inquiry - based learning were greatest at particular course levels , from using particular types of inquiry , or for particular types of assessment . we found 142 papers that described inquiry - based laboratory exercises that had been implemented in undergraduate biology courses between 2005 and 2012 ( see the supplemental material ) . on average , more than half of the exercises ( 58% ) were in the areas of biochemistry , cell biology , developmental biology , genetics , and molecular biology ( bcdgmb ; figure 1a ) , and the majority were used in courses for upper - level majors ( figure 1b ) . overall , most were guided - inquiry exercises rather than open - ended exercises or research experiences imbedded in a course ( figure 1c ) . however , bcdgmb exercises were used more often in upper - level courses and ecology and evolutionary biology ( eeb ) experiments were used proportionally more often in nonmajors courses ( table 1 and figure 2c ) . number of published inquiry - based laboratory studies in biology based on ( a ) subdiscipline in biology : bcdgmb , biochemistry , cell biology , developmental biology , genetics , and molecular biology ; eeb , ecology and evolutionary biology ; organismal , organismal biology , including plant and animal anatomy and physiology ( n = 173 ) ; ( b ) course level ( n = 135 ) ; and ( c ) inquiry type ( n = 143 ) . for the studies included in our sample , 73% presented assessment data ( see the supplemental material ) . of those studies that presented assessment data , almost two - thirds included more than one type of assessment data . proportionally more bcdgmb and research exercises included assessment data , whereas proportionally fewer eeb and guided - inquiry exercises included assessment data ( figure 3 , a and c ) . course levels did not vary significantly in the frequency of assessment data ( table 1 and figure 3b ) . for the studies with assessment data , qualitative assessment was more common than expected , assuming that the four different assessment types would occur with equal frequency ( p = 0.04 ; figure 3 ) . number of studies based on assessment type and ( a ) subdiscipline in biology ( n = 261 ) ; ( b ) course level ( n = 211 ) ; and ( c ) inquiry - type ( n = 231 ) . although a large portion of the studies included assessment data , of those that did , only 15% were controlled studies and only 12% were based on published instruments . published or validated instruments that were reported include the attitude and intentions survey ( based on silverstein , 1999 , and astrazeneca science teaching trust , 2002 ) , student assessment of their learning gains ( salg ; www.salgsite.org ) , science attitude survey ( moore , 1996 ) , and the classroom undergraduate research experience and summer undergraduate research experience surveys ( www.grinnell.edu/node/25703 ) . in other cases , studies used instruments that had been developed in the context of similar laboratory courses ( marshall , 2007 ; simmons et al . the majority of studies that used published instruments ( 11 out of 17 studies ) were using student self - assessment . although many studies presented assessment data for multiple semesters , relatively few presented assessment data from more than one institution ( campbell et al . all but one of the studies that included assessment data reported a positive effect of inquiry - based learning for at least one assessment measure , as would be expected for published experiments . in all controlled studies that measured learning gains , students in inquiry - based laboratory courses had significantly higher learning gains than students in non - inquiry courses . in addition , students in inquiry - based laboratory courses enjoyed them more and had more significant positive shifts in science attitudes as compared with students in non - inquiry laboratory courses . the one exception to the trend for a positive effect of inquiry - based learning was a study by basey and francis ( 2011 ) . they found that students had a significantly greater preference for scripted versions of laboratory exercises as compared with inquiry - based versions of the same exercises for one of three exercises used in a first - semester introductory biology laboratory course . effect size by assessment type , inquiry type , and course level some studies included more than one assessment for which an effect size could be calculated . total sample size by study differs for the assessment - type analysis , because two studies included more than one assessment type for which we could calculate an effect size . we found 142 papers that described inquiry - based laboratory exercises that had been implemented in undergraduate biology courses between 2005 and 2012 ( see the supplemental material ) . on average , more than half of the exercises ( 58% ) were in the areas of biochemistry , cell biology , developmental biology , genetics , and molecular biology ( bcdgmb ; figure 1a ) , and the majority were used in courses for upper - level majors ( figure 1b ) . however , bcdgmb exercises were used more often in upper - level courses and ecology and evolutionary biology ( eeb ) experiments were used proportionally more often in nonmajors courses ( table 1 and figure 2c ) . number of published inquiry - based laboratory studies in biology based on ( a ) subdiscipline in biology : bcdgmb , biochemistry , cell biology , developmental biology , genetics , and molecular biology ; eeb , ecology and evolutionary biology ; organismal , organismal biology , including plant and animal anatomy and physiology ( n = 173 ) ; ( b ) course level ( n = 135 ) ; and ( c ) inquiry type ( n = 143 ) . number of studies based on ( a ) inquiry type and level at which experiment was taught ( n = 144 ) ; ( b ) subdiscipline in biology and inquiry type ( n = 185 ) ; and ( c ) subdiscipline in biology and level at which experiment was taught ( n = 159 ) . summary of results of cross - tabulated tests of independence rows marked with * represent significant interactions between the two factors on the number of studies in our data set . for the studies included in our sample , 73% presented assessment data ( see the supplemental material ) . of those studies that presented assessment data , qualitative assessments ( such as interviews and observations ) were the most frequently reported ( 57% ) , with at least one qualitative approach presented in 59 of the 104 articles that contained assessment data ( see the supplemental material ) . proportionally more bcdgmb and research exercises included assessment data , whereas proportionally fewer eeb and guided - inquiry exercises included assessment data ( figure 3 , a and c ) . for the studies with assessment data , qualitative assessment was more common than expected , assuming that the four different assessment types would occur with equal frequency ( p = 0.04 ; figure 3 ) . number of studies based on assessment type and ( a ) subdiscipline in biology ( n = 261 ) ; ( b ) course level ( n = 211 ) ; and ( c ) inquiry - type ( n = 231 ) . although a large portion of the studies included assessment data , of those that did , only 15% were controlled studies and only 12% were based on published instruments . in other cases , studies used instruments that had been developed in the context of similar laboratory courses ( marshall , 2007 ; simmons et al . the majority of studies that used published instruments ( 11 out of 17 studies ) were using student self - assessment . most frequently , assessments covered the entire semester ( 61% of studies ) , rather than individual exercises . although many studies presented assessment data for multiple semesters , relatively few presented assessment data from more than one institution ( campbell et al . generally , data from multiple institutions came from national projects related biology laboratory education like the genome consortium for active teaching ( campbell et al . all but one of the studies that included assessment data reported a positive effect of inquiry - based learning for at least one assessment measure , as would be expected for published experiments . in all controlled studies that measured learning gains , students in inquiry - based laboratory courses had significantly higher learning gains than students in non - inquiry courses . in addition , students in inquiry - based laboratory courses enjoyed them more and had more significant positive shifts in science attitudes as compared with students in non - inquiry laboratory courses . the one exception to the trend for a positive effect of inquiry - based learning was a study by basey and francis ( 2011 ) . they found that students had a significantly greater preference for scripted versions of laboratory exercises as compared with inquiry - based versions of the same exercises for one of three exercises used in a first - semester introductory biology laboratory course . effect size by assessment type , inquiry type , and course level some studies included more than one assessment for which an effect size could be calculated . total sample size by study differs for the assessment - type analysis , because two studies included more than one assessment type for which we could calculate an effect size . ( 2004 ) , a large number of inquiry - based exercises for biology laboratory courses have been published in peer - reviewed , catalogued journals . our meta - analysis revealed several interesting patterns in the development , implementation , and dissemination of inquiry - based exercises in undergraduate biology laboratory courses . the greater number of published exercises in bcdgmb could be due to a variety of reasons , including a greater number of laboratory courses , the ease of developing inquiry - based exercises , higher rates of curriculum development , and greater dissemination of inquiry - based labs in the peer - reviewed literature in bcdgmb . whatever the explanation , our results suggest the need for greater emphasis on development and dissemination of inquiry - based laboratory exercises in ecology , evolutionary biology , and organismal biology . second , we found a greater emphasis on guided - inquiry exercises than open - ended inquiry and research - based approaches . moving from guided - inquiry toward research - based laboratory pedagogies in undergraduate science has been shown to help retain students in science majors and to prepare them for the workplace ( weaver et al . several national projects are working to advance course - based research experiences focusing on particular research projects ( e.g. in addition , the course - based undergraduate research network ( curenet ) is facilitating collaborations among faculty members interested in incorporating research in their courses ( auchincloss et al . this bias toward upper - level courses could be due to the fact that more upper - level laboratory courses are taught than introductory laboratory courses or that development of inquiry - based exercises for upper - level courses might be more straightforward . however , all colleges and universities teach introductory biology laboratory courses , whereas upper - level laboratory course offerings in particular subdisciplines of biology will vary from institution to institution and may be limited . our data suggest the need to continue the development and dissemination of inquiry - based laboratory curricula for introductory biology laboratory courses . for example , in a large first - year biology course , burke da silva ( 2012 ) found that not only can first - year students undertake research projects and produce quality outcomes , but even nonmajors find value in research projects . the vast majority of studies in our data set included some type of assessment data , and most studies with assessment data included multiple forms of assessment data . although most studies included assessment data , few used published , validated instruments for assessing their students learning . furthermore , the majority of studies using published instruments were using student self - assessment . the use of published , validated instruments will improve our ability to compare the effectiveness of inquiry - based learning in the laboratory across courses and institutions . if the results of these studies can be generalized across courses and institutions remains unclear due to differences in student demographics and faculty implementation of the curricula ( nrc , 2012 ) . , 2007 ; , we were unable to determine whether course or institutional contexts had an effect on student learning gains . ( 2014 ) suggests that differences across institutions do not appear to significantly affect learning gains . although many inquiry - based laboratory exercises will not be implemented in different courses at different institutions , comparisons across studies can be facilitated by the reporting of either effect sizes ( e.g. clearly , controlled studies allow us to draw stronger inferences about the impact of inquiry - based laboratory courses than studies that do not include a control ( handelsman et al . , a group of students who did not conduct the inquiry - based laboratory exercises ) . not surprisingly , the majority of studies in our data set supports the idea that students in inquiry - based laboratory courses make significantly greater learning gains than students in non - inquiry courses . in other words , the outcome of the average student in an inquiry - based laboratory course was 0.73 sds better than the outcome of the average student in a non - inquiry course . however , we would caution that this large effect size also might be indicative of a file - drawer effect in which studies that show no effect or negative effects of inquiry - based learning are not submitted for publication . the greatest effects seem to be found in introductory courses and courses that incorporate research ( table 2 ) . however , the number of studies in each category for which we were able to estimate effect sizes is very small . many more data are needed before we can draw any conclusions as to which students are benefited most by inquiry - based learning in laboratory courses , which approaches to laboratory instruction are most beneficial , and where student learning gains are most pronounced . overall , our meta - analysis of the literature suggests that inquiry - based learning in biology laboratory courses can increase student learning gains . however , whether the impact of individual innovations applies across institution types and course levels is unclear . with so much evidence supporting inquiry - based laboratory teaching and its enhancement of student learning , the question remains as to why inquiry and research - based pedagogies are not found more often in university science courses . in summary , the biology education community has made strides in developing , implementing , and disseminating new inquiry - based laboratory curricula for undergraduate courses . we need to redouble our efforts in curriculum development and dissemination , especially with respect to research - based curriculum at the introductory level . moreover , systematic assessment of current and future inquiry - based laboratory curricula across institutions using validated instruments is highly recommended .

sle is a chronic multisystem autoimmune disease with a broad range of clinical manifestations , including photosensitive skin rashes , discoid lesions , arthritis / arthralgia , nephritis , cardiac and pulmonary disease , and cns disorders . the disease pathogenesis is attributed to circulating antinuclear autoantibodies against a variety of nuclear antigens ( including dsdna , the ribonucleoprotein ( rnp ) complex ro , the rna - binding protein la , rnps , the rna molecule / protein complex sm , the c1 complement component subunit c1q , and phospholipids ) and the dysfunction of t and b lymphocytes and dendritic cells . although the pathogenesis is believed to lie in the dysregulation of the immune system , the involvement of various organ systems often leads to secondary morbidities resulting from renal failure , hypertension , or cns disorders , and more recently it is becoming increasingly clear that accelerated atherosclerosis associated with sle may contribute to premature mortality . atherosclerosis ( at ) is a chronic inflammatory disease of the arteries associated with various risk factors that promote lipid abnormalities ( i.e. , dyslipidemia ) , development and progression of atherosclerotic lesions , plaque rupture , and vascular thrombosis . at is enhanced in autoimmune diseases ; noninvasive investigations show increases in intima - media thickness , carotid plaque , and coronary artery calcifications in patients with antiphospholipid syndrome ( aps ) , systemic lupus erythematosus ( sle ) , and rheumatoid arthritis ( ra ) compared to controls . the reason for this accelerated process is still debatable and , although traditional risk factors ( such as hyperlipidemia , smoking , obesity , hypertension , diabetes mellitus , postmenopausal status , and sedentary lifestyle ) are more prevalent in those patients than in general population , they do not seem to fully explain that enhanced risk . experimental studies and human observations suggest that innate and adaptive immune responses participate in the pathogenesis of both at and autoimmune diseases . actually , some autoantibodies , including antioxidized low density lipoproteins ( antioxldl ) , anti-2-glycoprotein 1 ( anti2gpi ) , antiheat shock proteins 60/65 ( antihsp60/65 ) , and antioxldl/2gpi , have been shown to be associated to the pathogenesis of at [ 6 , 7 ] . however , their role in accelerated at in aps and sle patients is still controversial . identified additional factors for at in patients with sle include chronic inflammation and chronic exposure to steroid therapy . these factors can directly influence the development of at through a variety of mechanisms such as immune complex generation , complement activation , alteration of the oxidant - antioxidant balance locally within the vessel wall , and changes in the production and activity of a complex network of cytokines [ 810 ] ( figure 1 ) . characterization of the molecular and cellular basis of signalling abnormalities within the immune system that lead to auto reactivity and inflammation and their relationship to early atherosclerosis and cardiovascular disease ( cvd ) development remain critical for understanding the pathogenesis of sle . the vascular inflammatory response involves complex interaction between inflammatory cells ( neutrophils , lymphocytes , monocytes , and macrophages ) , endothelial cells ( ecs ) , vascular smooth muscle cells ( vsmcs ) , and extracellular matrix ( ecm ) . vascular injury is associated with increased expression of adhesion molecules by ecs and recruitment of inflammatory cells , growth factors , and cytokines , with consequent effects on ecs , vsmcs , and ecm . cytokines include tumour necrosis factor , interleukins , lymphokines , monokines , interferons , colony stimulating factors , and transforming growth factors . cytokines are produced by macrophages , t - cells and monocytes , as well as platelets , ecs and vsmcs . depending on their cellular source , cytokines are classified into type 1 cytokines , produced by th1 t - helper cells , that include il-2 , il-12 , ifn- , and tnf- ; and type 2 cytokines , produced by th2 t - helper cells that include il-4 , -5 , -6 , -10 , and -13 . the th2 cytokines play a role in distinct inflammatory processes , and may inhibit certain forms of autoimmunity . circulating cytokines interact with specific receptors on various cell types and activate signalling pathways leading to an inflammatory response involving cell adhesion , permeability , and apoptosis . cytokines are master regulators of the innate and adaptive immune response and , unsurprisingly , are known to regulate and , essentially , coordinate many stages of atherosclerosis [ 13 , 14 ] . many cytokines , such as interleukin ( il)-1 , il-6 , il-10 , interferon ifn , and tnf are expressed highly in atherosclerotic regions and exhibit pro- and antiatherogenic actions [ 1315 ] . innate cytokines such as il-1 or tnf may activate endothelial cells ( ecs ) , vascular smooth muscle cells ( vsmcs ) , monocytes / macrophages , lymphocytes ( t , b , nk ) , dendritic cells , and mast cells . these vascular cells can actively contribute to the inflammatory cytokine - dependent response in the vessel wall by production of cytokines or eliciting responses to cytokines , or can be involved in cytokine - mediated interaction with invading cells such as monocytes , t - cells , or mast cells . activation of these pathways results in accumulation of cells and increased ldl- and ecm - deposition which may facilitate subsequent invasions . numerous abnormalities of the cytokine network have been described in patients with sle as well as in murine lupus models . some of them were shown to play a pivotal physiopathological role in certain t - cell , b - cell or antigen presenting cell dysfunctions characteristic of the disease , while others are more likely to be innocent bystanders . several cytokines with relevance to cvd has been proven to be related to the pathogenesis of at in sle . in the following paragraphs we highlight some of the most likely plasmacytoid dendritic cells ( pdc ) activated by immune complexes containing nucleic acids secrete type i ifn ( ifn and ifn ) in sle . type i ifn causes differentiation of monocytes to myeloid - derived dendritic cell ( mdc ) and activation of autoreactive t and b cells . patients with sle have an increased expression of type i ifn - regulated genes because of a continuous production of ifn. recent reports have demonstrated that elevated levels of type i ifns ( cytokines with potent antiproliferative and antiangiogenic effects , and associated with active sle disease , and positivity for some autoantibodies ) could lead to endothelial dysfunction through the promotion of a reduction in the number of endothelial progenitor cells ( epcs , responsible for the neovascularization in sites of endothelial injury ) , thus contributing to the increased cv risk observed in sle . in that way , a recent study by denny and coworkers showed that sle patients displayed not only significant decreases in the number of circulating epcs , but also significant impairments in the capacity of epcs / cacs circulating angiogenic cells to differentiate into mature ecs and synthesize adequate levels of proangiogenic molecules vascular endothelial growth factor ( vegf ) and hepatic growth factor ( hgf ) . moreover , that study showed that lupus epcs / cacs had increased ifn expression . by contributing to endothelial disjunction / damage and inducing proinflammatory responses within the atherosclerotic plaque , the role of the type ii interferon ( ifn)whose expression is significantly increased in peripheral blood mononuclear cells ( pbmcs ) of sle patients in the progression of atherosclerosis has been well debated due to evidence conveying both pro- and antiatherogenic actions of the cytokine . since ifn , known to be a proinflammatory cytokine , can also display antiinflammatory properties , it is likely that it acts in both ways in at . these conflicting actions may well be gene - specific and it is known that approximately a quarter of all genes within the transcriptome of the macrophage , a key immune cell involved in at , is sensitive to ifn . ifn has been shown to influence many features of atherosclerosis such as foam cell formation , the adaptive th1-specific immune response and plaque development . in the global context of at nevertheless , the precise role of this type of ifn in the development of at in sle patients remains to be analyzed . ifns are often profoundly dysregulated in sle , and both ifn and ifn have been shown to induce b lymphocyte stimulator ( blys ) expression . blys ( also known as the b cell - activating factor belonging to the tnf family , or baff ) was identified as a novel tnf family ligand , and has proven to be a key factor in the selection and survival of b cells [ 2326 ] . the blys protein is expressed by a wide variety of cell types , including monocytes , activated neutrophils , t cells and dcs [ 2729 ] . although standing levels of blys are constitutively generated , its expression and secretion can be potentiated by inflammatory cytokines , such as il-2 , tnf , and ifn [ 2831 ] . high levels of blys may relax b cell selection and contribute to autoantibody production , exacerbating the sle disease state . it has been hypothesised a potential cooperative action of blys and ifns in the aetiology of sle . since blys is not known to have direct or immediate proinflammatory activities , changes in serum blys levels are unlikely to trigger acute inflammatory reactions and disease manifestations . however , it is possible than an increase in disease activity may lag behind increases in circulating blys levels due to indirect or nevertheless , the possible association between the overexpression of blys and the development and/or progression or at or cvd in sle patients remains elusive . tnf has differential effects on monocytes , on b cells , on t cells , and on dendritic cells , as well as on the process of programmed cell death . tnf is a growth factor for b lymphocytes , and b lymphocytes are able to produce significant amounts of tnf in an autocrine loop . tnf may also exert a significant influence on b cells by its capacity to induce il-6 . moreover , tnf stimulation leads to increased production of ifn , a cytokine with a clear - cut pathological role in sle , as previously described . tnf also constitutes an activating cytokine and a maturation factor of dendritic cells , which are essential in immune regulation and have also been implicated in autoimmunity in general , and in sle in particular . in addition , the elevated circulating levels of tnf found in sle patients have been found to be associated with high triglyceride and low hdl levels . moreover , in a recent study by rho and coworkers it was established a significant association between tnf expression levels and the severity of coronary calcium scores in sle patients . yet , that data should be further confirmed in a new cohort of patients , as a previous study by roman et al . found no association among tnf , il6 , or cd40l and the presence of carotid plaque in sle . nevertheless , because of its wide involvement in the activity of monocytes , dendritic cells , and lymphocytes as well as in the expression of other inflammatory cytokines involved in at development , tnf may be considered a major factor in sle - related cvd , acting both by contributing to hypertriglyceridaemia and by promoting atherosclerosis - related inflammation . interleukin-6 ( il-6 ) is a pleiotropic cytokine with a wide range of biological activities that plays an important role in immune regulation and inflammation . furthermore an association between il-6 and lupus was demonstrated in murine models of sle and blocking il-6 improved lupus in all models tested . il-6 is one of the most important b cell stimulating factors that induces the differentiation of t cells into effectors cells . immunoglobulin and antidna antibody production in vitro by b cells from lupus patients has been demonstrated to be promoted by il-6 and inhibited by antibodies against il-6 or the il-6 receptor . il-6 is involved in the recruitment of inflammatory cells and lipid homeostasis and is associated with increased cardiovascular mortality and prognosis in the general population . moreover , il-6 drives c - reactive protein ( crp ) production , which itself plays multiple roles , influencing key promoters of at ; moreover , it appears as an independent predictor of coronary events . however , the role of il-6 in the pathogenesis of sle - related at is also controversial . some authors found elevated il-6 levels only in cases with increased crp , concluding that it is part of the acute phase response . others defend the idea that the relationship between il-6 concentrations and the burden of at in sle patients represents more than an epiphenomenon , and that measurement of il-6 provides supplementary information in this cohort of sle patients . il-17 is a pro - inflammatory cytokine which participates in the defence against certain pathogens , primarily extra - cellular bacteria and fungi . il-17 is produced by several cell subsets including cd4 + t cells , cd8 + t cells , nk cells and neutrophils . in addition to its proinflammatory capacity , il-17 exerts its effects through the recruitment of monocytes and neutrophils by increasing the local production of chemokines ( il-8 , monocyte chemoattractant protein-1 , growth - related oncogene protein - alpha ) [ 4448 ] , the facilitation of t cell infiltration and activation by stimulating the expression of intercellular adhesion molecule-1 as well as the amplification of the immune response by inducing the production of il-6 , prostaglandin e2 , granulocyte - macrophage colony - stimulating factor and granulocyte colony - stimulating factor [ 50 , 51 ] . additionally , il-17 synergizes with other cytokines , in particular with il-1 , tnf , and ifn [ 5255 ] . th17 cells have been implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and multiple sclerosis , and recent evidence suggested that il-17-mediated inflammation might play a role in the pathogenesis of sle . also abnormally high levels of il-17 and il-23 have been reported in human sle sera , and more recently it has been provided evidence that il-17 production by t cells is increased in sle patients . that study further described that double negative ( c4-cd8- ) t cells , which are expanded in the peripheral blood of patients with sle , represent major producers of il-17 , and that they undergo a vigorous proliferative response following stimulation . a very recent study has demonstrated a concomitant presence of il-17 and ifn in patients and clinical specimens of coronary atherosclerosis , the presence of il-17/ifn dual - producing t cells within coronary plaques , and a synergistic effect of il-17 and ifn on elicitation of proinflammatory cytokine and chemokine production by cultured human vsmc . macrophage migration inhibitory factor ( mif ) has emerged as a potential link between sle and atherosclerosis development [ 10 , 62 ] . increased serum levels of mif have been detected in sle patients compared with healthy control individual . it can activate t cells , promote angiogenesis and induce proliferation of cells , while inhibiting p53 expression and apoptosis of the same cells [ 62 , 63 ] . mif can be induced by oxldl , which is an initiating factor in atherogenesis , and so expression of mif early on may enhance pro - inflammatory responses and lesion progression . the interaction between cd40 and cd40l is also an integral part of the inflammatory pathway in the vascular system . cd40 ligation on cells of the vascular wall promotes mononuclear cells recruitment and contributes to thrombosis in the setting of atherosclerosis . the co - stimulatory molecule cd40 ligand ( cd40l , also called scd154 ) is a member of the tnf family and participates in b cell differentiation and proliferation as well as in antibody isotype switching . the binding of cd40l to its receptor , cd40 , is thought to also be involved in atherogenesis and atherosclerotic plaque rupture [ 6769 ] . some reports indicated elevated serum concentrations of cd40l in patients with sle compared to matched control subjects [ 70 , 71 ] . cd40l has been found to be over expressed in t cells of patients with sle , and elevated concentrations of cd40 and cd40l have been found in atherosclerotic plaques in sle patients . an important outcome derived from the studies reported on this area is that only for a few cytokines there is sufficient consistent data allowing classifying them as typically proatherogenic ( il6 , il17,ifn , tnf , baff , mif , etc ) or antiatherogenic ( il-10 ) , and that some cytokines ( ifn , tnf , il4 , il-6 ) can exert pro- or antiatherogenic effects depending on the disease status . this knowledge can be used for improved early detection , prevention and treatment of atherosclerosis in sle . several genomic studies have been developed on sle pbmcs [ 7379 ] ( table 1 ) . genomic studies have consistently given strong support to the involvement of a dysregulation of ifn - dependent pathways in the pathogenesis of sle [ 80 , 81 ] . analysis of sle - upregulated genes showed a predominance of genes known to be upregulated in response to ifn . in some cases , expression results obtained using dna microarrays were also confirmed by independent methods such as quantitative real - time reverse transcription pcr [ 79 , 82 ] . additional changes that may be of great importance in determining the high thrombotic risk of sle patients include the upregulation of other gene families such as tnf and tnf receptor , chemokines and chemokine receptors , cell surface activation antigens , fc receptors , metalloproteinases , and defensins . interestingly , many of the expression changes observed in pbmcs isolated from sle patients were reproduced in healthy pbmcs cultured with ifn . the lack of detection of significant ifn transcripts in sle patient 's pbmcs supported that this cytokine may be mainly produced by plasmacytoid dendritic cells located in the patient 's tissues . recent searches for lupus genes through candidate single nucleotide polymorphism ( snp ) association scans , have further demonstrated that sle is a disease with complex genetic inheritance and no single causative gene . these studies have also given more proofs of the relationship between genetic profiles and development of at and cvd in sle patients . among them , polymorphisms in the region of the tnfaip3 gene were recently linked to sle . tnfaip3 encodes the deubiquitinating enzyme a20 , and endogenous inhibitor of the nuclear factor - kappab ( nfb ) pathway . nfb is a transcription factor that is activated by tnf or il-1/tlr signalling pathways , which induces transcription of proinflammatory genes . in at , nfb is activated at sites of the arterial wall that are prone to lesion development . snps in the tnfaip3 gene region may cause reduced expression or reduced activity of a20 , therefore contributing to an uncontrolled inflammatory response and autoimmunity and potentially accelerated at in these patients . the proteomic analysis of plasma samples from sle patients has allowed an important observation in order to understand the higher susceptibility of sle patients to suffer cv disorders . have studied by 2-de plasma samples from sle patients and healthy controls of initially unknown haptoglobin ( hp ) phenotype , and tryptic digests of the excised hpa polypeptide chain spots were analyzed by maldi - tof / ms there were remarkable interindividual differences in the hp patterns of sle patients compared with those of healthy controls . thus , hpa1f protein was only present in one of the patients studied , whereas the hpa2 isoform was detected in all but one sle patients studied , resulting in an hp2 allele frequency significantly higher than that in healthy controls . hp functions as an antioxidant and an essential endothelial protector by binding to free haemoglobin , avoiding oxidative stress . both the hemoglobin - binding and the antioxidant capacity of hpa1 is higher compared with that of hpa2 and hp genotype plays a critical role in the oxidative and inflammatory response to intraplaque haemorrhage . moreover , hp genotype modulates the balance of inflammatory ( th1 ) and antiinflammatory ( th2 ) cytokines produced by macrophages exposed to free haemoglobin , which may have implications in understanding interindividual differences in the inflammatory response to haemorrhage . moreover , large - artery elasticity index and small - artery elasticity index were significantly lower and systemic vascular resistance was higher in homozygotes for the 2 allele ( hp 2 - 2 ) compared with patients with hp 2 - 1 or hp 1 - 1 phenotypes . the knowledge of the hp phenotypes and their pmf by 2-de and ms in sle patients can help predicting or preventing cv disorders and determining a more precise prognosis and better treatment . there are still scarce data in that area , and studies performed are very heterogeneous . however , fundamental pathobiological mechanisms in atherogenesis development , and their association to autoimmune - mediated induction of cytokine expression have been identified in sle . nevertheless , genomic and proteomic areas are in continuous development and new information on genetic factors and gene and protein expression patterns in a near future are warranted . the pharmacological management of sle is challenging , owing to its unpredictable clinical course , the variable organ system involvement and the lack of clear understanding of disease pathogenesis . traditional management of sle has included the use of nonsteroidal antiinflammatory drugs , antimalarials , glucocorticoids , and immunosuppressive drugs such as azathioprine , methotrexate , cyclosporine a , cyclophosphamide , and mycophenolate mofetil [ 9395 ] . although many of these therapies have shown great efficacy , they are often associated with adverse effects . the development of safer therapies for sle has led to recent emphasis on targeting selected pathways that can be important in the inflammatory response in sle . in this context , a better understanding of lupus pathogenesis has led to the development of biological agents that are directed at biomarkers including , inhibitors of cytokines ( e.g. , tnf or il-10 ) , b - cell directed therapies , statins , and so forth . in sle , the loss of self - tolerance leads to persistence and activation of auto reactive b cells and t cells with the consecutive abnormal secretion of cytokines and production of autoantibodies , which are thus interesting target candidates for the development of novel sle therapies . several cytokines have been identified as major targets in sle , leading to the development of numerous monoclonal antibodies , some of them currently used in therapy or under clinical evaluation . a therapeutic option in patients with sle is inhibition of ifn. three general approaches could be used to target ifn : ( 1 ) reduce extra - cellular levels of ifn ; ( 2 ) reduce signalling from the ifn receptor ; ( 3 ) reduce the activity of the mediators of ifn activity . each approach has been shown to display pros and cons and implications for efficacy and safety needs to be weighed . nevertheless , recent data from a phase i clinical trial suggests that administration of neutralizing monoclonal antibodies against ifn can ameliorate disease activity . the foregoing evidence clearly implicates tnf in the accelerated atherogenesis and other cardiovascular events associated with autoimmune diseases such as rheumatoid arthritis ( ra ) and sle . several tnf antagonists are currently licensed for the treatment of ra , including infliximab , adalimumab ( a fully humanized monoclonal antibody ) and etanercept ( a fusion protein of human soluble tnf receptor and the fc component of human igg ) . all of these biologic agents have been shown to be effective in controlling disease activity , as well as reducing systemic inflammation [ 98100 ] , surrogate vascular and metabolic markers [ 101 , 102 ] , and cvd events . limited studies have evaluated the effect of antitnf therapy in sle , and are discussed in a recent revision by karim et al . . the study by aringer et al . looked at six patients with active sle treated with infliximab plus azathioprine or methotrexate . disease activity declined during therapy despite the fact that levels of anti - dsdna antibodies and cardiolipin increased in four out of six patients . there were associated infections including urinary tract infection in three patients , one of whom also had escherichia coli septicaemia , and a prolonged suspected viral fever in another patient . in the follow - up study aringer et al . also showed an increase in antibody detection to dna , histone , chromatin and igm cardiolipin , despite an overall reduction in disease activity . tnf antagonist therapy has also been associated with a lupus - like syndrome ; most of these case reports occurred in patients receiving either etanercept or infliximab . a small open trial used antiil-10 monoclonal antibodies in six patients with active sle . ten patients received intravenous il-10 monoclonal antibody 20 mg for 21 days and were followed for 6 months . the sledai score decreased significantly by day 21 , and was maintained at 6 months . another approach was recently developed , based on active immunotherapy , which consists of inducing antibodies able to neutralize the interaction of the self - cytokine to its receptor . in that way , in a model of ra , it was demonstrated that vaccination with a biologically inactive but immunogenic human tnf derivative ( keyhole limped hemocyanin - human tnf heterocomplex ) , led to the production of high titters of antibodies that neutralize human tnf bioactivity . other novel cytokines that might have a role , as implicated by vascular epidemiologic and pathophysiologic studies , could also be tested in the near future in the sle context , including il17 or il23 , which are being investigated in clinical trials for managing of ra . despite the positive results found in these studies with anticytokine therapies , numerous side effects as cytokine network dysregulation is highly complex in sle , further investigations are needed to evaluate whether this strategy may be advantageous in sle in the future . b - cell directed therapies are promising treatments for sle patients that do not respond to the first - line therapies . rituximab is a chimeric anticd20 monoclonal antibody which depletes b cells from the peripheral circulation , but allows regeneration from stem cells . cd20 is expressed at various stages of b - cell development from pre - b cells up to mature b cells , but not on plasma cells and hematopoietic stem cells . rituximab causes lysis of b cells mediated by complement and by fc - receptor - bearing cytotoxic cells , and also causes apoptosis . it is possible that rituximab has additional effects on the immune system [ 110112 ] . several groups have published uncontrolled studies of rituximab treatment in sle with positive results [ 113119 ] . adverse events most frequently were infections and a risk of relapse significantly elevated . a very recent study has investigated the changes in the lipid profile of sle patients after b - cell depletion therapy . they reported that rituximab acted positively on known risk factors for premature at such as hdl cholesterol , the total cholesterol / hdl ratio and tissue factor ( tf ) levels . this improvement in the lipid abnormalities coincided with decreased disease activity as measured by the bilag index and a fall in the antidsdna antibody levels . since rituximab has not known direct effects on lipid metabolism , it seems likely that the positive effects in the lipid profile of that set of patients is indirectly mediated through a reduction in the inflammatory activity of the disease . larger prospective studies should evaluate if this favourable effect contributes to a lower incidence of cv events . several studies have demonstrated increased levels of hypomethylated cpg - containing dna in sera from patients with sle , which may contribute to the initiation and/or perpetuation of the disease . thus , recent therapies have been focused on the use of synthetic oligodeoxynucleotides ( odn ) capable of neutralizing or inhibiting immune responses by inhibiting the activity elicited by this altered dna . among them , phosphorothioate odn mimic the immunosuppressive activity of the repetitive ttaggg motifs present in mammalian telomeres . these ttaggg multimers block the production of pro - inflammatory and t helper type 1 cytokines elicited when immune cells are activated by a wide variety of toll - like receptor ligands ( tlrs ) , polyclonal activators , and antigens . the suppressive activity of such odn has been attributed to their interference with the phosphorylation of signal transducer and activator of transcription 1 ( stat1 ) and stat4 , thereby blocking the inflammation mediated by stat - associated signalling cascades . these findings suggest that ttaggg multimers may find broad use in the treatment of diseases characterized by over - exuberant / persistent immune activation . intravenous immunoglobulin ( ivigs ) therapy has been also used for the treatment of sle . various mechanisms of ivigs have been shown to play a role in the modulation of sle : ivigs suppresses the expansion of autoreactive b lymphocytes through signalling the fcriib , idiotype - mediated inhibition of b cell receptors and neutralisation of cytokines such as the b cell survival factors ( b cell activation factor b cell activating factor belonging to the tnf family ( blys ) and a proliferation - inducing ligand ( april)- ) . in case reports and in open trials , high - dose ivigs ( 2 g / kg over a 5-day period ) has consistently been shown to be a beneficial and safe adjunct therapeutic agent for over 20 manifestations in patients with sle . the use of ivigs seems to be particularly successful in those patients with active disease and concomitant sepsis [ mj cuadrado , personal communication ] . statins may also play an important role in the treatment of sle patients regarding the prevention of cardiovascular disease as well as the immunomodulation over the chronic inflammatory activity of the disease . the pleiotropic properties of statins , in addition to their effects on the suppression of cholesterol synthesis , include direct influence on endothelium , plaque formation and thromboxan synthesis as well as direct and indirect immunomodulatory activities . statins have further demonstrated their antithrombotic / antiinflamatory effects on aps patients . in a preliminary study , our group evaluated the effects of fluvastatin treatment on monocyte expression and regulation of the main proteins involved in the characteristic procoagulant / proinflammatory state of aps patients . twenty patients with aps and previous history of thrombosis received fluvastatin ( 20 mg / day ) for one month . after one month of fluvastatin treatment , monocytes from aps patients showed a significant inhibition of tf , protease - activated receptors ( par)-1 and par-2 , vegf and flt1 expression . proteomic analysis showed significant changes in the protein patterns of monocytes from aps after fluvastatin administration , particularly on proteins involved in thrombotic development : inhibiting annexin ii and rhoa expression and increasing that of protein disulphide isomerase . the data of this study supported that fluvastatin have multiple profound effects in monocyte activity , which are directly related to thrombosis prevention in aps patients . a recent study by ferreira et al . demonstrated a surprisingly reduction of sle activity measured by sledai scale after atorvastatin therapy in addition to the improvement of the endothelial - dependent vasodilatation in sle patients after a 8 weeks controlled trial . in support for this observation , kotyla et al . observed similar reduction of sledai in the group of female patients treated with another statin : simvastatin . reduction of sledai was accompanied by a prominent suppression of tnf concentration in sera of patients treated . that phenomenon was observed just after 4 weeks of treatment with simvastatin at a dose of 20 mg . they also reported improvement of endothelial function , leading to the thesis that restoration of endothelial function was not restricted to the single compound ( atorvastatin ) but may be recognized as a class of drug effect . since tnf is believed to mediate endothelial damage , that authors also speculated that suppression of tnf levels after statin therapy might be one mechanism via which restoration of endothelial function occurs . because contradictory results were obtained in nzb / w mice that were given atorvastatin , either orally or intraperitoneally , and no significant effects of fluvastatin on cardiac events in renal transplant recipients with sle has been observed in a very recent study , the preliminary positive findings must be confirmed by multicentre and long - term studies to define whether statin treatment in sle patients is indeed associated with a relevant reduction of cardiovascular morbidity and mortality as well as with an amelioration of the inflammatory status and if this drug category should be broadly indicated for sle patients . in sum , cytokines identified as major players in the path physiology of sle , which have been selected as targets for the development of novel therapies ( including ifns , tnf or th1 cytokines ) , have also proved to influence the development of at and cvd in that autoimmune disease . therefore , therapies directed to reduce or eliminate their expression would give a double benefit for sle patients : the control of the disease , and the prevention of at and cvd development . in spite of the significant and continuous advances performed in the discovery of the cellular and molecular mechanisms responsible for sle - related at , relevant questions remain still unanswered : what are the direct links between chronic inflammation and the initiation and progression of vascular stiffening and at ? what are the safest and most effective pharmacologic approaches to limit inflammation even in the absence of disease - related activity ? cytokine upregulation constitute a major mechanism that links increased atheroma formation in patients with sle . however , because of the cytokines ' function overlap , their redundant activity , and their synergistic or antagonistic activity , ( which may depend on the cell type , the local environment , the presence of synergistic or competing factors , and tissue responsiveness to each cytokine ) , their pathophysiological role may be difficult to assess . a plethora of new treatment approaches using antibodies or small molecule inhibitors specifically targeting cytokines , cellular receptors , or signalling mechanisms has emerged in recent years . all of them have shown limited effectiveness , and , in a similar way to what happened with the traditional treatments administered to sle patients , showed a number of adverse effects . nevertheless , the knowledge continues increasing , and there are two lessons to learn from the experience to date : ( 1 ) that no single treatment is effective and safe enough ; and ( 2 ) that every single patient shows a cellular and molecular profile that physicians should seriously take into consideration . anti - inflammatory therapeutic strategies in the setting of sle - related at need to consider a network of interactive cytokines rather than individual proinflammatory factors in isolation . genetic profiles may also be relevant to the severity of the disease and the response to each specific drug . probably the best therapeutic strategy would imply a combination of immunosuppressant and antiinflammatory drugs , which should further be administered taking into account the cellular and molecular profile specific of each sle patient .

systemic lupus erythematosus ( sle ) , a chronic multisystem autoimmune disease with a broad range of clinical manifestations , is associated with accelerated atherosclerosis ( at ) and increased risk of cardiovascular complications . relevant factors directly influencing the development of at comprise immune complex generation , complement activation , and changes in the production and activity of a complex network of cytokines , including type i and ii interferons , b lymphocyte stimulator ( blys ) , tnf , il-6 , il-17 and migration macrophage inhibitor ( mif ) . autoantibodies , also responsible for cytokine expression and activation , play a supplementary key role in the development of at . genomic and proteomic studies have contributed to the discovery of genes and proteins involved in at , including some that may be suitable to be used as biomarkers . all that data has allowed the development of new drugs , most of them evaluated in clinical trials : inhibitors of ifn and tnf , b cell directed therapies , synthetic oligodeoxynucleotides , intravenous immunoglobulin , or statins . the focus of the present paper is to summarize recent evidence showing the role of cytokines in the development of at in sle and the rationale , and safety concerns , in the use of combined therapy to prevent at and cardiovascular disease .

1. Introduction 2. Cytokines as Immune Mediators Involved in Atherosclerosis and CVD Development 3. Cytokines Involved in the Pathogenesis of Atherosclerosis in SLE 4. Genomics and Proteomics Biomarkers for SLE Atherosclerosis and Cytokine Involvement 5. SLE Treatment and Its Influence on Cytokine Expression and Atherosclerosis Development 6. Conclusions

sle is a chronic multisystem autoimmune disease with a broad range of clinical manifestations , including photosensitive skin rashes , discoid lesions , arthritis / arthralgia , nephritis , cardiac and pulmonary disease , and cns disorders . the disease pathogenesis is attributed to circulating antinuclear autoantibodies against a variety of nuclear antigens ( including dsdna , the ribonucleoprotein ( rnp ) complex ro , the rna - binding protein la , rnps , the rna molecule / protein complex sm , the c1 complement component subunit c1q , and phospholipids ) and the dysfunction of t and b lymphocytes and dendritic cells . although the pathogenesis is believed to lie in the dysregulation of the immune system , the involvement of various organ systems often leads to secondary morbidities resulting from renal failure , hypertension , or cns disorders , and more recently it is becoming increasingly clear that accelerated atherosclerosis associated with sle may contribute to premature mortality . atherosclerosis ( at ) is a chronic inflammatory disease of the arteries associated with various risk factors that promote lipid abnormalities ( i.e. at is enhanced in autoimmune diseases ; noninvasive investigations show increases in intima - media thickness , carotid plaque , and coronary artery calcifications in patients with antiphospholipid syndrome ( aps ) , systemic lupus erythematosus ( sle ) , and rheumatoid arthritis ( ra ) compared to controls . actually , some autoantibodies , including antioxidized low density lipoproteins ( antioxldl ) , anti-2-glycoprotein 1 ( anti2gpi ) , antiheat shock proteins 60/65 ( antihsp60/65 ) , and antioxldl/2gpi , have been shown to be associated to the pathogenesis of at [ 6 , 7 ] . these factors can directly influence the development of at through a variety of mechanisms such as immune complex generation , complement activation , alteration of the oxidant - antioxidant balance locally within the vessel wall , and changes in the production and activity of a complex network of cytokines [ 810 ] ( figure 1 ) . characterization of the molecular and cellular basis of signalling abnormalities within the immune system that lead to auto reactivity and inflammation and their relationship to early atherosclerosis and cardiovascular disease ( cvd ) development remain critical for understanding the pathogenesis of sle . vascular injury is associated with increased expression of adhesion molecules by ecs and recruitment of inflammatory cells , growth factors , and cytokines , with consequent effects on ecs , vsmcs , and ecm . many cytokines , such as interleukin ( il)-1 , il-6 , il-10 , interferon ifn , and tnf are expressed highly in atherosclerotic regions and exhibit pro- and antiatherogenic actions [ 1315 ] . innate cytokines such as il-1 or tnf may activate endothelial cells ( ecs ) , vascular smooth muscle cells ( vsmcs ) , monocytes / macrophages , lymphocytes ( t , b , nk ) , dendritic cells , and mast cells . these vascular cells can actively contribute to the inflammatory cytokine - dependent response in the vessel wall by production of cytokines or eliciting responses to cytokines , or can be involved in cytokine - mediated interaction with invading cells such as monocytes , t - cells , or mast cells . some of them were shown to play a pivotal physiopathological role in certain t - cell , b - cell or antigen presenting cell dysfunctions characteristic of the disease , while others are more likely to be innocent bystanders . several cytokines with relevance to cvd has been proven to be related to the pathogenesis of at in sle . in the following paragraphs we highlight some of the most likely plasmacytoid dendritic cells ( pdc ) activated by immune complexes containing nucleic acids secrete type i ifn ( ifn and ifn ) in sle . type i ifn causes differentiation of monocytes to myeloid - derived dendritic cell ( mdc ) and activation of autoreactive t and b cells . patients with sle have an increased expression of type i ifn - regulated genes because of a continuous production of ifn. recent reports have demonstrated that elevated levels of type i ifns ( cytokines with potent antiproliferative and antiangiogenic effects , and associated with active sle disease , and positivity for some autoantibodies ) could lead to endothelial dysfunction through the promotion of a reduction in the number of endothelial progenitor cells ( epcs , responsible for the neovascularization in sites of endothelial injury ) , thus contributing to the increased cv risk observed in sle . in that way , a recent study by denny and coworkers showed that sle patients displayed not only significant decreases in the number of circulating epcs , but also significant impairments in the capacity of epcs / cacs circulating angiogenic cells to differentiate into mature ecs and synthesize adequate levels of proangiogenic molecules vascular endothelial growth factor ( vegf ) and hepatic growth factor ( hgf ) . by contributing to endothelial disjunction / damage and inducing proinflammatory responses within the atherosclerotic plaque , the role of the type ii interferon ( ifn)whose expression is significantly increased in peripheral blood mononuclear cells ( pbmcs ) of sle patients in the progression of atherosclerosis has been well debated due to evidence conveying both pro- and antiatherogenic actions of the cytokine . these conflicting actions may well be gene - specific and it is known that approximately a quarter of all genes within the transcriptome of the macrophage , a key immune cell involved in at , is sensitive to ifn . in the global context of at nevertheless , the precise role of this type of ifn in the development of at in sle patients remains to be analyzed . ifns are often profoundly dysregulated in sle , and both ifn and ifn have been shown to induce b lymphocyte stimulator ( blys ) expression . blys ( also known as the b cell - activating factor belonging to the tnf family , or baff ) was identified as a novel tnf family ligand , and has proven to be a key factor in the selection and survival of b cells [ 2326 ] . although standing levels of blys are constitutively generated , its expression and secretion can be potentiated by inflammatory cytokines , such as il-2 , tnf , and ifn [ 2831 ] . however , it is possible than an increase in disease activity may lag behind increases in circulating blys levels due to indirect or nevertheless , the possible association between the overexpression of blys and the development and/or progression or at or cvd in sle patients remains elusive . moreover , tnf stimulation leads to increased production of ifn , a cytokine with a clear - cut pathological role in sle , as previously described . in addition , the elevated circulating levels of tnf found in sle patients have been found to be associated with high triglyceride and low hdl levels . moreover , in a recent study by rho and coworkers it was established a significant association between tnf expression levels and the severity of coronary calcium scores in sle patients . found no association among tnf , il6 , or cd40l and the presence of carotid plaque in sle . nevertheless , because of its wide involvement in the activity of monocytes , dendritic cells , and lymphocytes as well as in the expression of other inflammatory cytokines involved in at development , tnf may be considered a major factor in sle - related cvd , acting both by contributing to hypertriglyceridaemia and by promoting atherosclerosis - related inflammation . interleukin-6 ( il-6 ) is a pleiotropic cytokine with a wide range of biological activities that plays an important role in immune regulation and inflammation . il-6 is involved in the recruitment of inflammatory cells and lipid homeostasis and is associated with increased cardiovascular mortality and prognosis in the general population . however , the role of il-6 in the pathogenesis of sle - related at is also controversial . others defend the idea that the relationship between il-6 concentrations and the burden of at in sle patients represents more than an epiphenomenon , and that measurement of il-6 provides supplementary information in this cohort of sle patients . in addition to its proinflammatory capacity , il-17 exerts its effects through the recruitment of monocytes and neutrophils by increasing the local production of chemokines ( il-8 , monocyte chemoattractant protein-1 , growth - related oncogene protein - alpha ) [ 4448 ] , the facilitation of t cell infiltration and activation by stimulating the expression of intercellular adhesion molecule-1 as well as the amplification of the immune response by inducing the production of il-6 , prostaglandin e2 , granulocyte - macrophage colony - stimulating factor and granulocyte colony - stimulating factor [ 50 , 51 ] . additionally , il-17 synergizes with other cytokines , in particular with il-1 , tnf , and ifn [ 5255 ] . th17 cells have been implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and multiple sclerosis , and recent evidence suggested that il-17-mediated inflammation might play a role in the pathogenesis of sle . also abnormally high levels of il-17 and il-23 have been reported in human sle sera , and more recently it has been provided evidence that il-17 production by t cells is increased in sle patients . the co - stimulatory molecule cd40 ligand ( cd40l , also called scd154 ) is a member of the tnf family and participates in b cell differentiation and proliferation as well as in antibody isotype switching . cd40l has been found to be over expressed in t cells of patients with sle , and elevated concentrations of cd40 and cd40l have been found in atherosclerotic plaques in sle patients . an important outcome derived from the studies reported on this area is that only for a few cytokines there is sufficient consistent data allowing classifying them as typically proatherogenic ( il6 , il17,ifn , tnf , baff , mif , etc ) or antiatherogenic ( il-10 ) , and that some cytokines ( ifn , tnf , il4 , il-6 ) can exert pro- or antiatherogenic effects depending on the disease status . genomic studies have consistently given strong support to the involvement of a dysregulation of ifn - dependent pathways in the pathogenesis of sle [ 80 , 81 ] . additional changes that may be of great importance in determining the high thrombotic risk of sle patients include the upregulation of other gene families such as tnf and tnf receptor , chemokines and chemokine receptors , cell surface activation antigens , fc receptors , metalloproteinases , and defensins . these studies have also given more proofs of the relationship between genetic profiles and development of at and cvd in sle patients . in at , nfb is activated at sites of the arterial wall that are prone to lesion development . snps in the tnfaip3 gene region may cause reduced expression or reduced activity of a20 , therefore contributing to an uncontrolled inflammatory response and autoimmunity and potentially accelerated at in these patients . have studied by 2-de plasma samples from sle patients and healthy controls of initially unknown haptoglobin ( hp ) phenotype , and tryptic digests of the excised hpa polypeptide chain spots were analyzed by maldi - tof / ms there were remarkable interindividual differences in the hp patterns of sle patients compared with those of healthy controls . both the hemoglobin - binding and the antioxidant capacity of hpa1 is higher compared with that of hpa2 and hp genotype plays a critical role in the oxidative and inflammatory response to intraplaque haemorrhage . however , fundamental pathobiological mechanisms in atherogenesis development , and their association to autoimmune - mediated induction of cytokine expression have been identified in sle . traditional management of sle has included the use of nonsteroidal antiinflammatory drugs , antimalarials , glucocorticoids , and immunosuppressive drugs such as azathioprine , methotrexate , cyclosporine a , cyclophosphamide , and mycophenolate mofetil [ 9395 ] . the development of safer therapies for sle has led to recent emphasis on targeting selected pathways that can be important in the inflammatory response in sle . in this context , a better understanding of lupus pathogenesis has led to the development of biological agents that are directed at biomarkers including , inhibitors of cytokines ( e.g. , tnf or il-10 ) , b - cell directed therapies , statins , and so forth . in sle , the loss of self - tolerance leads to persistence and activation of auto reactive b cells and t cells with the consecutive abnormal secretion of cytokines and production of autoantibodies , which are thus interesting target candidates for the development of novel sle therapies . several cytokines have been identified as major targets in sle , leading to the development of numerous monoclonal antibodies , some of them currently used in therapy or under clinical evaluation . three general approaches could be used to target ifn : ( 1 ) reduce extra - cellular levels of ifn ; ( 2 ) reduce signalling from the ifn receptor ; ( 3 ) reduce the activity of the mediators of ifn activity . the foregoing evidence clearly implicates tnf in the accelerated atherogenesis and other cardiovascular events associated with autoimmune diseases such as rheumatoid arthritis ( ra ) and sle . several tnf antagonists are currently licensed for the treatment of ra , including infliximab , adalimumab ( a fully humanized monoclonal antibody ) and etanercept ( a fusion protein of human soluble tnf receptor and the fc component of human igg ) . limited studies have evaluated the effect of antitnf therapy in sle , and are discussed in a recent revision by karim et al . tnf antagonist therapy has also been associated with a lupus - like syndrome ; most of these case reports occurred in patients receiving either etanercept or infliximab . in that way , in a model of ra , it was demonstrated that vaccination with a biologically inactive but immunogenic human tnf derivative ( keyhole limped hemocyanin - human tnf heterocomplex ) , led to the production of high titters of antibodies that neutralize human tnf bioactivity . other novel cytokines that might have a role , as implicated by vascular epidemiologic and pathophysiologic studies , could also be tested in the near future in the sle context , including il17 or il23 , which are being investigated in clinical trials for managing of ra . despite the positive results found in these studies with anticytokine therapies , numerous side effects as cytokine network dysregulation is highly complex in sle , further investigations are needed to evaluate whether this strategy may be advantageous in sle in the future . b - cell directed therapies are promising treatments for sle patients that do not respond to the first - line therapies . several studies have demonstrated increased levels of hypomethylated cpg - containing dna in sera from patients with sle , which may contribute to the initiation and/or perpetuation of the disease . thus , recent therapies have been focused on the use of synthetic oligodeoxynucleotides ( odn ) capable of neutralizing or inhibiting immune responses by inhibiting the activity elicited by this altered dna . these ttaggg multimers block the production of pro - inflammatory and t helper type 1 cytokines elicited when immune cells are activated by a wide variety of toll - like receptor ligands ( tlrs ) , polyclonal activators , and antigens . various mechanisms of ivigs have been shown to play a role in the modulation of sle : ivigs suppresses the expansion of autoreactive b lymphocytes through signalling the fcriib , idiotype - mediated inhibition of b cell receptors and neutralisation of cytokines such as the b cell survival factors ( b cell activation factor b cell activating factor belonging to the tnf family ( blys ) and a proliferation - inducing ligand ( april)- ) . the use of ivigs seems to be particularly successful in those patients with active disease and concomitant sepsis [ mj cuadrado , personal communication ] . statins may also play an important role in the treatment of sle patients regarding the prevention of cardiovascular disease as well as the immunomodulation over the chronic inflammatory activity of the disease . in a preliminary study , our group evaluated the effects of fluvastatin treatment on monocyte expression and regulation of the main proteins involved in the characteristic procoagulant / proinflammatory state of aps patients . proteomic analysis showed significant changes in the protein patterns of monocytes from aps after fluvastatin administration , particularly on proteins involved in thrombotic development : inhibiting annexin ii and rhoa expression and increasing that of protein disulphide isomerase . demonstrated a surprisingly reduction of sle activity measured by sledai scale after atorvastatin therapy in addition to the improvement of the endothelial - dependent vasodilatation in sle patients after a 8 weeks controlled trial . because contradictory results were obtained in nzb / w mice that were given atorvastatin , either orally or intraperitoneally , and no significant effects of fluvastatin on cardiac events in renal transplant recipients with sle has been observed in a very recent study , the preliminary positive findings must be confirmed by multicentre and long - term studies to define whether statin treatment in sle patients is indeed associated with a relevant reduction of cardiovascular morbidity and mortality as well as with an amelioration of the inflammatory status and if this drug category should be broadly indicated for sle patients . in sum , cytokines identified as major players in the path physiology of sle , which have been selected as targets for the development of novel therapies ( including ifns , tnf or th1 cytokines ) , have also proved to influence the development of at and cvd in that autoimmune disease . therefore , therapies directed to reduce or eliminate their expression would give a double benefit for sle patients : the control of the disease , and the prevention of at and cvd development . in spite of the significant and continuous advances performed in the discovery of the cellular and molecular mechanisms responsible for sle - related at , relevant questions remain still unanswered : what are the direct links between chronic inflammation and the initiation and progression of vascular stiffening and at ? however , because of the cytokines ' function overlap , their redundant activity , and their synergistic or antagonistic activity , ( which may depend on the cell type , the local environment , the presence of synergistic or competing factors , and tissue responsiveness to each cytokine ) , their pathophysiological role may be difficult to assess . a plethora of new treatment approaches using antibodies or small molecule inhibitors specifically targeting cytokines , cellular receptors , or signalling mechanisms has emerged in recent years . all of them have shown limited effectiveness , and , in a similar way to what happened with the traditional treatments administered to sle patients , showed a number of adverse effects . genetic profiles may also be relevant to the severity of the disease and the response to each specific drug .

serijska merenja natriuretskih peptida kod pacijenata sa akutnim koronarnim sindromom su pokazala da su njihove vrednosti u kasnijoj , hroninoj fazi bolji prediktor prognoze i pouzdaniji indikator funkcije leve komore nego one u akutnoj fazi . stoga je cilj ovog rada da se ustanove vrednosti nt - probnp - a 6 meseci nakon akutnog infarkta miokarda ( aim ) , njihova udruenost sa tradicionalnim faktorima rizika , karakteristikama intrahospitalnog i ranog postinfarktnog toka , kao i njegov prognostiki znaaj i optimalne prediktivne vrednosti , tokom narednog jednogodinjeg praenja . . koncentracije nt - probnp - a u litijum - heparinskoj plazmi su odreivane korienjem jedno - stepenog enzimskog imunoeseja , baziranog na sendvi principu , na ureaju dimension rxl clinical chemistry system ( dade behring - siemens ) . je 521 ( 3351095 ) pg / ml . najvie vrednosti nt - probnp - a bile su znaajno pozitivno udruene sa sranim dogaajima tokom prvih 6 meseci nakon aim ; nasuprot tome , negativna udruenost sa reperfuzionom terapijom u leenju indeks infarkta potvrdila je njen dugotrajni povoljan efekat . je nezavisan prognostiki znaaj nt - probnp - a u predvianju novonastale srane insuficijencije ( si ) ( p=0,014 ) , kao i novih koronarnih dogaaja ( nkd ) ( p=0,035 ) . analizom roc krive dobijene su optimalne prediktivne vrednosti nt - probnp - a : 800 pg / ml za si i 516 pg / ml za nkd . vrednosti nt - probnp - a 6 meseci nakon aim su znaajno udruene sa karakteristikama infarktnog i ranog postinfarktnog toka i nezavisno predviaju najvanije nove srane dogaaje tokom narednih godinu dana . during the last decade , b type - natriuretic peptides have moved on from bench to bedside very quickly . originally , they were introduced in clinical practice as a diagnostic tool for heart failure ( hf ) ( 1 ) . later , their independent prognostic value was also shown , especially concerning mortality and heart failure , in patients with stable and unstable coronary artery disease ( cad ) ( 25 ) . on the other hand , data about acute coronary events prediction are still conflicting ; in contrast to the peace trial ( 2 ) , in which neither bnp nor nt - probnp significantly increased the risk of myocardial infarction ( mi ) , the heart and soul study found an independent association of both markers with the individual outcomes of heart failure , myocardial infarction and cardiovascular death ( 6 ) . also , nt - probnp was found to be a useful biomarker for distinguishing patients with long - standing hypertension who are at risk of heart failure , allowing optimization and proper treatment of these patients ( 7 ) . patients with previous myocardial infarction represent a heterogenous group , whose prognosis differs significantly . since traditional risk factors have less prognostic value in this secondary prevention population , they are important candidates for neurohumoral testing . serial analyses of nt - probnp in patients with non - st segment elevation acute coronary syndromes ( frisc - ii substudy ) showed that levels measured during a chronic , relatively stable phase are a better predictor of mortality than those measured during an acute , unstable phase ( 8) . also , assessment of nt - probnp level 6 months after st - elevation mi was a better indicator of infarct size and left ventricular function measured by cardiac magnetic resonance than baseline ( admission ) nt - pro bnp values ( 9 ) . although previously thought to be equally effective for diagnostic and prognostic purposes ( 10 ) , recently published data from the heart and soul study found nt - probnp to be superior to bnp , when added to clinical risk factors , for net reclassification of the risk for major adverse cardiac events in patients with stable cad ( 6 ) . meta - analysis of nine prospective studies , which indicated strong association between the circulating concentration of nt - probnp and long - term prognosis of patients with stable cad , pointed out that although most of the included studies grouped the population according to the median or quartiles of nt - probnp , the specific nt - probnp , levels varied greatly among different studies , making it impossible to give a precise cut - point ( 11 ) . therefore , we designed this study to assess the association of nt - probnp measured 6 months after ami , with traditional risk factors , characteristics of inhospital and early postinfarction course , as well as its prognostic value and optimal cut - points in the next one - year follow - up . we enrolled prospectively 114 consecutive ambulatory patients , who were treated for ami six months ago in our coronary care unit . of them , 14 patients met exclusion criteria based on data from the literature about other potential sources of natriuretic peptides such as : age > 70 , renal failure , liver disease , known malignant disease , chronic systemic illness or trauma or surgery in the last 2 months . finally , 100 participants gave informed consent for study enrollment and underwent a baseline study appointment , which included a medical history interview , physical examination , and resting echocardiogram . fasting venous blood samples were drawn and edta plasma was frozen at 80 c until measurement . patients were followed - up for the next one year , for the occurrence of new cardiac events ; the relevant endpoints were new - onset hf and new coronary events ( nce ) , defined as a composite of myocardial reinfarction or unstable angina ( according to standard definitions ) ( 12 ) . new - onset heart failure was defined as a clinical syndrome involving at least 2 of the following : paroxysmal nocturnal dyspnoea , orthopnea , elevated jugular venous pressure , pulmonary rales and third heart sound . these clinical signs and symptoms had to be accompanied by intravenous diuretic or vasodilator therapy . the study protocol was approved by the ethical committee of school of medicine , university of belgrade and conformed to applicable institutional and national guidelines for research on human subjects , as well as to the declaration of helsinki . samples of edta anticoagulated venous blood were obtained in the morning and centrifuged at 2000 rpm . the nt - probnp concentrations in lithium - heparin plasma were determined using a one step enzyme immunoassay based on the sandwich principle on a dimension rxl clinical chemistry system ( dade behring - siemens ) with commercial reagents ( 13 ) . the interassay coefficient of variation was 3.1% at a concentration of 50 pg / ml ( n=60 ) , 2.5% at a concentration of 12000 pg / ml ( n=60 ) . fasting baseline glucose , creatinine , and serum lipids ( total cholesterol , hdl cholesterol and triglycerides ) were determined by use of routine methods of the respective study centers . two dimensional ultrasound resting echocardiography was performed using a commercially available imaging system ( vivid 4 , general electrics health care ) for each subject by an expert sonographer , blinded to patient data , at study entry . determination of systolic ejection fraction ( ef ) of the left ventricle was done by planimetry of the left ventricle ( biplane simpson method ) ( 14 ) . since the distribution of nt - probnp values was skewed , their original values were described as medians with interquartile range and were log - transformed to enable the application of parametric tests . normally distributed data were expressed as mean standard deviation ( sd ) ; categorical data were summarized as frequencies and percentages . since this investigation is a longitudinal study in which patients were followed for one year ( and therefore a control group to themselves ) , there was no healthy control group . logistic regression analysis was used to investigate the association of nt - probnp values ( as highest vs. other 3 quartiles ) with demographic and risk factors , as well as with in - hospital and early postinfarction course . cox proportional hazards regression model , adjusted for variables known to be important predictors of hf or nce was used for prognostic analysis , i.e. to examine the independent association of nt - probnp with new cardiac events during the one - year follow - up . receiver - operating characteristic ( roc ) analysis was used to estimate the overall prognostic accuracy of nt - probnp and to define its optimal predictive cut - off values . finally , kaplan - meier survival curves with log rank tests were used for comparisons of the occurence of new cardiac events in patient groups stratified according to optimal cut - points . we enrolled prospectively 114 consecutive ambulatory patients , who were treated for ami six months ago in our coronary care unit . of them , 14 patients met exclusion criteria based on data from the literature about other potential sources of natriuretic peptides such as : age > 70 , renal failure , liver disease , known malignant disease , chronic systemic illness or trauma or surgery in the last 2 months . finally , 100 participants gave informed consent for study enrollment and underwent a baseline study appointment , which included a medical history interview , physical examination , and resting echocardiogram . fasting venous blood samples were drawn and edta plasma was frozen at 80 c until measurement . patients were followed - up for the next one year , for the occurrence of new cardiac events ; the relevant endpoints were new - onset hf and new coronary events ( nce ) , defined as a composite of myocardial reinfarction or unstable angina ( according to standard definitions ) ( 12 ) . new - onset heart failure was defined as a clinical syndrome involving at least 2 of the following : paroxysmal nocturnal dyspnoea , orthopnea , elevated jugular venous pressure , pulmonary rales and third heart sound . these clinical signs and symptoms had to be accompanied by intravenous diuretic or vasodilator therapy . the study protocol was approved by the ethical committee of school of medicine , university of belgrade and conformed to applicable institutional and national guidelines for research on human subjects , as well as to the declaration of helsinki . samples of edta anticoagulated venous blood were obtained in the morning and centrifuged at 2000 rpm . the nt - probnp concentrations in lithium - heparin plasma were determined using a one step enzyme immunoassay based on the sandwich principle on a dimension rxl clinical chemistry system ( dade behring - siemens ) with commercial reagents ( 13 ) . the interassay coefficient of variation was 3.1% at a concentration of 50 pg / ml ( n=60 ) , 2.5% at a concentration of 12000 pg / ml ( n=60 ) . fasting baseline glucose , creatinine , and serum lipids ( total cholesterol , hdl cholesterol and triglycerides ) were determined by use of routine methods of the respective study centers . two dimensional ultrasound resting echocardiography was performed using a commercially available imaging system ( vivid 4 , general electrics health care ) for each subject by an expert sonographer , blinded to patient data , at study entry . determination of systolic ejection fraction ( ef ) of the left ventricle was done by planimetry of the left ventricle ( biplane simpson method ) ( 14 ) . since the distribution of nt - probnp values was skewed , their original values were described as medians with interquartile range and were log - transformed to enable the application of parametric tests . normally distributed data were expressed as mean standard deviation ( sd ) ; categorical data were summarized as frequencies and percentages . since this investigation is a longitudinal study in which patients were followed for one year ( and therefore a control group to themselves ) , there was no healthy control group . logistic regression analysis was used to investigate the association of nt - probnp values ( as highest vs. other 3 quartiles ) with demographic and risk factors , as well as with in - hospital and early postinfarction course . cox proportional hazards regression model , adjusted for variables known to be important predictors of hf or nce was used for prognostic analysis , i.e. to examine the independent association of nt - probnp with new cardiac events during the one - year follow - up . receiver - operating characteristic ( roc ) analysis was used to estimate the overall prognostic accuracy of nt - probnp and to define its optimal predictive cut - off values . finally , kaplan - meier survival curves with log rank tests were used for comparisons of the occurence of new cardiac events in patient groups stratified according to optimal cut - points . baseline characteristics of study population and association of nt - probnp with demographic and traditional risk factors , characteristics of in - hospital and early postinfarction course the final study group consisted of 100 ambulatory patients ( 81 men and 19 women , mean age 599 years ) who had survived an ami six months earlier . at least one risk factor was present in 88% of patients ; most of them had hypertension ( 68% ) and hyperlipidaemia ( 67% ) , followed by smoking ( 60% ) , family history ( 41% ) and diabetes ( 20% ) . st segment elevation at index event was present in 83% of patients and reperfusion therapy was given to 67% of patients . the localization of infarction was anterior in 42% of patients and 19% of patients had heart failure ( killip class > 1 ) during initial hospitalization . multivessel disease was found at coronary angiography in 41% of patients . during the first 6 months after ami ( early postinfarction course ) , 12% of patients had heart failure ( nyha > i ) , 8% had a new coronary event and 6% of patients were surgically revascularized ( all coronary events and procedures happened at least 2 months before study inclusion ) ; beta blockers were used by 75% and ace inhibitors by 79% of patients . median ( iqr ) level of nt - probnp was 521 ( 335,1095 ) pg / ml . factors significantly associated with the highest nt - probnp quartile among demographic and risk factors , as well as in - hospital and early postinfarction course are presented in table i. older age was independently associated with highest nt - probnp values , together with all cardiac events during the first 6 month after ami , and the strongest association was found , as expected , with actual heart failure . negative association was observed with reperfusion therapy for index ami ( during initial hospitalization ) . independent associations of the highest nt - probnp quartile with demographic and risk factors ( model 1 ) , in - hospital characteristics ( model 2 ) and early postinfarction ( 6 months ) course ( model 3 ) . stemi indicates st - elevation myocardial infarction ; nyha indicates new york heart association ( classification of heart failure ) . adjusted for sex , diabetes , hypertension , smoking , hyperlipidaemia and family history of cad . adjusted for infarct localization , peak creatine - kinase , killip class and multivessel disease . because of the known relationship of nt - probnp and heart failure , only patients without any signs and symptoms of heart failure , as well as without radiographic signs of hf , were included in the prognostic analysis ( 88 patients , mean age 579 , female 14 , male 74 , mean ef 488 ; median ( iqr ) nt - probnp 490 ( 296,950 ) pg / ml ) . during one - year follow - up ( 1114 months ) , 2 patients died ( 2.3% ) , 10 ( 11.3% ) patients had nce and 15 ( 17% ) had new - onset hf . multivariate cox regression analysis revealed the independent prognostic value of nt - probnp ( as log - transformed continuous variable ) for new - onset hf and nce prediction ( table ii ) . independent predictors of new coronary events ( nce ) and new - onset heart failure ( hf)*. adjusted for age , risk factors , reperfusion therapy and ef calculation of the areas under the roc curves , as a measure of overall prognostic accuracy , demonstrated that nt - probnp performed better for new - onset hf than for nce , although the accuracy was satisfactory for both events ( figure 1 ) . therefore , further analysis of the roc curve , in order to assess the optimal predictive cut - off values of nt - probnp , was done . the value with the highest sensitivity and specificity was 800 pg / ml for new - onset hf ( sensitivity 78% , specificity 79% , negative predictive value 92% ) and 516 pg / ml for nce ( sensitivity 75% , specificity 58% , negative predictive value 91% ) . receiver operating characteristic ( roc ) curves showing prognostic accuracy of ntprobnp for new - onset heart failure prediction ( a. ) and new coronary events prediction ( b. ) , together with the optimal cut - off values . when patients were stratified into two groups , according to optimized cut - off values , kaplan - meier analysis showed a highly significant difference in the rates of new - onset hf and a clear trend toward higher incidence of nce ( figure 2 ) . kaplan - meier survival curves without new - onset heart failure ( a. ) and new coronary events ( nce ) ( b. ) , according to optimal cut - off values of nt - probnp . because of the known relationship of nt - probnp and heart failure , only patients without any signs and symptoms of heart failure , as well as without radiographic signs of hf , were included in the prognostic analysis ( 88 patients , mean age 579 , female 14 , male 74 , mean ef 488 ; median ( iqr ) nt - probnp 490 ( 296,950 ) pg / ml ) . during one - year follow - up ( 1114 months ) , 2 patients died ( 2.3% ) , 10 ( 11.3% ) patients had nce and 15 ( 17% ) had new - onset hf . multivariate cox regression analysis revealed the independent prognostic value of nt - probnp ( as log - transformed continuous variable ) for new - onset hf and nce prediction ( table ii ) . independent predictors of new coronary events ( nce ) and new - onset heart failure ( hf)*. adjusted for age , risk factors , reperfusion therapy and ef calculation of the areas under the roc curves , as a measure of overall prognostic accuracy , demonstrated that nt - probnp performed better for new - onset hf than for nce , although the accuracy was satisfactory for both events ( figure 1 ) . therefore , further analysis of the roc curve , in order to assess the optimal predictive cut - off values of nt - probnp , was done . the value with the highest sensitivity and specificity was 800 pg / ml for new - onset hf ( sensitivity 78% , specificity 79% , negative predictive value 92% ) and 516 pg / ml for nce ( sensitivity 75% , specificity 58% , negative predictive value 91% ) . receiver operating characteristic ( roc ) curves showing prognostic accuracy of ntprobnp for new - onset heart failure prediction ( a. ) and new coronary events prediction ( b. ) , together with the optimal cut - off values . when patients were stratified into two groups , according to optimized cut - off values , kaplan - meier analysis showed a highly significant difference in the rates of new - onset hf and a clear trend toward higher incidence of nce ( figure 2 ) . kaplan - meier survival curves without new - onset heart failure ( a. ) and new coronary events ( nce ) ( b. ) , according to optimal cut - off values of nt - probnp . the main findings of our study are : a. ) nt - probnp values are higher in outpatients with previous mi than in the healthy population , as well as in the patients with stable cad ; b. ) none of the traditional risk factors for cad ( apart from age ) was independently associated with nt - probnp ; conversely , all cardiac events during the first 6 months after ami were associated with highest nt - probnp values , the strongest one being the association with heart failure . negative association with reperfusion therapy for index infarction during initial hospitalization confirmed its long - term beneficial effect , by reducing the infarct size ; c. ) nt - probnp accurately predicted new - onset hf in the next one - year follow - up and , to a lesser extent , nce as well ; d. ) the optimal nt - probnp values were : 800 pg / ml for new - onset hf and 516 pg / ml for nce . patients with previous mi are usually classified as chronic cad patients , together with those with stable angina or previous coronary artery bypass grafting ( cabg ) . this may not stand true because the existence of previous myocardial infarction is usually associated with worse angiographic findings ( more widespread cad ) , as well as more frequent appearance of heart failure and recurrent ischemic events , which influences quality of life and life expectancy ( 15 , 16 ) . our data support these findings by showing a significantly higher nt - probnp median value ( 521 ; iqr 335,1095 pg / ml ) compared to the one described for a healthy population by melander et al . ( 17 ) ( 61 ; iqr 34,110 pg / ml ) , ( p<0.001 ) , as well the one described for patients with chronic stable cad ( 6 ) ( 174 ; iqr 74,460 pg / ml ) , ( p<0.01 ) . apart from increased ventricular stretch and wall tension , natriuretic peptides synthesis has been shown in atherosclerotic coronary plaques as well ( 18 ) and nt - probnp levels might reveal the prevalence and severity of coronary stenoses ( 19 , 20 ) . multivessel disease was present in almost half of the patients in our study , possibly contributing to the higher median nt - pro - bnp level . the reason for choosing 6 months after ami for nt - probnp measurement was based on the results of lindahl et al . ( 8) showing that its levels decrease markedly in the first 24 h and then decrease more gradually over the following 6 months . distribution of nt - probnp levels , shown on figure 1 , justifies the common use of the highest quartile for correlations and prognostic purposes . highest nt - probnp values in our study were expectedly associated with age , hf and recurrent ischemic event in the early postinfarction period , as well as with index st - elevation mi which usually represents larger myocardial necrosis and subsequent left ventricular remodelling compared to non - st - elevation mi . in contrast , reperfusion therapy for index event was expectedly associated with lower nt - probnp values , since it definitely reduces the infarct size and consequent remodeling of the left ventricle . highest nt - probnp values were also found in revascularized patients , which can be partially explained by transitory periprocedural ischemia directly stimulating its synthesis ( 21 ) , but other potential stimuli related to revascularization are also possible , such as inflammation and modified neuroendocrine function . having in mind the fact that about 19% of men and 26% of women older than 45 years having a myocardial infarction will die within one year , whereby survivors will have a 1.5 to 15 times higher risk for illness and death compared to the general population ( 22 ) , use of a simple blood test for nt - probnp level that could improve risk stratification and possibly tailor the therapy becomes essential . furthermore , it has been shown that beneficial effects of adequate therapeutic protocol for heart failure were accompanied by a significant reduction of bnp ( 23 ) . patients included in the prognostic analysis in this study were those surviving 6 months after myocardial infarction , free of coronary events or interventions for at least two months , and with relatively preserved left ventricular function , therefore representing a homogenous intermediate risk group of postinfarction patients . in the next one - year follow - up , multivariate cox regression analysis revealed the independent prognostic value of nt - probnp for new - onset hf prediction ( together with reccurent coronary event ) , as well as for new coronary events prediction ( together with diabetes and statin non - use ) . this is in accordance with the recently published data on participants in cardiac rehabilitation , of whom 50% had ami and 50% had coronary artery bypass surgery ( 24 ) . they showed that 12-months nt - probnp levels after an acute cardiovascular event are strongly associated with subsequent long - term events and may provide numerically better reclassification of risk for an adverse cardiovascular event compared to nt - probnp levels shortly after the acute event , after adjustment for the established risk factors . in our study , patients with high nt - probnp values had 3 times higher risk for incident hf . similar findings have been shown among ambulatory patients with stable cad and preserved left ventricular function , among which more than half had a history of prior mi ( 25 ) . the overall prognostic accuracy ( auc ) for incident hf of nt - probnp in our study was 0.77 and the optimal cut - off value was 800 pg / ml . compared to the limit of the highest nt - probnp quartile in our stable patients ( 950 pg / ml ) , it was associated with a higher sensitivity ( 78% vs 63% ) , which is important in the secondary prevention populations , especially as that was at an expense of somewhat lower specificity ( 79% vs 84% ) . negative predictive values were high for both cut - offs and almost the same ( ~90% ) . although to a lesser extent , nt - probnp was also predictive of nce in our study ( auc=0.72 ) . the explanation could be in transitory hypoxia , stimulating cardiac natriuretic peptides expression by increasing the tissue content of natriuretic peptide messenger rna , as shown by goetze et al . also , silent ischemia was found to increase natriuretic peptides in patients with type 2 diabetes ( 27 ) . one of the first authors reporting an independent association of nt - probnp with the coronary events in fully adjusted models of secondary prevention population were investigators of the hope study ( 28 ) . previously mentioned study on ambulatory patients with stable cad and preserved left ventricular function also observed an association between an elevated nt - probnp level and the risk of mi , although the magnitude was less than for the other cardiovascular events and was significantly attenuated after adjusting for the other prognostic markers ( 25 ) . on the contrary , mishra et al . ( 6 ) found nt - probnp to be significantly associated with mi , fully adjusted for all relevant clinical , labaratory and echocardiographic variables . the optimal cut - off value in our study was 516 pg / ml , which is very close to the one mentioned by bibbins - domingo et al . ( 25 ) ( 500 pg / ml ) to indicate patients with highest risk for future events among those with stable cad . the fact that kaplan - meier analysis showed only a trend toward higher risk of nce when patients were stratified according to optimal cut - off value may reflect the relatively small number of nce in this population and possibly become significant in a larger study population , with more nce . since current recommendations for the use of natriuretic peptides ( 29 ) leave the question of optimal time for risk stratification after acute coronary syndrome open , our results propose nt - probnp values 6 months after ami to be reliable and useful for identification of patients who could profit from more intensive monitoring and timely treatment options toward reducing future adverse cardiovascular events . the absence of association with traditional risk factors , together with its prognostic significance may reinforce its role in the secondary prevention after ami . due to the limited sample size , the results should be prospectively validated in a larger group of patients , including more women . second , most highrisk patients were excluded because they fulfilled the exclusion criteria ( i.e. other potential sources of nt - probnp secretion ) or died during the first 6 months after ami , so the results may not be generalized to the whole post - myocardial infarction population . finally , minimization of the potential biases could be obtained by measuring each nt - probnp sample in triplicate or duplicate . due to the limited sample size , the results should be prospectively validated in a larger group of patients , including more women . second , most highrisk patients were excluded because they fulfilled the exclusion criteria ( i.e. other potential sources of nt - probnp secretion ) or died during the first 6 months after ami , so the results may not be generalized to the whole post - myocardial infarction population . finally , minimization of the potential biases could be obtained by measuring each nt - probnp sample in triplicate or duplicate . in this secondary prevention population of postmyocardial infarction patients , highest nt - probnp values were associated mostly with heart failure and recurrent ischemic events in the first 6 months after ami . long term beneficial effect of reperfusion therapy was confirmed by significantly lower nt - probnp values 6 months after ami . nt - probnp accurately predicted new - onset hf in the next one - year followup , at the optimal cut - off value of 800 pg / ml and , to a lesser extent nce , at 516 pg / ml .

summarybackgroundsince serial analyses of nt - probnp in patients with acute coronary syndromes have shown that levels measured during a chronic , later phase are a better predictor of prognosis and indicator of left ventricular function than the levels measured during an acute phase , we sought to assess the association of nt - probnp , measured 6 months after acute myocardial infarction ( ami ) , with traditional risk factors , characteristics of in - hospital and early postinfarction course , as well as its prognostic value and optimal cut - points in the ensuing 1-year follow-up.methodsfasting venous blood samples were drawn from 100 ambulatory patients and nt - probnp concentrations in lithium - heparin plasma were determined using a one - step enzyme immunoassay based on the sandwich principle on a dimension rxl clinical chemistry system ( dade behring - siemens ) . patients were followed - up for the next 1 year , for the occurrence of new cardiac events.resultsmedian ( iqr ) level of nt - probnp was 521 ( 3351095 ) pg / ml . highest values were mostly associated with cardiac events during the first 6 months after ami . negative association with reperfusion therapy for index infarction confirmed its long - term beneficial effect . in the next one - year follow - up of stable patients , multivariate cox regression analysis revealed the independent prognostic value of nt - probnp for new - onset heart failure prediction ( p=0.014 ) , as well as for new coronary events prediction ( p=0.035 ) . calculation of the aucs revealed the optimal nt - probnp cut - points of 800 pg / ml and 516 pg / ml , respectively.conclusionsnt-probnp values 6 months after ami are mainly associated with the characteristics of early infarction and postinfarction course and can predict new cardiac events in the next one - year follow - up .

Uvod Metode Rezultati Zakljuak Introduction Material and Methods Study population and design Blood sampling procedures and biochemical assays for measurement of NT-proBNP levels Echocardiography Statistical analysis Results Prognostic significance of NT-proBNP ROC analysis and optimized NT-proBNP cut-off values Discussion Limitations of the study Conclusions

koncentracije nt - probnp - a u litijum - heparinskoj plazmi su odreivane korienjem jedno - stepenog enzimskog imunoeseja , baziranog na sendvi principu , na ureaju dimension rxl clinical chemistry system ( dade behring - siemens ) . je 521 ( 3351095 ) pg / ml . analizom roc krive dobijene su optimalne prediktivne vrednosti nt - probnp - a : 800 pg / ml za si i 516 pg / ml za nkd . later , their independent prognostic value was also shown , especially concerning mortality and heart failure , in patients with stable and unstable coronary artery disease ( cad ) ( 25 ) . on the other hand , data about acute coronary events prediction are still conflicting ; in contrast to the peace trial ( 2 ) , in which neither bnp nor nt - probnp significantly increased the risk of myocardial infarction ( mi ) , the heart and soul study found an independent association of both markers with the individual outcomes of heart failure , myocardial infarction and cardiovascular death ( 6 ) . also , nt - probnp was found to be a useful biomarker for distinguishing patients with long - standing hypertension who are at risk of heart failure , allowing optimization and proper treatment of these patients ( 7 ) . serial analyses of nt - probnp in patients with non - st segment elevation acute coronary syndromes ( frisc - ii substudy ) showed that levels measured during a chronic , relatively stable phase are a better predictor of mortality than those measured during an acute , unstable phase ( 8) . also , assessment of nt - probnp level 6 months after st - elevation mi was a better indicator of infarct size and left ventricular function measured by cardiac magnetic resonance than baseline ( admission ) nt - pro bnp values ( 9 ) . although previously thought to be equally effective for diagnostic and prognostic purposes ( 10 ) , recently published data from the heart and soul study found nt - probnp to be superior to bnp , when added to clinical risk factors , for net reclassification of the risk for major adverse cardiac events in patients with stable cad ( 6 ) . meta - analysis of nine prospective studies , which indicated strong association between the circulating concentration of nt - probnp and long - term prognosis of patients with stable cad , pointed out that although most of the included studies grouped the population according to the median or quartiles of nt - probnp , the specific nt - probnp , levels varied greatly among different studies , making it impossible to give a precise cut - point ( 11 ) . therefore , we designed this study to assess the association of nt - probnp measured 6 months after ami , with traditional risk factors , characteristics of inhospital and early postinfarction course , as well as its prognostic value and optimal cut - points in the next one - year follow - up . fasting venous blood samples were drawn and edta plasma was frozen at 80 c until measurement . patients were followed - up for the next one year , for the occurrence of new cardiac events ; the relevant endpoints were new - onset hf and new coronary events ( nce ) , defined as a composite of myocardial reinfarction or unstable angina ( according to standard definitions ) ( 12 ) . new - onset heart failure was defined as a clinical syndrome involving at least 2 of the following : paroxysmal nocturnal dyspnoea , orthopnea , elevated jugular venous pressure , pulmonary rales and third heart sound . the nt - probnp concentrations in lithium - heparin plasma were determined using a one step enzyme immunoassay based on the sandwich principle on a dimension rxl clinical chemistry system ( dade behring - siemens ) with commercial reagents ( 13 ) . logistic regression analysis was used to investigate the association of nt - probnp values ( as highest vs. other 3 quartiles ) with demographic and risk factors , as well as with in - hospital and early postinfarction course . to examine the independent association of nt - probnp with new cardiac events during the one - year follow - up . receiver - operating characteristic ( roc ) analysis was used to estimate the overall prognostic accuracy of nt - probnp and to define its optimal predictive cut - off values . finally , kaplan - meier survival curves with log rank tests were used for comparisons of the occurence of new cardiac events in patient groups stratified according to optimal cut - points . patients were followed - up for the next one year , for the occurrence of new cardiac events ; the relevant endpoints were new - onset hf and new coronary events ( nce ) , defined as a composite of myocardial reinfarction or unstable angina ( according to standard definitions ) ( 12 ) . new - onset heart failure was defined as a clinical syndrome involving at least 2 of the following : paroxysmal nocturnal dyspnoea , orthopnea , elevated jugular venous pressure , pulmonary rales and third heart sound . the nt - probnp concentrations in lithium - heparin plasma were determined using a one step enzyme immunoassay based on the sandwich principle on a dimension rxl clinical chemistry system ( dade behring - siemens ) with commercial reagents ( 13 ) . logistic regression analysis was used to investigate the association of nt - probnp values ( as highest vs. other 3 quartiles ) with demographic and risk factors , as well as with in - hospital and early postinfarction course . to examine the independent association of nt - probnp with new cardiac events during the one - year follow - up . receiver - operating characteristic ( roc ) analysis was used to estimate the overall prognostic accuracy of nt - probnp and to define its optimal predictive cut - off values . finally , kaplan - meier survival curves with log rank tests were used for comparisons of the occurence of new cardiac events in patient groups stratified according to optimal cut - points . baseline characteristics of study population and association of nt - probnp with demographic and traditional risk factors , characteristics of in - hospital and early postinfarction course the final study group consisted of 100 ambulatory patients ( 81 men and 19 women , mean age 599 years ) who had survived an ami six months earlier . during the first 6 months after ami ( early postinfarction course ) , 12% of patients had heart failure ( nyha > i ) , 8% had a new coronary event and 6% of patients were surgically revascularized ( all coronary events and procedures happened at least 2 months before study inclusion ) ; beta blockers were used by 75% and ace inhibitors by 79% of patients . median ( iqr ) level of nt - probnp was 521 ( 335,1095 ) pg / ml . factors significantly associated with the highest nt - probnp quartile among demographic and risk factors , as well as in - hospital and early postinfarction course are presented in table i. older age was independently associated with highest nt - probnp values , together with all cardiac events during the first 6 month after ami , and the strongest association was found , as expected , with actual heart failure . negative association was observed with reperfusion therapy for index ami ( during initial hospitalization ) . independent associations of the highest nt - probnp quartile with demographic and risk factors ( model 1 ) , in - hospital characteristics ( model 2 ) and early postinfarction ( 6 months ) course ( model 3 ) . because of the known relationship of nt - probnp and heart failure , only patients without any signs and symptoms of heart failure , as well as without radiographic signs of hf , were included in the prognostic analysis ( 88 patients , mean age 579 , female 14 , male 74 , mean ef 488 ; median ( iqr ) nt - probnp 490 ( 296,950 ) pg / ml ) . during one - year follow - up ( 1114 months ) , 2 patients died ( 2.3% ) , 10 ( 11.3% ) patients had nce and 15 ( 17% ) had new - onset hf . multivariate cox regression analysis revealed the independent prognostic value of nt - probnp ( as log - transformed continuous variable ) for new - onset hf and nce prediction ( table ii ) . independent predictors of new coronary events ( nce ) and new - onset heart failure ( hf)*. adjusted for age , risk factors , reperfusion therapy and ef calculation of the areas under the roc curves , as a measure of overall prognostic accuracy , demonstrated that nt - probnp performed better for new - onset hf than for nce , although the accuracy was satisfactory for both events ( figure 1 ) . therefore , further analysis of the roc curve , in order to assess the optimal predictive cut - off values of nt - probnp , was done . the value with the highest sensitivity and specificity was 800 pg / ml for new - onset hf ( sensitivity 78% , specificity 79% , negative predictive value 92% ) and 516 pg / ml for nce ( sensitivity 75% , specificity 58% , negative predictive value 91% ) . receiver operating characteristic ( roc ) curves showing prognostic accuracy of ntprobnp for new - onset heart failure prediction ( a. ) , together with the optimal cut - off values . when patients were stratified into two groups , according to optimized cut - off values , kaplan - meier analysis showed a highly significant difference in the rates of new - onset hf and a clear trend toward higher incidence of nce ( figure 2 ) . kaplan - meier survival curves without new - onset heart failure ( a. ) , according to optimal cut - off values of nt - probnp . because of the known relationship of nt - probnp and heart failure , only patients without any signs and symptoms of heart failure , as well as without radiographic signs of hf , were included in the prognostic analysis ( 88 patients , mean age 579 , female 14 , male 74 , mean ef 488 ; median ( iqr ) nt - probnp 490 ( 296,950 ) pg / ml ) . during one - year follow - up ( 1114 months ) , 2 patients died ( 2.3% ) , 10 ( 11.3% ) patients had nce and 15 ( 17% ) had new - onset hf . multivariate cox regression analysis revealed the independent prognostic value of nt - probnp ( as log - transformed continuous variable ) for new - onset hf and nce prediction ( table ii ) . independent predictors of new coronary events ( nce ) and new - onset heart failure ( hf)*. adjusted for age , risk factors , reperfusion therapy and ef calculation of the areas under the roc curves , as a measure of overall prognostic accuracy , demonstrated that nt - probnp performed better for new - onset hf than for nce , although the accuracy was satisfactory for both events ( figure 1 ) . therefore , further analysis of the roc curve , in order to assess the optimal predictive cut - off values of nt - probnp , was done . the value with the highest sensitivity and specificity was 800 pg / ml for new - onset hf ( sensitivity 78% , specificity 79% , negative predictive value 92% ) and 516 pg / ml for nce ( sensitivity 75% , specificity 58% , negative predictive value 91% ) . receiver operating characteristic ( roc ) curves showing prognostic accuracy of ntprobnp for new - onset heart failure prediction ( a. ) and new coronary events prediction ( b. ) , together with the optimal cut - off values . when patients were stratified into two groups , according to optimized cut - off values , kaplan - meier analysis showed a highly significant difference in the rates of new - onset hf and a clear trend toward higher incidence of nce ( figure 2 ) . kaplan - meier survival curves without new - onset heart failure ( a. ) , according to optimal cut - off values of nt - probnp . nt - probnp values are higher in outpatients with previous mi than in the healthy population , as well as in the patients with stable cad ; b. ) none of the traditional risk factors for cad ( apart from age ) was independently associated with nt - probnp ; conversely , all cardiac events during the first 6 months after ami were associated with highest nt - probnp values , the strongest one being the association with heart failure . negative association with reperfusion therapy for index infarction during initial hospitalization confirmed its long - term beneficial effect , by reducing the infarct size ; c. ) nt - probnp accurately predicted new - onset hf in the next one - year follow - up and , to a lesser extent , nce as well ; d. ) the optimal nt - probnp values were : 800 pg / ml for new - onset hf and 516 pg / ml for nce . this may not stand true because the existence of previous myocardial infarction is usually associated with worse angiographic findings ( more widespread cad ) , as well as more frequent appearance of heart failure and recurrent ischemic events , which influences quality of life and life expectancy ( 15 , 16 ) . our data support these findings by showing a significantly higher nt - probnp median value ( 521 ; iqr 335,1095 pg / ml ) compared to the one described for a healthy population by melander et al . ( 17 ) ( 61 ; iqr 34,110 pg / ml ) , ( p<0.001 ) , as well the one described for patients with chronic stable cad ( 6 ) ( 174 ; iqr 74,460 pg / ml ) , ( p<0.01 ) . apart from increased ventricular stretch and wall tension , natriuretic peptides synthesis has been shown in atherosclerotic coronary plaques as well ( 18 ) and nt - probnp levels might reveal the prevalence and severity of coronary stenoses ( 19 , 20 ) . the reason for choosing 6 months after ami for nt - probnp measurement was based on the results of lindahl et al . distribution of nt - probnp levels , shown on figure 1 , justifies the common use of the highest quartile for correlations and prognostic purposes . highest nt - probnp values in our study were expectedly associated with age , hf and recurrent ischemic event in the early postinfarction period , as well as with index st - elevation mi which usually represents larger myocardial necrosis and subsequent left ventricular remodelling compared to non - st - elevation mi . in contrast , reperfusion therapy for index event was expectedly associated with lower nt - probnp values , since it definitely reduces the infarct size and consequent remodeling of the left ventricle . highest nt - probnp values were also found in revascularized patients , which can be partially explained by transitory periprocedural ischemia directly stimulating its synthesis ( 21 ) , but other potential stimuli related to revascularization are also possible , such as inflammation and modified neuroendocrine function . having in mind the fact that about 19% of men and 26% of women older than 45 years having a myocardial infarction will die within one year , whereby survivors will have a 1.5 to 15 times higher risk for illness and death compared to the general population ( 22 ) , use of a simple blood test for nt - probnp level that could improve risk stratification and possibly tailor the therapy becomes essential . patients included in the prognostic analysis in this study were those surviving 6 months after myocardial infarction , free of coronary events or interventions for at least two months , and with relatively preserved left ventricular function , therefore representing a homogenous intermediate risk group of postinfarction patients . in the next one - year follow - up , multivariate cox regression analysis revealed the independent prognostic value of nt - probnp for new - onset hf prediction ( together with reccurent coronary event ) , as well as for new coronary events prediction ( together with diabetes and statin non - use ) . they showed that 12-months nt - probnp levels after an acute cardiovascular event are strongly associated with subsequent long - term events and may provide numerically better reclassification of risk for an adverse cardiovascular event compared to nt - probnp levels shortly after the acute event , after adjustment for the established risk factors . similar findings have been shown among ambulatory patients with stable cad and preserved left ventricular function , among which more than half had a history of prior mi ( 25 ) . the overall prognostic accuracy ( auc ) for incident hf of nt - probnp in our study was 0.77 and the optimal cut - off value was 800 pg / ml . compared to the limit of the highest nt - probnp quartile in our stable patients ( 950 pg / ml ) , it was associated with a higher sensitivity ( 78% vs 63% ) , which is important in the secondary prevention populations , especially as that was at an expense of somewhat lower specificity ( 79% vs 84% ) . one of the first authors reporting an independent association of nt - probnp with the coronary events in fully adjusted models of secondary prevention population were investigators of the hope study ( 28 ) . previously mentioned study on ambulatory patients with stable cad and preserved left ventricular function also observed an association between an elevated nt - probnp level and the risk of mi , although the magnitude was less than for the other cardiovascular events and was significantly attenuated after adjusting for the other prognostic markers ( 25 ) . the optimal cut - off value in our study was 516 pg / ml , which is very close to the one mentioned by bibbins - domingo et al . since current recommendations for the use of natriuretic peptides ( 29 ) leave the question of optimal time for risk stratification after acute coronary syndrome open , our results propose nt - probnp values 6 months after ami to be reliable and useful for identification of patients who could profit from more intensive monitoring and timely treatment options toward reducing future adverse cardiovascular events . the absence of association with traditional risk factors , together with its prognostic significance may reinforce its role in the secondary prevention after ami . other potential sources of nt - probnp secretion ) or died during the first 6 months after ami , so the results may not be generalized to the whole post - myocardial infarction population . other potential sources of nt - probnp secretion ) or died during the first 6 months after ami , so the results may not be generalized to the whole post - myocardial infarction population . in this secondary prevention population of postmyocardial infarction patients , highest nt - probnp values were associated mostly with heart failure and recurrent ischemic events in the first 6 months after ami . long term beneficial effect of reperfusion therapy was confirmed by significantly lower nt - probnp values 6 months after ami . nt - probnp accurately predicted new - onset hf in the next one - year followup , at the optimal cut - off value of 800 pg / ml and , to a lesser extent nce , at 516 pg / ml .

, several research groups studied this subject , and one of the largest studies involved 65 patients . over a four - week period , subjects were administered either a chocolate bar or a placebo bar , and no difference in acne severity was seen . this belief was reflected in textbooks , patient information brochures , and the medical literature . however , later researchers noted methodological flaws in the original study , including the fact that the placebo bar contained a similar total sugar and fat content as the chocolate bar . a randomized controlled trial of australian males demonstrated significant improvement of acne severity after 12 weeks of adherence to a low - glycemic - load diet , compared to the high - glycemic - load diet control group . follow - up studies have noted that switching to a low glycemic diet led to better insulin sensitivity , lower androgen bioavailability , and altered skin sebum production . it is not known , however , what role individual differences , duration of dietary changes , and other factors play in this association . therefore , studies such as di landro et al.s have documented the opposing view that weekly consumption of cakes , sweets , and chocolate foods high in glycemic load was not associated with a higher risk of acne . in addition , researchers examining the link between dairy consumption and acne have noted some association . studies have demonstrated this link in three separate populations , though in each instance the correlation results were considered relatively weak . closer evaluation of this link revealed that only skim milk showed a statistically significant correlation with acne , perhaps due to its increased processing and/or decreased estrogen content in comparison to whole milk . a number of other dietary factors have been studied for their potential role in improving acne . for example , an inverse relationship has been found between acne severity and consumption of omega-3-rich fish with the mechanism of action postulated as omega-3-mediated reduction of inflammatory acne . while some promising results have been noted from in vitro or animal studies , studies in humans are limited , and each of these dietary factors requires further investigation before recommendations may be made to patients . these include foods or supplements containing vitamin a , omega-3 fatty acids , zinc , antioxidants , and fiber . . given that the scientific literature on this topic is rapidly evolving , it would be expected that patients might receive conflicting advice . patients have long held certain beliefs about diet and acne , including the common perception that fried , greasy foods would lead to oily skin and acne . however , it is not known whether patients are aware of the research findings that support a link between specific dietary factors and acne . patients seek out and receive medical information from multiple sources , and current patient beliefs about diet and acne are not known . this study was designed to be an initial , exploratory survey study of current patient perceptions about the link between diet and acne . the aim was to identify common beliefs , misconceptions , and current information sources regarding the connection between diet and acne . as in other exploratory survey studies , these findings would help indicate directions for future research and would be of benefit to clinicians when counseling patients . patients presenting to an academic dermatology clinic for the treatment of acne or acne scarring were asked if they wished to participate in a survey study about the link between diet and acne . if so , they completed a self - administered questionnaire prior to the physician visit . subjects were asked to classify their acne from mild to severe , based on their personal perception of the disease s impact . demographic data , such as age , gender , weight , height , and education level , was included in the questionnaire . of the 50 questionnaires administered , 49 were completed in full and were included in the analysis . the mean age of patients was 28.5 years ( range , 16 to 45 years ) ; 38 were female and 11 were male . three respondents had an under - weight bmi of less than 18.5 ; 30 respondents had a normal bmi between 18.524.9 ; 13 respondents were overweight with bmi 2529.9 ; and 2 respondents qualified as obese with bmi greater than 30.0 . a majority , 45 of 49 respondents , was educated at the college level ( table ia ) . respondents reported the following acne severity distribution : 13 patients had mild acne , 1 patient had mild - moderate , 29 patients had moderate , 1 had moderate - severe , and 5 had severe acne ( table ib ) . among our population only 3 of 38 ( 8% ) female patients and 1 of 11 ( 9% ) male patients believed that diet had no effect on acne . of female patients , 30 of 38 ( 81% ) patients believed that diet affected acne either somewhat , a lot , or was the complete cause of acne . among male patients , a majority , 35 of 49 patients ( 71% ) , had changed their diets in order to help their acne ( table iia ) . thirteen of the 35 ( 37% ) tried dietary changes before medications , compared to the 14 patients that first attempted medications . of those 14 , 9 tried over - the - counter medications first ( 25% ) and 5 tried prescription first ( 14% ) . four patients tried a combination of dietary changes with prescription medications ( 11% ) ; 2 combined dietary changes with over - the - counter medications ( 6% ) ; and the remaining tried a combination of all three or a combination of over - the - counter and prescription medications ( table iib ) . the dietary item most frequently implicated to worsen acne was fried , greasy foods ( 71% ) . other responses included chocolate ( 53% ) , followed by dairy products ( 47% ) . this was followed by soda drinks ( 35% ) , caffeine ( 27% ) , and refined carbohydrates ( 27% ) . among the named categories of dietary items believed to aggravate acne , sugar was named by the lowest percentage of respondents ( 16% ) . the category listed by most respondents as helping acne was that of vitamin a ( 41% ) , followed by antioxidants ( 33% ) , fish / omega-3s ( 29% ) , and zinc ( 27% ) ( table iiib ) . twenty percent reported acne improvement with less fried or greasy foods , followed by 14% reporting improvement with less dairy products and 14% reporting improvement with less refined carbohydrates ( table iiic ) . the most widely utilized source of information was a google search ( 49% ) , followed by dermatologist ( 43% ) and then family members and tv ( tied at 41% ) . medical websites , specifically sites such as webmd or that of the american academy of dermatology , were used by 31% , while primary care physician ( pcp ) was used by 18% ( table iva ) . respondents were split evenly between those satisfied with the information provided on their sources ( 49% ) and those not satisfied ( 51% ) ( table ivb ) . this trend was found in almost all categories of sources listed except for : dermatologist , family members , medical websites , and pcp . of the 24 out of 49 satisfied respondents , 18 ( 75% ) marked dermatologist as one of their sources of diet and acne information . not satisfied with their information , only 3 ( 12% ) had marked dermatologist as a source of information . similarly , 10 of 24 satisfied respondents ( 42% ) marked medical websites as one of their sources , whereas 5 of 25 unsatisfied respondents ( 20% ) reported the use of medical websites . these findings reverse when looking at family members as a source of information . among unsatisfied respondents , 52% had marked family members as a source of diet and acne education , as compared to 29% of satisfied respondents ( table ivc ) . in fact , only 5% of female patients and only 9% of male patients believed that diet had no effect on acne . a majority of respondents , 71% , had changed their diet in order to help their acne . of the foods listed , the highest percentage of respondents ( 71% ) chose a category that patients have historically described as an acne trigger food : that of fried / greasy foods . this is a popular , long - held belief , as anecdotally , patients sometimes make the connection between greasy foods and increased oil production on the face . the strongest research evidence , however , supports a link between a high glycemic index ( gi ) diet and acne . while fried or greasy foods may contribute to a high gi diet , so would sugar , soda drinks , and refined carbohydrates . these categories , however , were listed by much lower percentages of respondents ( 16% , 35% , and 27% , respectively ) . this is an important point to emphasize to our patients . while fried , greasy foods are a concern , so are a number of other foods . as dermatologists , we should be emphasizing that even innocuous foods , such as white bread , pasta , and other refined carbohydrates , when part of an overall high gi diet , may worsen acne . chocolate as a dietary factor triggering acne is also a long - held belief , and studies evaluating its role in acne were performed in the 1960s . at this time , though , there is no evidence that a factor specific to chocolate can trigger acne . rather , research points to its sugar content and contribution to a high gi diet as a possible trigger . ideally , then , chocolate , sugar , and refined carbohydrates would have similar ratings by patients , as they may all contribute to a high gi index diet . in fact , while 53% reported chocolate as a possible trigger food , only 16% reported sugar as a potential trigger . while chocolate bars may serve as an acne trigger , sugar and other sugary foods are just as concerning . of note , although some studies have shown a link to dairy , less than half ( 47% ) of our respondents believed that dairy products could aggravate acne . an interesting finding is that a significant proportion of respondents believed that certain foods or supplements could help acne . this may be due to reports of high - dose vitamin a therapy used in the past as acne therapy , and the fact that acne medications such as tretinoin and isotretinoin are related to vitamin a. our survey did not distinguish between standard vitamin a supplements and high - dose supplementation , and therefore it is not known whether a belief in vitamin a therapy extends to standard supplement dosing . when vitamin a is removed from the analysis , the highest category becomes antioxidants ( 33% ) , followed by fish or omega-3s ( 29% ) , zinc ( 27% ) , and dietary fiber ( 20% ) . for each of these nutrients , while some promising results in the treatment of acne have been noted from in vitro or animal studies , studies in humans are limited . at this point , these can not be recommended routinely , but their use by patients can not be dismissed entirely either . in conclusion , while there is limited research performed in the area of foods or supplements that may help in the treatment of acne , many of our respondents believe they may help . in an era of rapid dissemination of information , patients are sometimes made aware of promising findings from small studies . this highlights the need for dermatologists to stay abreast of the latest dietary findings , and in particular not to dismiss these out of hand . in addition , dermatologists must be cognizant of the fact that a significant proportion of patients may believe that certain foods or supplements have the ability to combat acne , and therefore must be able to provide information on the limited evidence that exists to support their use . in order to disseminate accurate , evidence - based information in our survey study , less than half ( 43% ) relied on dermatologists for information . the remaining respondents turned to a variety of sources , including the internet ( google search and medical websites ) , mass media ( magazines , television , and newspaper ) , and personal relationships ( mainly family members and friends ) . the sources of information utilized by patients have changed markedly . in a survey study from 1999 , general information on acne was obtained most frequently from family physicians ( 71% ) . in our survey the mass media continues to play a role in providing health information . in the 1999 survey study , magazines and television were utilized by 44% each . in our survey study , magazines were utilized by 27% , and television was utilized by 41% of respondents . the most notable change has been the rise of the internet as a tool for health information . our respondents utilized google most frequently ( 49% ) as a source of information , with medical websites utilized by 31% . in the 1999 study , it is clear that the internet has had a significant impact on health information - seeking behavior . what is less clear is how physicians should respond to this change when educating our patients . in this study , more patients obtained information from the internet than from their primary care physician or their dermatologist . however , a significant number of those satisfied with their diet and acne education had marked pcp ( 25% ) or dermatologist ( 75% ) as one of their sources of information . conversely , the majority of respondents not satisfied with their information source did not mark dermatologist or pcp as a source of information ( 86% ) . thus , with so many acne patients making dietary changes , and many of them turning to the internet for information to guide them , it becomes even more imperative that physicians be educated on the topic of acne and diet and be able to serve as an authoritative source . for those patients who seek further information , we also need to be able to refer patients to credible sources of online health information , with specific recommendations for high - quality , up - to - date health information websites . our study suggests that education from physicians and medical websites is correlated with higher satisfaction in patients . physicians must also counsel patients on the drawbacks of utilizing search engines for health information , as opposed to utilizing well - regarded medical websites . if relying on a google search , patients may be directed to commercial sites or sites containing inaccurate information . finally , we need to ensure , as a profession , that health education materials are easily available on the internet and that such information is accurate , evidence - based , free from commercial bias , and reflective of the most current research findings . due to the design of this exploratory survey study , patients were recruited from the population of patients seeking treatment for acne at a single academic institution . most , 31 of 49 , were of normal weight ( 63% ) , with only 15 of 49 ( 31% ) categorized as overweight or obese . population , in which 69% are overweight or obese ; 29% are college educated ; and 51% are female . another weakness of the study may be the lack of a quantitative definition of acne severity . in the past , multiple quantitative classification systems have been proposed , though these methods have been inconsistently accurate in the clinical setting . given the variable expressions of acne , as well as its psychosocial factors difficult to quantitate , a more important assessment of severity may be the patient s perception of its impact , which we elicited in the study . in fact , only 5% of female patients and only 9% of male patients believed that diet had no effect on acne . a majority of respondents , 71% , had changed their diet in order to help their acne . of the foods listed , the highest percentage of respondents ( 71% ) chose a category that patients have historically described as an acne trigger food : that of fried / greasy foods . this is a popular , long - held belief , as anecdotally , patients sometimes make the connection between greasy foods and increased oil production on the face . the strongest research evidence , however , supports a link between a high glycemic index ( gi ) diet and acne . while fried or greasy foods may contribute to a high gi diet , so would sugar , soda drinks , and refined carbohydrates . these categories , however , were listed by much lower percentages of respondents ( 16% , 35% , and 27% , respectively ) . this is an important point to emphasize to our patients . while fried , greasy foods are a concern , so are a number of other foods . as dermatologists , we should be emphasizing that even innocuous foods , such as white bread , pasta , and other refined carbohydrates , when part of an overall high gi diet , may worsen acne . chocolate as a dietary factor triggering acne is also a long - held belief , and studies evaluating its role in acne were performed in the 1960s . at this time , though , there is no evidence that a factor specific to chocolate can trigger acne . rather , research points to its sugar content and contribution to a high gi diet as a possible trigger . ideally , then , chocolate , sugar , and refined carbohydrates would have similar ratings by patients , as they may all contribute to a high gi index diet . in fact , while 53% reported chocolate as a possible trigger food , only 16% reported sugar as a potential trigger . while chocolate bars may serve as an acne trigger , sugar and other sugary foods are just as concerning . of note , although some studies have shown a link to dairy , less than half ( 47% ) of our respondents believed that dairy products could aggravate acne . an interesting finding is that a significant proportion of respondents believed that certain foods or supplements could help acne . this may be due to reports of high - dose vitamin a therapy used in the past as acne therapy , and the fact that acne medications such as tretinoin and isotretinoin are related to vitamin a. our survey did not distinguish between standard vitamin a supplements and high - dose supplementation , and therefore it is not known whether a belief in vitamin a therapy extends to standard supplement dosing . when vitamin a is removed from the analysis , the highest category becomes antioxidants ( 33% ) , followed by fish or omega-3s ( 29% ) , zinc ( 27% ) , and dietary fiber ( 20% ) . for each of these nutrients , while some promising results in the treatment of acne have been noted from in vitro or animal studies , studies in humans are limited . at this point , these can not be recommended routinely , but their use by patients can not be dismissed entirely either . in conclusion , while there is limited research performed in the area of foods or supplements that may help in the treatment of acne , many of our respondents believe they may help . in an era of rapid dissemination of information , patients are sometimes made aware of promising findings from small studies . this highlights the need for dermatologists to stay abreast of the latest dietary findings , and in particular not to dismiss these out of hand . in addition , dermatologists must be cognizant of the fact that a significant proportion of patients may believe that certain foods or supplements have the ability to combat acne , and therefore must be able to provide information on the limited evidence that exists to support their use . in order to disseminate accurate , evidence - based information , we must understand what sources patients utilize for medical information . in our survey study , the remaining respondents turned to a variety of sources , including the internet ( google search and medical websites ) , mass media ( magazines , television , and newspaper ) , and personal relationships ( mainly family members and friends ) . the sources of information utilized by patients have changed markedly . in a survey study from 1999 , general information on acne was obtained most frequently from family physicians ( 71% ) . in our survey the mass media continues to play a role in providing health information . in the 1999 survey study , magazines and television were utilized by 44% each . in our survey study , magazines were utilized by 27% , and television was utilized by 41% of respondents . the most notable change has been the rise of the internet as a tool for health information . our respondents utilized google most frequently ( 49% ) as a source of information , with medical websites utilized by 31% . in the 1999 study , it is clear that the internet has had a significant impact on health information - seeking behavior . what is less clear is how physicians should respond to this change when educating our patients . in this study , more patients obtained information from the internet than from their primary care physician or their dermatologist . however , a significant number of those satisfied with their diet and acne education had marked pcp ( 25% ) or dermatologist ( 75% ) as one of their sources of information . conversely , the majority of respondents not satisfied with their information source did not mark dermatologist or pcp as a source of information ( 86% ) . thus , with so many acne patients making dietary changes , and many of them turning to the internet for information to guide them , it becomes even more imperative that physicians be educated on the topic of acne and diet and be able to serve as an authoritative source . for those patients who seek further information , we also need to be able to refer patients to credible sources of online health information , with specific recommendations for high - quality , up - to - date health information websites . our study suggests that education from physicians and medical websites is correlated with higher satisfaction in patients . physicians must also counsel patients on the drawbacks of utilizing search engines for health information , as opposed to utilizing well - regarded medical websites . if relying on a google search , patients may be directed to commercial sites or sites containing inaccurate information . finally , we need to ensure , as a profession , that health education materials are easily available on the internet and that such information is accurate , evidence - based , free from commercial bias , and reflective of the most current research findings . due to the design of this exploratory survey study , patients were recruited from the population of patients seeking treatment for acne at a single academic institution . most , 31 of 49 , were of normal weight ( 63% ) , with only 15 of 49 ( 31% ) categorized as overweight or obese . population , in which 69% are overweight or obese ; 29% are college educated ; and 51% are female . another weakness of the study may be the lack of a quantitative definition of acne severity . in the past , multiple quantitative classification systems have been proposed , though these methods have been inconsistently accurate in the clinical setting . given the variable expressions of acne , as well as its psychosocial factors difficult to quantitate , a more important assessment of severity may be the patient s perception of its impact , which we elicited in the study . this survey study was designed to explore patient beliefs regarding the link between diet and acne . in this small sample , this belief impacted behavior , as the majority had changed their diets in order to improve their acne . due to the study design , the results from this patient sample can not be applied to the general population . however , even in this highly educated patient population , there was a persistence of long - held beliefs that certain foods may serve as acne triggers ( i.e. , fried or greasy foods and chocolate ) and less recognition of the foods that research has identified as potential triggers ( i.e. , sugars , refined carbohydrates , and dairy ) . many respondents also reported a belief that certain foods or supplements could help acne , an area with some promising preliminary results but one that requires further investigation before any conclusions may be drawn . especially given the multiplicity of information sources utilized by patients , as well as the ongoing research in this area and subsequent evolving recommendations , dermatologists must serve as an authoritative information source . dermatologists must be able to provide a balanced overview of this topic to their patients and be able to direct patients to credible , balanced sources for additional dietary change recommendations . these findings indicate a need for further study on the best practices in providing health information , especially as medical recommendations are evolving . our population was highly educated , and the majority was of normal weight . further research in this area must target a more diverse population . even within this skewed population , however , we noted widely conflicting beliefs among acne patients . there is a clear need for easily available , evidence - based , and up - to - date information on the link between diet and acne .

background : in the past , medical literature reflected that diet was not a proven cause of acne . however , studies in recent years have substantiated a link between certain dietary factors and acne . it is unclear whether patients are aware of recent research findings.objectives:acne patients were surveyed to explore beliefs regarding the link between diet and acne , to determine whether these beliefs translated into behavior change and to identify health information sources.patients/methods:upon institutional review board ( irb ) approval , surveys were administered to 50 acne patients at an academic dermatology clinic in 2014 , with 49 completed in full and included in this analysis.results:ninety-two percent of respondents believed that diet could affect acne . seventy - one percent attempted to change their diet to improve acne . seventy - one percent believed acne to be caused by fried or greasy foods , although chocolate ( 53% ) , dairy ( 47% ) , and soda drinks ( 35% ) were highly implicated . patients obtained information from google searches ( 49% ) , dermatologists ( 43% ) , family members and tv ( 41% each ) , and medical websites ( 31%).conclusions : in this exploratory study , patients reported utilizing a diversity of information sources , a majority from the internet . in those surveyed , there was a persistence of long - held belief that fried / greasy foods and chocolate may serve as acne triggers , and less belief in trigger foods supported by recent research , including refined carbohydrates and sugar . given the multiplicity of beliefs and utilized sources among acne patients in our survey , there is a need to establish up - to - date and reliable methods to educate patients on diet and acne .

Introduction Materials and methods Results Discussion These beliefs are translating to behavior change The majority of patients believed that certain foods may worsen acne Patients also believe that certain foods may help acne Information sources Study weaknesses Conclusion

, several research groups studied this subject , and one of the largest studies involved 65 patients . over a four - week period , subjects were administered either a chocolate bar or a placebo bar , and no difference in acne severity was seen . this belief was reflected in textbooks , patient information brochures , and the medical literature . however , later researchers noted methodological flaws in the original study , including the fact that the placebo bar contained a similar total sugar and fat content as the chocolate bar . a randomized controlled trial of australian males demonstrated significant improvement of acne severity after 12 weeks of adherence to a low - glycemic - load diet , compared to the high - glycemic - load diet control group . follow - up studies have noted that switching to a low glycemic diet led to better insulin sensitivity , lower androgen bioavailability , and altered skin sebum production . it is not known , however , what role individual differences , duration of dietary changes , and other factors play in this association . therefore , studies such as di landro et al.s have documented the opposing view that weekly consumption of cakes , sweets , and chocolate foods high in glycemic load was not associated with a higher risk of acne . in addition , researchers examining the link between dairy consumption and acne have noted some association . closer evaluation of this link revealed that only skim milk showed a statistically significant correlation with acne , perhaps due to its increased processing and/or decreased estrogen content in comparison to whole milk . a number of other dietary factors have been studied for their potential role in improving acne . while some promising results have been noted from in vitro or animal studies , studies in humans are limited , and each of these dietary factors requires further investigation before recommendations may be made to patients . these include foods or supplements containing vitamin a , omega-3 fatty acids , zinc , antioxidants , and fiber . patients have long held certain beliefs about diet and acne , including the common perception that fried , greasy foods would lead to oily skin and acne . however , it is not known whether patients are aware of the research findings that support a link between specific dietary factors and acne . patients seek out and receive medical information from multiple sources , and current patient beliefs about diet and acne are not known . this study was designed to be an initial , exploratory survey study of current patient perceptions about the link between diet and acne . the aim was to identify common beliefs , misconceptions , and current information sources regarding the connection between diet and acne . patients presenting to an academic dermatology clinic for the treatment of acne or acne scarring were asked if they wished to participate in a survey study about the link between diet and acne . demographic data , such as age , gender , weight , height , and education level , was included in the questionnaire . of the 50 questionnaires administered , 49 were completed in full and were included in the analysis . a majority , 45 of 49 respondents , was educated at the college level ( table ia ) . respondents reported the following acne severity distribution : 13 patients had mild acne , 1 patient had mild - moderate , 29 patients had moderate , 1 had moderate - severe , and 5 had severe acne ( table ib ) . among our population only 3 of 38 ( 8% ) female patients and 1 of 11 ( 9% ) male patients believed that diet had no effect on acne . of female patients , 30 of 38 ( 81% ) patients believed that diet affected acne either somewhat , a lot , or was the complete cause of acne . among male patients , a majority , 35 of 49 patients ( 71% ) , had changed their diets in order to help their acne ( table iia ) . the dietary item most frequently implicated to worsen acne was fried , greasy foods ( 71% ) . other responses included chocolate ( 53% ) , followed by dairy products ( 47% ) . this was followed by soda drinks ( 35% ) , caffeine ( 27% ) , and refined carbohydrates ( 27% ) . among the named categories of dietary items believed to aggravate acne , sugar was named by the lowest percentage of respondents ( 16% ) . the category listed by most respondents as helping acne was that of vitamin a ( 41% ) , followed by antioxidants ( 33% ) , fish / omega-3s ( 29% ) , and zinc ( 27% ) ( table iiib ) . twenty percent reported acne improvement with less fried or greasy foods , followed by 14% reporting improvement with less dairy products and 14% reporting improvement with less refined carbohydrates ( table iiic ) . the most widely utilized source of information was a google search ( 49% ) , followed by dermatologist ( 43% ) and then family members and tv ( tied at 41% ) . respondents were split evenly between those satisfied with the information provided on their sources ( 49% ) and those not satisfied ( 51% ) ( table ivb ) . this trend was found in almost all categories of sources listed except for : dermatologist , family members , medical websites , and pcp . of the 24 out of 49 satisfied respondents , 18 ( 75% ) marked dermatologist as one of their sources of diet and acne information . not satisfied with their information , only 3 ( 12% ) had marked dermatologist as a source of information . similarly , 10 of 24 satisfied respondents ( 42% ) marked medical websites as one of their sources , whereas 5 of 25 unsatisfied respondents ( 20% ) reported the use of medical websites . these findings reverse when looking at family members as a source of information . among unsatisfied respondents , 52% had marked family members as a source of diet and acne education , as compared to 29% of satisfied respondents ( table ivc ) . in fact , only 5% of female patients and only 9% of male patients believed that diet had no effect on acne . a majority of respondents , 71% , had changed their diet in order to help their acne . of the foods listed , the highest percentage of respondents ( 71% ) chose a category that patients have historically described as an acne trigger food : that of fried / greasy foods . this is a popular , long - held belief , as anecdotally , patients sometimes make the connection between greasy foods and increased oil production on the face . the strongest research evidence , however , supports a link between a high glycemic index ( gi ) diet and acne . while fried or greasy foods may contribute to a high gi diet , so would sugar , soda drinks , and refined carbohydrates . these categories , however , were listed by much lower percentages of respondents ( 16% , 35% , and 27% , respectively ) . while fried , greasy foods are a concern , so are a number of other foods . as dermatologists , we should be emphasizing that even innocuous foods , such as white bread , pasta , and other refined carbohydrates , when part of an overall high gi diet , may worsen acne . chocolate as a dietary factor triggering acne is also a long - held belief , and studies evaluating its role in acne were performed in the 1960s . at this time , though , there is no evidence that a factor specific to chocolate can trigger acne . ideally , then , chocolate , sugar , and refined carbohydrates would have similar ratings by patients , as they may all contribute to a high gi index diet . while chocolate bars may serve as an acne trigger , sugar and other sugary foods are just as concerning . of note , although some studies have shown a link to dairy , less than half ( 47% ) of our respondents believed that dairy products could aggravate acne . an interesting finding is that a significant proportion of respondents believed that certain foods or supplements could help acne . this may be due to reports of high - dose vitamin a therapy used in the past as acne therapy , and the fact that acne medications such as tretinoin and isotretinoin are related to vitamin a. our survey did not distinguish between standard vitamin a supplements and high - dose supplementation , and therefore it is not known whether a belief in vitamin a therapy extends to standard supplement dosing . when vitamin a is removed from the analysis , the highest category becomes antioxidants ( 33% ) , followed by fish or omega-3s ( 29% ) , zinc ( 27% ) , and dietary fiber ( 20% ) . for each of these nutrients , while some promising results in the treatment of acne have been noted from in vitro or animal studies , studies in humans are limited . in conclusion , while there is limited research performed in the area of foods or supplements that may help in the treatment of acne , many of our respondents believe they may help . in an era of rapid dissemination of information , patients are sometimes made aware of promising findings from small studies . this highlights the need for dermatologists to stay abreast of the latest dietary findings , and in particular not to dismiss these out of hand . in addition , dermatologists must be cognizant of the fact that a significant proportion of patients may believe that certain foods or supplements have the ability to combat acne , and therefore must be able to provide information on the limited evidence that exists to support their use . in order to disseminate accurate , evidence - based information in our survey study , less than half ( 43% ) relied on dermatologists for information . the remaining respondents turned to a variety of sources , including the internet ( google search and medical websites ) , mass media ( magazines , television , and newspaper ) , and personal relationships ( mainly family members and friends ) . the sources of information utilized by patients have changed markedly . in our survey the mass media continues to play a role in providing health information . in the 1999 survey study , magazines and television were utilized by 44% each . in our survey study , magazines were utilized by 27% , and television was utilized by 41% of respondents . the most notable change has been the rise of the internet as a tool for health information . our respondents utilized google most frequently ( 49% ) as a source of information , with medical websites utilized by 31% . in the 1999 study , it is clear that the internet has had a significant impact on health information - seeking behavior . in this study , more patients obtained information from the internet than from their primary care physician or their dermatologist . however , a significant number of those satisfied with their diet and acne education had marked pcp ( 25% ) or dermatologist ( 75% ) as one of their sources of information . conversely , the majority of respondents not satisfied with their information source did not mark dermatologist or pcp as a source of information ( 86% ) . thus , with so many acne patients making dietary changes , and many of them turning to the internet for information to guide them , it becomes even more imperative that physicians be educated on the topic of acne and diet and be able to serve as an authoritative source . for those patients who seek further information , we also need to be able to refer patients to credible sources of online health information , with specific recommendations for high - quality , up - to - date health information websites . our study suggests that education from physicians and medical websites is correlated with higher satisfaction in patients . physicians must also counsel patients on the drawbacks of utilizing search engines for health information , as opposed to utilizing well - regarded medical websites . if relying on a google search , patients may be directed to commercial sites or sites containing inaccurate information . finally , we need to ensure , as a profession , that health education materials are easily available on the internet and that such information is accurate , evidence - based , free from commercial bias , and reflective of the most current research findings . due to the design of this exploratory survey study , patients were recruited from the population of patients seeking treatment for acne at a single academic institution . most , 31 of 49 , were of normal weight ( 63% ) , with only 15 of 49 ( 31% ) categorized as overweight or obese . another weakness of the study may be the lack of a quantitative definition of acne severity . in the past , multiple quantitative classification systems have been proposed , though these methods have been inconsistently accurate in the clinical setting . given the variable expressions of acne , as well as its psychosocial factors difficult to quantitate , a more important assessment of severity may be the patient s perception of its impact , which we elicited in the study . in fact , only 5% of female patients and only 9% of male patients believed that diet had no effect on acne . a majority of respondents , 71% , had changed their diet in order to help their acne . of the foods listed , the highest percentage of respondents ( 71% ) chose a category that patients have historically described as an acne trigger food : that of fried / greasy foods . this is a popular , long - held belief , as anecdotally , patients sometimes make the connection between greasy foods and increased oil production on the face . the strongest research evidence , however , supports a link between a high glycemic index ( gi ) diet and acne . while fried or greasy foods may contribute to a high gi diet , so would sugar , soda drinks , and refined carbohydrates . these categories , however , were listed by much lower percentages of respondents ( 16% , 35% , and 27% , respectively ) . while fried , greasy foods are a concern , so are a number of other foods . as dermatologists , we should be emphasizing that even innocuous foods , such as white bread , pasta , and other refined carbohydrates , when part of an overall high gi diet , may worsen acne . chocolate as a dietary factor triggering acne is also a long - held belief , and studies evaluating its role in acne were performed in the 1960s . at this time , though , there is no evidence that a factor specific to chocolate can trigger acne . ideally , then , chocolate , sugar , and refined carbohydrates would have similar ratings by patients , as they may all contribute to a high gi index diet . while chocolate bars may serve as an acne trigger , sugar and other sugary foods are just as concerning . of note , although some studies have shown a link to dairy , less than half ( 47% ) of our respondents believed that dairy products could aggravate acne . an interesting finding is that a significant proportion of respondents believed that certain foods or supplements could help acne . this may be due to reports of high - dose vitamin a therapy used in the past as acne therapy , and the fact that acne medications such as tretinoin and isotretinoin are related to vitamin a. our survey did not distinguish between standard vitamin a supplements and high - dose supplementation , and therefore it is not known whether a belief in vitamin a therapy extends to standard supplement dosing . when vitamin a is removed from the analysis , the highest category becomes antioxidants ( 33% ) , followed by fish or omega-3s ( 29% ) , zinc ( 27% ) , and dietary fiber ( 20% ) . for each of these nutrients , while some promising results in the treatment of acne have been noted from in vitro or animal studies , studies in humans are limited . in conclusion , while there is limited research performed in the area of foods or supplements that may help in the treatment of acne , many of our respondents believe they may help . in an era of rapid dissemination of information , patients are sometimes made aware of promising findings from small studies . this highlights the need for dermatologists to stay abreast of the latest dietary findings , and in particular not to dismiss these out of hand . in addition , dermatologists must be cognizant of the fact that a significant proportion of patients may believe that certain foods or supplements have the ability to combat acne , and therefore must be able to provide information on the limited evidence that exists to support their use . in our survey study , the remaining respondents turned to a variety of sources , including the internet ( google search and medical websites ) , mass media ( magazines , television , and newspaper ) , and personal relationships ( mainly family members and friends ) . the sources of information utilized by patients have changed markedly . in our survey the mass media continues to play a role in providing health information . in the 1999 survey study , magazines and television were utilized by 44% each . in our survey study , magazines were utilized by 27% , and television was utilized by 41% of respondents . the most notable change has been the rise of the internet as a tool for health information . our respondents utilized google most frequently ( 49% ) as a source of information , with medical websites utilized by 31% . in the 1999 study , it is clear that the internet has had a significant impact on health information - seeking behavior . in this study , more patients obtained information from the internet than from their primary care physician or their dermatologist . however , a significant number of those satisfied with their diet and acne education had marked pcp ( 25% ) or dermatologist ( 75% ) as one of their sources of information . conversely , the majority of respondents not satisfied with their information source did not mark dermatologist or pcp as a source of information ( 86% ) . thus , with so many acne patients making dietary changes , and many of them turning to the internet for information to guide them , it becomes even more imperative that physicians be educated on the topic of acne and diet and be able to serve as an authoritative source . for those patients who seek further information , we also need to be able to refer patients to credible sources of online health information , with specific recommendations for high - quality , up - to - date health information websites . our study suggests that education from physicians and medical websites is correlated with higher satisfaction in patients . physicians must also counsel patients on the drawbacks of utilizing search engines for health information , as opposed to utilizing well - regarded medical websites . if relying on a google search , patients may be directed to commercial sites or sites containing inaccurate information . finally , we need to ensure , as a profession , that health education materials are easily available on the internet and that such information is accurate , evidence - based , free from commercial bias , and reflective of the most current research findings . due to the design of this exploratory survey study , patients were recruited from the population of patients seeking treatment for acne at a single academic institution . most , 31 of 49 , were of normal weight ( 63% ) , with only 15 of 49 ( 31% ) categorized as overweight or obese . another weakness of the study may be the lack of a quantitative definition of acne severity . in the past , multiple quantitative classification systems have been proposed , though these methods have been inconsistently accurate in the clinical setting . given the variable expressions of acne , as well as its psychosocial factors difficult to quantitate , a more important assessment of severity may be the patient s perception of its impact , which we elicited in the study . this survey study was designed to explore patient beliefs regarding the link between diet and acne . in this small sample , this belief impacted behavior , as the majority had changed their diets in order to improve their acne . however , even in this highly educated patient population , there was a persistence of long - held beliefs that certain foods may serve as acne triggers ( i.e. , fried or greasy foods and chocolate ) and less recognition of the foods that research has identified as potential triggers ( i.e. , sugars , refined carbohydrates , and dairy ) . many respondents also reported a belief that certain foods or supplements could help acne , an area with some promising preliminary results but one that requires further investigation before any conclusions may be drawn . especially given the multiplicity of information sources utilized by patients , as well as the ongoing research in this area and subsequent evolving recommendations , dermatologists must serve as an authoritative information source . these findings indicate a need for further study on the best practices in providing health information , especially as medical recommendations are evolving . our population was highly educated , and the majority was of normal weight . further research in this area must target a more diverse population . even within this skewed population , however , we noted widely conflicting beliefs among acne patients . there is a clear need for easily available , evidence - based , and up - to - date information on the link between diet and acne .

sequence analysis included a total of 335 patients ( 191 female , 144 male ) with definite or suspected clinical diagnosis of stgd1 who underwent routine genetic testing at the institute of human genetics regensburg . a positive family history of stgd1 was reported for 35 cases ; 131 individuals had a negative family history , and no further information on the familial status of retinal degeneration was available for 169 patients . information on ethnicity was mainly provided by the patients themselves or was estimated from their first or last name . a total of 283 patients were caucasian ( 80% ) , 30 patients were from the middle east , 1 patient was from asia , and for 21 patients ethnicity could not unanimously be determined . a control group of 779 probands free of retinal disease was recruited from the lower franconian region in germany and used to assess the role of common variants in stgd1 pathology . in accordance with the german genetic diagnostics act , written informed consent this study adhered to the tenets of the declaration of helsinki . for dna extractions , mutational analyses of the coding exons and the flanking intronic sequences [ 12 bp/+6 bp covering the key donor ( ( c|a ) a g g t ( a|g ) a g t ) and acceptor ( y10 n ( c|t ) a g ( g|a ) ) splice signals at the exon intron boundaries ] of the abca4 gene ( nm_00350.2 ) were mainly performed ( 186 patients ) by a custom - designed genechip customseq resequencing array ( retchip ) ( affymetrix , santa clara , ca , usa ) . multiplex - pcr products were amplified by exon flanking oligonucleotide primer pairs ( oligonucleotide sequences will be provided upon request ) . the data were analyzed with the affymetrix genechip analysis software ( gseq ) and the seqc module ( jsi medical systems , kippenheim , germany ) as previously described . for 149 patients , mutation testing was performed by next generation sequencing ( ngs ) on an ion torrent semiconductor personal sequencing machine ( life technologies , darmstadt , germany ) upon multiplex - pcr amplification of the fragments with the stargardt mastr kit ( multiplicom , niel , belgium ) . regions with a depth of sequence coverage below 30 were reanalyzed by sanger chain - terminating dideoxynucleotide sequencing on an abi 3130xl instrument ( applied biosystems , carlsbad , ca , usa ) . next generation sequencing data were analyzed within the clc genomics workbench ( clc bio , aarhus , denmark ) . potentially disease - relevant variants identified by retchip or ngs were routinely confirmed by sanger sequencing . to test for deep intronic sequence variants in 236 patients , primer pairs abca4-exon30.1 fwd ( 5-tct gaa gtt ccc aca tag tac ttc c-3)/rev ( 5-gat tgt gtt tga gtg gct agg g-3 ) and abca4-exon36.1 fwd ( 5-act acc acc ttc ctg aca acc aa-3)/rev ( 5-cta cat ctc tct cca tag gct cag a-3 ) were used to amplify and sequence a 607- and 535-bp fragment within abca4 intervening sequences 30 and 36 , respectively . all variants are described according to current nomenclature guidelines ( http://www.hgvs.org/mutnomen ; in the public domain ) . variants were classified as recommended by the american college of medical genetics and genomics ( acmg ) standards and guidelines and the association for molecular pathology ( amp ) clinical practice guidelines and reports without discrimination between benign or likely benign and were based on population data , computational data , previous publications , and functional data . variant frequency in non - finnish europeans was taken from the browser of the exome aggregation consortium ( exac ) . a variant was classified as benign if the minor allele frequency ( maf ) was 0.005 except for variant c.2588g > c . variants with maf below 0.005 or with an unknown frequency were further tested for disease relevance by prediction algorithms of the programs mutationtaster ( mutation taster , http://www.mutationtaster.org/ ; in the public domain ) and polyphen-2 ( polyphen2 , http://genetics.bwh.harvard.edu/pph/ ; in the public domain ) . a sequence variant was classified as a variant of uncertain significance ( vus ) when none or only one of the two programs predicted a possible pathogenic effect . exceptions to this rule included missense mutations well documented in the literature to be disease associated in stgd1 patients . when both programs , mutationtaster and polyphen-2 , predicted a pathogenic effect , the variants were classified as stop and frameshift mutations as well as variants with experimental evidence of abca4 dysfunction were classified as pathogenic . in addition , rare variants were further analyzed for a potential effect on correct splicing by using the interface provided by the software package alamut ( interactive biosoftware , rouen , france ; alamut visual 2.7.1 ) , which combines five algorithms ( splicesitefinder , maxentscan , nnsplice , genesplicer , and human splicing finder ) . variants were selected for further testing by in vitro minigene splicing assays when the score differences calculated by four prediction programs were > 10% . genotyping of controls was performed on a humanexome bead chip ( illumina , san diego , ca , usa ) by the international amd genomics consortium . association testing and genetic risk score ( grs ) calculations were performed as described by grassmann et al . in brief , logistic regression analysis was used to assess the association of common alleles ( maf > 0.01 ) present in the coding regions of the abca4 gene . since variants rs1800717 and rs6666652 are highly correlated ( pairwise r = 1 , based on the eur panel in the 1000 genomes project , build 20130502 , http://www.internationalgenome.org/ ; in the public domain ) , only rs6666652 was used in the analysis . variants with a maf 0.01 and rare deep intronic variants were not included in further calculations . initially , we determined the odds ratios ( or ) , 95% confidence intervals ( 95% ci ) , and p values for each common variant . we adjusted the resulting p values according to the false discovery rate ( fdr ) and considered variants with an fdr < 0.05 to be statistically significant . a grs was calculated from significantly associated variants for all controls and patients from a multivariate logistic regression model . this was done by multiplying the number of risk - modulating alleles ( which can be either 0 , 1 , or 2 ) with the respective logarithmic odds ratio ( log odds , computed from the multivariate model ) ; then the sum of all weighted genotypes was calculated . finally , a constant ( 1.0503 ) was subtracted from the score to center the risk score distribution of the controls on zero . exons harboring spliceogenic variants and control exons were pcr amplified from genomic dna using a 7:1 ratio of taq : pfu polymerase and oligonucleotide primer pairs flanked by restriction sites for ecori and bamhi ( supplementary table s1 ) . hek293 cells were transfected in duplicate with 3 g construct dna using transit - lt1 transfection reagent ( mirus bio , madison , wi , usa ) and harvested after 48 hours ; total rna was extracted using rneasy micro kit ( qiagen ) . pcr was performed with the revertaidh first strand cdna synthesis kit ( thermo fisher scientific , waltham , ma , usa ) and a pspl3-specific sd6 ( 5-tct gag tca cct gga caa cc-3 ) and sa4 ( 5-cac ctg agg agt gaa ttg gtc g-3 ) primer pair in combination with oligonucleotide primers for the housekeeping genes actb ( forward 5-gac atc cgc aaa gac ctg ta-3 ; reverse ( 5-cag gag agc aat gat ctt ga-3 ) and gusb ( forward 5-gat cca cct ctg atg ttc ac-3 ; reverse ( 5-tat tcc cca gca ctc tcg tc-3 ) . polymerase chain reaction products were separated by electrophoresis on a 2% agarose gel , excised , cloned , and sequenced . fragment analysis was done by pcr amplification with fluorescein amidite ( fam)labeled sd6 using phusion flash high - fidelity pcr master mix ( thermo fisher scientific ) . samples and a liz size standard ( applied biosystems , darmstadt , germany ) were run on an abi 3130xl sequencer and analyzed with the peak scanner software ( applied biosystems ) . sequence analysis included a total of 335 patients ( 191 female , 144 male ) with definite or suspected clinical diagnosis of stgd1 who underwent routine genetic testing at the institute of human genetics regensburg . a positive family history of stgd1 was reported for 35 cases ; 131 individuals had a negative family history , and no further information on the familial status of retinal degeneration was available for 169 patients . information on ethnicity was mainly provided by the patients themselves or was estimated from their first or last name . a total of 283 patients were caucasian ( 80% ) , 30 patients were from the middle east , 1 patient was from asia , and for 21 patients ethnicity could not unanimously be determined . a control group of 779 probands free of retinal disease was recruited from the lower franconian region in germany and used to assess the role of common variants in stgd1 pathology . in accordance with the german genetic diagnostics act , written informed consent mutational analyses of the coding exons and the flanking intronic sequences [ 12 bp/+6 bp covering the key donor ( ( c|a ) a g g t ( a|g ) a g t ) and acceptor ( y10 n ( c|t ) a g ( g|a ) ) splice signals at the exon intron boundaries ] of the abca4 gene ( nm_00350.2 ) were mainly performed ( 186 patients ) by a custom - designed genechip customseq resequencing array ( retchip ) ( affymetrix , santa clara , ca , usa ) . multiplex - pcr products were amplified by exon flanking oligonucleotide primer pairs ( oligonucleotide sequences will be provided upon request ) . the data were analyzed with the affymetrix genechip analysis software ( gseq ) and the seqc module ( jsi medical systems , kippenheim , germany ) as previously described . for 149 patients , mutation testing was performed by next generation sequencing ( ngs ) on an ion torrent semiconductor personal sequencing machine ( life technologies , darmstadt , germany ) upon multiplex - pcr amplification of the fragments with the stargardt mastr kit ( multiplicom , niel , belgium ) . regions with a depth of sequence coverage below 30 were reanalyzed by sanger chain - terminating dideoxynucleotide sequencing on an abi 3130xl instrument ( applied biosystems , carlsbad , ca , usa ) . next generation sequencing data were analyzed within the clc genomics workbench ( clc bio , aarhus , denmark ) . potentially disease - relevant variants identified by retchip or ngs were routinely confirmed by sanger sequencing . to test for deep intronic sequence variants in 236 patients , primer pairs abca4-exon30.1 fwd ( 5-tct gaa gtt ccc aca tag tac ttc c-3)/rev ( 5-gat tgt gtt tga gtg gct agg g-3 ) and abca4-exon36.1 fwd ( 5-act acc acc ttc ctg aca acc aa-3)/rev ( 5-cta cat ctc tct cca tag gct cag a-3 ) were used to amplify and sequence a 607- and 535-bp fragment within abca4 intervening sequences 30 and 36 , respectively . all variants are described according to current nomenclature guidelines ( http://www.hgvs.org/mutnomen ; in the public domain ) . variants were classified as recommended by the american college of medical genetics and genomics ( acmg ) standards and guidelines and the association for molecular pathology ( amp ) clinical practice guidelines and reports without discrimination between benign or likely benign and were based on population data , computational data , previous publications , and functional data . variant frequency in non - finnish europeans was taken from the browser of the exome aggregation consortium ( exac ) . a variant was classified as benign if the minor allele frequency ( maf ) was 0.005 except for variant c.2588g > c . variants with maf below 0.005 or with an unknown frequency were further tested for disease relevance by prediction algorithms of the programs mutationtaster ( mutation taster , http://www.mutationtaster.org/ ; in the public domain ) and polyphen-2 ( polyphen2 , http://genetics.bwh.harvard.edu/pph/ ; in the public domain ) . a sequence variant was classified as a variant of uncertain significance ( vus ) when none or only one of the two programs predicted a possible pathogenic effect . exceptions to this rule included missense mutations well documented in the literature to be disease associated in stgd1 patients . when both programs , mutationtaster and polyphen-2 , predicted a pathogenic effect , the variants were classified as stop and frameshift mutations as well as variants with experimental evidence of abca4 dysfunction were classified as pathogenic . in addition , rare variants were further analyzed for a potential effect on correct splicing by using the interface provided by the software package alamut ( interactive biosoftware , rouen , france ; alamut visual 2.7.1 ) , which combines five algorithms ( splicesitefinder , maxentscan , nnsplice , genesplicer , and human splicing finder ) . variants were selected for further testing by in vitro minigene splicing assays when the score differences calculated by four prediction programs were > 10% . genotyping of controls was performed on a humanexome bead chip ( illumina , san diego , ca , usa ) by the international amd genomics consortium . association testing and genetic risk score ( grs ) calculations were performed as described by grassmann et al . in brief , logistic regression analysis was used to assess the association of common alleles ( maf > 0.01 ) present in the coding regions of the abca4 gene . since variants rs1800717 and rs6666652 are highly correlated ( pairwise r = 1 , based on the eur panel in the 1000 genomes project , build 20130502 , http://www.internationalgenome.org/ ; in the public domain ) , only rs6666652 was used in the analysis . variants with a maf 0.01 and rare deep intronic variants were not included in further calculations . initially , we determined the odds ratios ( or ) , 95% confidence intervals ( 95% ci ) , and p values for each common variant . we adjusted the resulting p values according to the false discovery rate ( fdr ) and considered variants with an fdr < 0.05 to be statistically significant . a grs was calculated from significantly associated variants for all controls and patients from a multivariate logistic regression model . this was done by multiplying the number of risk - modulating alleles ( which can be either 0 , 1 , or 2 ) with the respective logarithmic odds ratio ( log odds , computed from the multivariate model ) ; then the sum of all weighted genotypes was calculated . finally , a constant ( 1.0503 ) was subtracted from the score to center the risk score distribution of the controls on zero . exons harboring spliceogenic variants and control exons were pcr amplified from genomic dna using a 7:1 ratio of taq : pfu polymerase and oligonucleotide primer pairs flanked by restriction sites for ecori and bamhi ( supplementary table s1 ) . hek293 cells were transfected in duplicate with 3 g construct dna using transit - lt1 transfection reagent ( mirus bio , madison , wi , usa ) and harvested after 48 hours ; total rna was extracted using rneasy micro kit ( qiagen ) . pcr was performed with the revertaidh first strand cdna synthesis kit ( thermo fisher scientific , waltham , ma , usa ) and a pspl3-specific sd6 ( 5-tct gag tca cct gga caa cc-3 ) and sa4 ( 5-cac ctg agg agt gaa ttg gtc g-3 ) primer pair in combination with oligonucleotide primers for the housekeeping genes actb ( forward 5-gac atc cgc aaa gac ctg ta-3 ; reverse ( 5-cag gag agc aat gat ctt ga-3 ) and gusb ( forward 5-gat cca cct ctg atg ttc ac-3 ; reverse ( 5-tat tcc cca gca ctc tcg tc-3 ) . polymerase chain reaction products were separated by electrophoresis on a 2% agarose gel , excised , cloned , and sequenced . fragment analysis was done by pcr amplification with fluorescein amidite ( fam)labeled sd6 using phusion flash high - fidelity pcr master mix ( thermo fisher scientific ) . samples and a liz size standard ( applied biosystems , darmstadt , germany ) were run on an abi 3130xl sequencer and analyzed with the peak scanner software ( applied biosystems ) . mutational analysis of the abca4 gene was performed in 335 stgd1 patients and identified 189 unique sequence variations , of which 171 were listed in the non - finnish european exac cohort as a rare variant with maf 0.005 . of the 171 rare abca4 variants , 148 were classified as pathogenic or likely pathogenic and 23 as vus ( supplementary table s2 ) . twenty - eight percent ( 48/171 ) of the rare alleles constitute novel as yet unpublished variants ( table 1 ) . missense mutations represent 59.6% ( 102/171 ) followed by nonsense and frameshift mutations ( 15.8% ; 27/171 ) , splice site mutations ( 13.5% ; 23/171 ) and synonymous variants ( 8.8% ; 15/171 ) . of note , variant c.2588g > c is present with a maf > 0.005 ( 0.0063 ; exac ) but was classified as pathogenic based on its known deleterious effect on abca4 protein function . novel rare sequence variants in the abca4 gene the most common pathogenic or likely pathogenic mutations in our cohort were c.5882g > a ( 51 of 396 mutant alleles ) , c.[3113c > t(;)1622t > c ] ( 36/396 ) , c.2588g > c ( 28/396 ) , and c.5461 - 10t > c ( 23/396 ) , which is consistent with our earlier report of frequent mutations in 144 german stgd1 patients . together , these sequence changes account for 34.8% ( 138/396 ) of disease - associated variants . ninety - nine mutations were identified in only one patient ( supplementary table s2 ) . twenty - eight sequence variants were considered to have a potential impact on splicing of the mature abca4 transcript based on results from the bioinformatic analysis ( supplementary table s2 ) . experimental evidence for splicing defects was previously published for five of these ( c.768g > t , c.2588g > c , c.4253 + 5g > a , c.5461 - 10t > c , c.5714 + 5g > a ) ( supplementary table s2 ) . here , functional pspl3-based minigene assays were performed for another 22 variants ( table 2 ; supplementary fig . the analysis of cdnas derived from transfected hek293 cells revealed aberrant pre - mrna splicing for all of the 13 variants affecting canonical gt - ag splice sequences and for c.4773 + 3a > g located in the conserved donor splice site of abca4 exon 33 . this includes three mutations changing the canonical gt to the weaker gc 5 splice site , which is present in 1% of authentic introns . the absence of functional donor or acceptor splice sites led to exon skipping , intron retention , and use of cryptic splice sites ( table 2 ; supplementary fig . s1 ) , likely leading to truncated protein products or nonsense - mediated mrna decay ( nmd ) . functional analysis of putative splice mutations in the abca4 gene of the eight exonic sequence changes included in the minigene assay , three stop mutations and four missense mutations were predicted to create cryptic canonical splice sites , whereas the synonymous mutation c.3813g > a could weaken the donor splice site of exon 25 ( table 2 ) . synonymous mutation c.3813g > a and missense mutations c.4469g > a , c.4919g > a , and c.6647c > t led to incorrectly spliced transcripts that match the predictions from the bioinformatic analysis ( table 2 ; supplementary fig . s1 ) . hence , these four variants initially classified as likely pathogenic or vus were reclassified as being pathogenic ( supplementary table s2 ) . sequence variants c.122g > a / p.(w41 * ) , c.179c > t / p.(a60v ) , c.3808g > t / p.(e1270 * ) , and c.5189g > a / p.(w1730 * ) revealed no effect on mrna splicing in the in vitro assay . two or more likely pathogenic or pathogenic changes in the abca4 gene were found in 151 of the 335 stgd1 patients . in another seven patients a deleterious mutation was detected in addition to a vus , eventually raising the overall detection rate of two expected mutations to 47.2% ( supplementary table s3 ) . this includes 14 cases with null mutations : c.[2692g > t(;)5461 - 10t > c(;)5606c > t ] , c.[5018 + 2t > c(;)6386 + 2c > g ] , c.[296dupa];[5018 + 2t > c ] , c.[3813g > a(;)3813g > a ] , c.[4234c > t(;)5714 + 5g > a ] , c.[4773 + 3a > g(;)5461 - 10t > c ] , c.[5461 - 10t > c];[5461 - 10t > c ] , c.[67 - 1g > c];[67 - 1g > c ] , c.[1a > g ; 6089g > a];[5714 + 5g > a ] , c.[5714 + 5g > a(;)5714 + 5g > a ] , c.[5917delg];[5917delg ] , and c.[5714 + 5g > a];[5917delg ] . the latter two combinations were found in two patients ( i d 86 and i d 255 ) , and an additional 58 cases were carrying a null and a missense mutation ( supplementary table s3 ) . one pathogenic or likely pathogenic mutation was identified in 82 individuals , one vus in 13 cases resulting in a detection rate of 28.4% for carriers of a single abca4 mutation ( supplementary table s3 ) . complex alleles verified by testing the parent(s ) of the respective patients were c.[1a > g ; 6089g > a ] in patient 219 , c.[1609c > t ; 5881g > a ] in patient 314 , c.[1622t > c ; 3113c > t ] in patients 1 , 13 , 106 , 124 , 273 , 282 , and 311 , c.[2588g > c ; 3289a > t ] in patient 75 , and c.[2588g > c ; 5153t > g ] and c.[5318c > t ; 6148g > c ] together in patient 69 . in a total of 89 patients ( 26.6% ) no disease - associated variant was found in the abca4 gene . to investigate the frequency of deep intronic variants in the abca4 locus in our patient cohort , we sequenced genomic regions encompassing putative alternative exons in intron 30 and 36 , namely , exon 30.1 , exon 36.01 , and exon 36.1 . ten different sequence variants were found in 236 patients , of which four ( c.4539 + 2028c > t , c.4540 - 2036c > g ) were defined as rare variants with a maf 0.005 in the 1000 genomes catalog , phase 3 ( table 3 ; supplementary fig . these changes were each identified once in the 472 alleles analyzed . among these , c.4539 + 2028c > t and c.5196 + 1056a > g have been described previously by braun et al . ( 2013 ) as v5 and v3 , respectively , and were later also found in another cohort of stgd1 patients . deep intronic variants c.4540 - 2036c > a and c.5196 + 1013a > g are reported for the first time . the c.5196 + 1056a > g ( v3 ) variant is predicted to strengthen a cryptic donor splice site . pcr analysis using keratinocyte mrna of patients carrying the v3 variant revealed the inclusion of a 177-bp segment into the abca4 transcript ( termed alternative exon 36.01 ) , thus providing experimental evidence for a loss - of - function effect of this variation . patient 230 in our cohort carried the v3 variant and a second variant ( c.2588g > c ) annotated as pathogenic . this suggests that the v3 mutation is indeed the second disease - associated allele in our patient . a novel sequence change , c.5196 + 1013a > g , was found in patient 79 in combination with c.1654g > a , a likely pathogenic missense mutation [ p.(v552i ) ] . similar to the v3 variant , c.5196 + 1013a > g is predicted to strengthen a cryptic donor splice site and thus may also result in deleterious effects upon splicing . based on bioinformatics , variants c.4539 + 2028c > t ( v5 ) and c.4540 - 2036c > a are not predicted to affect splicing . the c.4539 + 2028c > t ( v5 ) sequence change was identified in patient 42 with no other probable mutant abca4 allele ; variant c.4540 - 2036c > a was detected in patient 159 in addition to pathogenic mutation c.[1622t six previously reported intronic sequence changes ( c.4539 + 2001g > a , c.4539 + 2064c > t , c.5196 + 1136c > a , c.5196 + 1137g > a , c.5196 + 1159g > a , c.5196 + 1216c > a ) were not found in our patient cohort ( table 3 ) . deep intronic variants in the abca4 gene we noticed a striking difference in the frequencies of common abca4 variants ( maf 0.01 ) in the patient versus the control group ( supplementary table s2 ; fig . 1 ) . this led us to investigate whether such variants may exert an effect on the risk for developing stgd1 . genotype information on 16 common abca4 variants was available for the 335 patients and 781 german controls from lower franconia . we found statistically significant association for six variations ( c.1268a > g , c.4203c > a , c.5603a > t , c.5682g > c , c.5843c > t , c.6249c > t ) ( fdr < 0.05 , supplementary table s4 ) . to estimate the independence of the associated variants , we fit a multivariate logistic regression model incorporating these variations . in the multivariate model , association of variant c.4203c > a ( rs1801666 ) with disease risk was no longer statistically significant , probably due to linkage disequilibrium with the strongly associated variant c.5603a > t ( rs1801466 , r = 0.38 ) . the remaining five risk - modulating variants were found to be independently associated with disease risk ( table 4 ) . repeating the analysis excluding the data from 52 samples of non - european or unknown ancestry , similar results were obtained , including the significant and independent association of the five risk - modulating variants ( c.1268a > g , c.5603a > t , c.5682g > c , c.5843c > t , c.6249c > t ) with similar effect sizes . frequency of common abca4 variants in patients with stargardt disease and european non - finnish controls . six variants that were found to be significantly enriched in either patients or controls are framed . association of common abca4 variants with stargardt disease assuming that common pathogenic mutations emerged on common haplotypes tagged by associated variants , our analysis may measure their impact on disease risk rather than that of the common associated variant . we therefore evaluated the correlation between common mutations in our cohort ( c.1622t > c , c.2588g > c , c.3113c > t , c.4234c > t , c.5461 - 10t > c , c.5714 + 5g > a , c.5882g > a , c.5917delg ) and the five risk - modulating variants ( c.1268a > g , c.5603a > t , c.5682g > c , three frequent mutations ( c.2588g > c , c.5461 - 10t > c , c.5882g > a ) were found to be potentially correlated ( r > 0.05 ) with common stgd1-associated variants . subsequently , the association analysis was repeated by excluding individuals carrying any of three correlated pathogenic mutations c.5882g > a , c.2588g > c , and c.5461 - 10t > c from the analysis . still , stgd1 association of common variants c.1268a > g and c.5603a > t remained highly significant ( p values of 3.45e-05 and 4.82e-05 , respectively ) . similarly , removing patients carrying one of the eight most common stgd1 mutations ( c.5882g > a , c.3113c > t , c.1622t > c , c.2588g > c , c.5461 - 10t > c , c.5714 + 5g > a , c.4234c > t , or c.5917delg ) from the analysis resulted in a statistically significant association for the common variants c.5603a > t and c.1268a > g ( p values of 0.000273 and 0.0109 , respectively ) ( supplementary table s5 ) . for variant c.6249c > t , the association was no longer significant after removal of patients carrying common mutations , although the variant still revealed a similar effect size . the latter finding suggests that loss of significance is likely due to lack of statistical power caused by the reduced number of individuals in the subsequent analyses ( from n = 335 to n = 247 to n = 197 patients , supplementary table s5 ) . in contrast , variant c.5682g > c showed a reduced effect size and also a lack of statistically significant association in both subsequent analyses . to further evaluate the role of these common variants in retinal pathology , a grs was calculated for the stgd1 patients in our cohort and the 781 control individuals . this was computed as the sum of risk - increasing and protective alleles weighted by the relative effect size of each of the risk - modulating variants from the multivariate logistic regression model . an alpha estimate of 1.0503 was used to center the grs on zero for the controls . patients diagnosed with stgd1 have a significantly higher mean grs ( 0.418 ; p value < 10 , 95% ci : 0.34790.4887 ) compared to controls ( 0.0 , 95% ci : 0.0466 to 0.0466 ) ( fig . importantly , patients with one , two , or more disease - associated abca4 mutations have a significantly higher grs than those with none ( p = 0.0011 and 0.0024 ; respectively ) ( fig . 2 ) , implying a risk - increasing effect of common abca4 alleles when combined with a single defective copy of the abca4 gene . genetic risk score ( grs ) for patients and controls stratified according to the number of abca4 mutations or disease status . an alpha estimate of 1.0503 was used to center the grs on zero for the controls . mutational analysis of the abca4 gene was performed in 335 stgd1 patients and identified 189 unique sequence variations , of which 171 were listed in the non - finnish european exac cohort as a rare variant with maf 0.005 . of the 171 rare abca4 variants , 148 were classified as pathogenic or likely pathogenic and 23 as vus ( supplementary table s2 ) . twenty - eight percent ( 48/171 ) of the rare alleles constitute novel as yet unpublished variants ( table 1 ) . missense mutations represent 59.6% ( 102/171 ) followed by nonsense and frameshift mutations ( 15.8% ; 27/171 ) , splice site mutations ( 13.5% ; 23/171 ) and synonymous variants ( 8.8% ; 15/171 ) . of note , variant c.2588g > c is present with a maf > 0.005 ( 0.0063 ; exac ) but was classified as pathogenic based on its known deleterious effect on abca4 protein function . novel rare sequence variants in the abca4 gene the most common pathogenic or likely pathogenic mutations in our cohort were c.5882g > a ( 51 of 396 mutant alleles ) , c.[3113c > t(;)1622t > c ] ( 36/396 ) , c.2588g > c ( 28/396 ) , and c.5461 - 10t > c ( 23/396 ) , which is consistent with our earlier report of frequent mutations in 144 german stgd1 patients . together , these sequence changes account for 34.8% ( 138/396 ) of disease - associated variants . ninety - nine mutations were identified in only one patient ( supplementary table s2 ) . twenty - eight sequence variants were considered to have a potential impact on splicing of the mature abca4 transcript based on results from the bioinformatic analysis ( supplementary table s2 ) . experimental evidence for splicing defects was previously published for five of these ( c.768g > t , c.2588g > c , c.4253 + 5g > a , c.5461 - 10t > c , c.5714 + 5g > a ) ( supplementary table s2 ) . here , functional pspl3-based minigene assays were performed for another 22 variants ( table 2 ; supplementary fig . the analysis of cdnas derived from transfected hek293 cells revealed aberrant pre - mrna splicing for all of the 13 variants affecting canonical gt - ag splice sequences and for c.4773 + 3a > g located in the conserved donor splice site of abca4 exon 33 . this includes three mutations changing the canonical gt to the weaker gc 5 splice site , which is present in 1% of authentic introns . the absence of functional donor or acceptor splice sites led to exon skipping , intron retention , and use of cryptic splice sites ( table 2 ; supplementary fig . s1 ) , likely leading to truncated protein products or nonsense - mediated mrna decay ( nmd ) . functional analysis of putative splice mutations in the abca4 gene of the eight exonic sequence changes included in the minigene assay , three stop mutations and four missense mutations were predicted to create cryptic canonical splice sites , whereas the synonymous mutation c.3813g > a could weaken the donor splice site of exon 25 ( table 2 ) . synonymous mutation c.3813g > a and missense mutations c.4469g > a , c.4919g > a , and c.6647c > t led to incorrectly spliced transcripts that match the predictions from the bioinformatic analysis ( table 2 ; supplementary fig . s1 ) . hence , these four variants initially classified as likely pathogenic or vus were reclassified as being pathogenic ( supplementary table s2 ) . sequence variants c.122g > a / p.(w41 * ) , c.179c > t / p.(a60v ) , c.3808g > t / p.(e1270 * ) , and c.5189g > a / p.(w1730 * ) revealed no effect on mrna splicing in the in vitro assay . two or more likely pathogenic or pathogenic changes in the abca4 gene were found in 151 of the 335 stgd1 patients . in another seven patients a deleterious mutation was detected in addition to a vus , eventually raising the overall detection rate of two expected mutations to 47.2% ( supplementary table s3 ) . this includes 14 cases with null mutations : c.[2692g > t(;)5461 - 10t > c(;)5606c > t ] , c.[5018 + 2t > c(;)6386 + 2c > g ] , c.[296dupa];[5018 + 2t > c ] , c.[3813g > a(;)3813g > a ] , c.[4234c > t(;)5714 + 5g > a ] , c.[4773 + 3a > g(;)5461 - 10t > c ] , c.[5461 - 10t > c];[5461 - 10t > c ] , c.[67 - 1g > c];[67 - 1g > c ] , c.[1a > g ; 6089g > a];[5714 + 5g > a ] , c.[5714 + 5g > a(;)5714 + 5g > a ] , c.[5917delg];[5917delg ] , and c.[5714 + 5g > a];[5917delg ] . the latter two combinations were found in two patients ( i d 86 and i d 255 ) , and an additional 58 cases were carrying a null and a missense mutation ( supplementary table s3 ) . one pathogenic or likely pathogenic mutation was identified in 82 individuals , one vus in 13 cases resulting in a detection rate of 28.4% for carriers of a single abca4 mutation ( supplementary table s3 ) . complex alleles verified by testing the parent(s ) of the respective patients were c.[1a > g ; 6089g > a ] in patient 219 , c.[1609c > t ; 5881g > a ] in patient 314 , c.[1622t > c ; 3113c > t ] in patients 1 , 13 , 106 , 124 , 273 , 282 , and 311 , c.[2588g > c ; 3289a > t ] in patient 75 , and c.[2588g > c ; 5153t > g ] and c.[5318c > t ; 6148g > c ] together in patient 69 . in a total of 89 patients ( 26.6% ) to investigate the frequency of deep intronic variants in the abca4 locus in our patient cohort , we sequenced genomic regions encompassing putative alternative exons in intron 30 and 36 , namely , exon 30.1 , exon 36.01 , and exon 36.1 . ten different sequence variants were found in 236 patients , of which four ( c.4539 + 2028c > t , c.4540 - 2036c > a , c.5196 + 1013a > g , c.5196 + 1056a > g ) were defined as rare variants with a maf 0.005 in the 1000 genomes catalog , phase 3 ( table 3 ; supplementary fig . s2 ) . these changes were each identified once in the 472 alleles analyzed . among these , c.4539 + 2028c > t and c.5196 + 1056a > g have been described previously by braun et al . ( 2013 ) as v5 and v3 , respectively , and were later also found in another cohort of stgd1 patients . deep intronic variants c.4540 - 2036c > a and c.5196 + 1013a > g are reported for the first time . the c.5196 + 1056a > g ( v3 ) variant is predicted to strengthen a cryptic donor splice site . pcr analysis using keratinocyte mrna of patients carrying the v3 variant revealed the inclusion of a 177-bp segment into the abca4 transcript ( termed alternative exon 36.01 ) , thus providing experimental evidence for a loss - of - function effect of this variation . patient 230 in our cohort carried the v3 variant and a second variant ( c.2588g > c ) annotated as pathogenic . this suggests that the v3 mutation is indeed the second disease - associated allele in our patient . a novel sequence change , c.5196 + 1013a > g , was found in patient 79 in combination with c.1654g > a , a likely pathogenic missense mutation [ p.(v552i ) ] . similar to the v3 variant , c.5196 + 1013a > g is predicted to strengthen a cryptic donor splice site and thus may also result in deleterious effects upon splicing . based on bioinformatics , variants c.4539 + 2028c > t ( v5 ) and c.4540 - 2036c > a are not predicted to affect splicing . the c.4539 + 2028c > t ( v5 ) sequence change was identified in patient 42 with no other probable mutant abca4 allele ; variant c.4540 - 2036c > a was detected in patient 159 in addition to pathogenic mutation c.[1622t c(;)3113c > t ] and likely pathogenic mutation c.3261a > c . a disease association of these intronic variants in our patients is thus unclear . six previously reported intronic sequence changes ( c.4539 + 2001g > a , c.4539 + 2064c > t , c.5196 + 1136c > a , c.5196 + 1137g > a , c.5196 + 1159g > a , c.5196 + 1216c > a ) were not found in our patient cohort ( table 3 ) . we noticed a striking difference in the frequencies of common abca4 variants ( maf 0.01 ) in the patient versus the control group ( supplementary table s2 ; fig . 1 ) . this led us to investigate whether such variants may exert an effect on the risk for developing stgd1 . genotype information on 16 common abca4 variants was available for the 335 patients and 781 german controls from lower franconia . for each variant , we found statistically significant association for six variations ( c.1268a > g , c.4203c > a , c.5603a > t , c.5682g > c , c.5843c > t , c.6249c > t ) ( fdr < 0.05 , supplementary table s4 ) . to estimate the independence of the associated variants , we fit a multivariate logistic regression model incorporating these variations . in the multivariate model , association of variant c.4203c > a ( rs1801666 ) with disease risk was no longer statistically significant , probably due to linkage disequilibrium with the strongly associated variant c.5603a > t ( rs1801466 , r = 0.38 ) . the remaining five risk - modulating variants were found to be independently associated with disease risk ( table 4 ) . repeating the analysis excluding the data from 52 samples of non - european or unknown ancestry , similar results were obtained , including the significant and independent association of the five risk - modulating variants ( c.1268a > g , c.5603a > t , c.5682g > c , c.5843c > t , c.6249c > t ) with similar effect sizes . frequency of common abca4 variants in patients with stargardt disease and european non - finnish controls . six variants that were found to be significantly enriched in either patients or controls are framed . association of common abca4 variants with stargardt disease assuming that common pathogenic mutations emerged on common haplotypes tagged by associated variants , our analysis may measure their impact on disease risk rather than that of the common associated variant . we therefore evaluated the correlation between common mutations in our cohort ( c.1622t > c , c.2588g > c , c.3113c > t , c.4234c > t , c.5461 - 10t > c , c.5714 + 5g > a , c.5882g > a , c.5917delg ) and the five risk - modulating variants ( c.1268a > g , c.5603a > t , c.5682g > c , three frequent mutations ( c.2588g > c , c.5461 - 10t > c , c.5882g > a ) were found to be potentially correlated ( r > 0.05 ) with common stgd1-associated variants . subsequently , the association analysis was repeated by excluding individuals carrying any of three correlated pathogenic mutations c.5882g > a , c.2588g > c , and c.5461 - 10t > c from the analysis . still , stgd1 association of common variants c.1268a > g and c.5603a > t remained highly significant ( p values of 3.45e-05 and 4.82e-05 , respectively ) . similarly , removing patients carrying one of the eight most common stgd1 mutations ( c.5882g > a , c.3113c > t , c.1622t > c , c.2588g > c , c.5461 - 10t > c , c.5714 + 5g > a , c.4234c > t , or c.5917delg ) from the analysis resulted in a statistically significant association for the common variants c.5603a > t and c.1268a > g ( p values of 0.000273 and 0.0109 , respectively ) ( supplementary table s5 ) . for variant c.6249c > t , the association was no longer significant after removal of patients carrying common mutations , although the variant still revealed a similar effect size . the latter finding suggests that loss of significance is likely due to lack of statistical power caused by the reduced number of individuals in the subsequent analyses ( from n = 335 to n = 247 to n = 197 patients , supplementary table s5 ) . in contrast , variant c.5682g > c showed a reduced effect size and also a lack of statistically significant association in both subsequent analyses . to further evaluate the role of these common variants in retinal pathology , a grs was calculated for the stgd1 patients in our cohort and the 781 control individuals . this was computed as the sum of risk - increasing and protective alleles weighted by the relative effect size of each of the risk - modulating variants from the multivariate logistic regression model . an alpha estimate of 1.0503 was used to center the grs on zero for the controls . patients diagnosed with stgd1 have a significantly higher mean grs ( 0.418 ; p value < 10 , 95% ci : 0.34790.4887 ) compared to controls ( 0.0 , 95% ci : 0.0466 to 0.0466 ) ( fig . importantly , patients with one , two , or more disease - associated abca4 mutations have a significantly higher grs than those with none ( p = 0.0011 and 0.0024 ; respectively ) ( fig . 2 ) , implying a risk - increasing effect of common abca4 alleles when combined with a single defective copy of the abca4 gene . genetic risk score ( grs ) for patients and controls stratified according to the number of abca4 mutations or disease status . an alpha estimate of 1.0503 was used to center the grs on zero for the controls . almost 20 years after the identification of abca4 as the causative gene for autosomal recessive stgd1 , the genetics of this common form of hereditary macular degeneration still have not fully been clarified . our study describes the findings of mutational profiling of the abca4 gene in the largest cohort of stgd1 patients published so far . a total of 171 unique rare sequence variants , 48 of which constitute novel , so far unreported , mutations , were identified in the coding region , and an additional four rare deep intronic variants were detected in intron 30 and 36 of the abca4 gene . in 47% of patients , this is at the lower end of the mutation detection rate in stgd1 patients , with other studies identifying biallelic mutations between 55% and 80% , but may be explained by a combination of factors . first , clinical heterogeneity and variable diagnostic criteria inherent to multiclinic retrospective cohort studies may have led to uncertainties in clinical diagnosis in some patients of our cohort . in addition , small deletions / insertions may have been missed due to limitations in the detection accuracy of this type of mutation by the resequencing array and ion torrent semiconductor technology . finally , large rearrangements within the abca4 locus that have been shown to occur in 0.3% to 2.2% of the analyzed chromosomes in previous reports would not have been detected by the mutational screening in this study . an important question relates to the high proportion of carriers of single abca4 mutations consistently observed in numerous studies regardless of the ethnic background of the patients . a single mutant abca4 allele was found in almost 30% of the 335 individuals in our patient group , a number representing the upper end of the reported ranges . mutations in noncoding regions of the abca4 gene locus have been proposed as a common source for a second causative mutation . more specifically , several studies have described the occurrence of deep intronic variants in intron 30 and 36 in a significant proportion of patients ( 7.917.9% ) with single abca4 mutations . indeed , there is some experimental evidence that a few of these variants activate / strengthen cryptic splice sites leading to the inclusion of alternative exons in the mature transcript . in our cohort , of 95 stgd patients with a single pathogenic / likely pathogenic abca4 mutation , only 2 were found to carry an additional rare intronic variant in intron 30 or 36 ( 2.1% ) . two other novel rare intronic variants were detected in a patient with two causative abca4 mutations and a patient with no abca4 mutations , respectively . from these data , we conclude that causative abca4 mutations in intron 30 and 36 may contribute to stgd1 pathogenesis only in a minority of cases . similarly , in a small german cohort of 46 patients with single heterozygous abca4 variants , no mutations were found in these intronic regions . to further add to the discussion on the impact of deep intronic variants to stgd1 disease , the full sequence analysis of the entire abca4 locus including exonic as well as intronic sequences will be needed independent of the patient 's mutation status . it was estimated that approximately 95% of multiexon transcripts undergo alternative splicing . while the key components and mechanisms involved in mrna processing are reasonably well understood , high - confidence predictions on splicing effects of defined sequence changes remain inaccurate . in this context , it is of note that the extent and variability of alternative splicing in the human retina were found to be remarkable , although the functional consequences of the alternative transcripts remain obscure . approximately 13% of novel mrna junctions were expressed in the retina at levels similar to or higher than the reference transcript . deep rna sequencing of three healthy human retinas revealed at least 11 alternatively spliced exons and 71 alternative exon / intron junctions , implying the possibility of additional deep intronic regions as candidates to potentially harbor mutations affecting the correct splicing of the abca4 transcript . considering that 7.6% of stgd1 patients of a belgian cohort carried a founder deep intronic c.4539 + 2001g > a mutation , it is conceivable that a significant fraction of the german stgd1 patients carry as yet unknown mutations in the noncoding regions of the abca4 gene . to address the strikingly high frequency of carriers with only a single identified abca4 disease allele , we assessed the contribution of common abca4 variants on disease development . to this end , our findings reveal that variants c.1268a > g ( or 0.6682 ; ci 0.5310.836 ) , c.5843c > t ( or 0.1678 ; 0.0630.377 ) , and c.6249c > t ( or 0.6483 ; ci 0.4071.001 ) exert a protective effect , while variants c.5603a > t ( or 1.9652 ; ci 1.3352.909 ) and c.5682g > c ( or 1.3973 ; ci 1.0821.800 ) confer an increased risk for developing stgd1 . these results are in agreement with and extend previous studies that identified c.5603a > t as a risk - increasing allele and c.1268a > g as a protective variant . our data now facilitate a grs estimate that coalesces the total effect of protective and risk - increasing alleles similarly to the model developed to assess the genetic risk for amd . interestingly , the grs in the group of patients with only a single disease - associated abca4 mutation is significantly higher than in patients with no abca4 mutation , suggesting that common variants are involved in conferring risk for stgd1 . simultaneous loss of significant association with stgd1 and effect size was observed for common variant c.5682g > c when the three most frequent pathogenic mutations were excluded from the analysis . this could be due to linkage disequilibrium between certain pathogenic mutations and c.5682g > c , implying that the common abca4 allele either has no effect on the pathogenicity of the rare variant or adds to the damaging effect of the mutation . furthermore , c.5682g > c may act as a risk - increasing allele only in trans when combined with specific pathogenic mutations . the assumption that the presence of a common genetic variant with an adverse effect on abca4 function actually triggers the deleterious consequences of the pathogenic mutation could explain why some of the common mutations have a frequency of > 1% in certain populations but do probably not cause stgd1 in the respective carriers . for example , the allele frequency of pathogenic mutation c.5882g > a / p.(g1961e ) is 1.4% in the south asian population ( exac ) and even higher in somalia , although a higher prevalence of stgd1 in these countries has not been reported . alternatively , a protective abca4 allele may ameliorate a pathogenic mutation in trans , although only in combination with a less severe abca4 allele . in this context , it should be noted that protective variant c.6249c > t is five times more frequent in the african than in the european or asian populations . fourteen stgd1 patients in our study each harbor two null mutations in the abca4 gene . compound heterozygosity / homozygosity could be confirmed in seven of these patients . in accordance with the hypothesis by maugeri et al . , the presence of the severe mutations in our patients would strongly suggest a cone rod dystrophy or an atypical retinitis pigmentosa phenotype , thus reflecting the greatly reduced or even absent functional activity of abca4 . a review of the clinical data , however , verified that all 14 patients presented with typical symptoms of stgd1 disease with age of onset ranging between 4 and 60 years . more precisely , eight patients presented with initial symptoms at the age of 11 or younger ; two patients were under 20 ; and the remaining four patients were 30 to 60 years old when they first noticed vision problems . in another report , one - third of the nontruncating abca4 alleles were found to cause more severe disease than premature truncations . thus , specific common abca4 variants may alleviate deleterious mutations leading to asymptomatic , mild , or less severe disease and vice versa . taken together , future studies aiming at investigation of the influence of common variations and rare mutations on disease risk are necessary , ideally in a prospective cohort setting . in summary , despite certain shortcomings in terms of possible uncertainties in clinical diagnosis and the paucity of detecting large copy number variations ( cnvs ) or mutations in noncoding regions , the present study further broadens our knowledge about the genetic basis of stgd1 disease . first , we expanded the mutational spectrum of stgd1 by identifying an additional 48 novel pathogenic / likely pathogenic gene variants in abca4 . our findings also add to the clarification of the role of deep intronic variants in putative alternative exons within intron 30 and 36 , which seem to represent only a minor proportion of disease - associated variants . finally , an in - depth analysis of common variants at the abca4 gene locus revealed risk - altering properties for several common variants , particularly in those patients who carry only a single disease - associated abca4 allele . furthermore , grs data can be used to ascertain the risk of a patient with a single abca4 mutation and may further refine our understanding of abca4-associated retinal pathology .

purposestargardt disease ( stgd1 ) is an autosomal recessive retinopathy , caused by mutations in the retina - specific atp - binding cassette transporter ( abca4 ) gene . to establish the mutational spectrum and to assess effects of selected deep intronic and common genetic variants on disease , we performed a comprehensive sequence analysis in a large cohort of german stgd1 patients.methodsdna samples of 335 stgd1 patients were analyzed for abca4 mutations in its 50 coding exons and adjacent intronic sequences by resequencing array technology or next generation sequencing ( ngs ) . parts of intron 30 and 36 were screened by sanger chain - terminating dideoxynucleotide sequencing . an in vitro splicing assay was used to test selected variants for their splicing behavior . by logistic regression analysis we assessed the association of common abca4 alleles while a multivariate logistic regression model calculated a genetic risk score ( grs).resultsour analysis identified 148 pathogenic or likely pathogenic mutations , of which 48 constitute so far unpublished abca4-associated disease alleles . four rare deep intronic variants were found once in 472 alleles analyzed . in addition , we identified six risk - modulating common variants . genetic risk score estimates suggest that defined common abca4 variants influence disease risk in carriers of a single pathogenic abca4 allele.conclusionsour study adds to the mutational spectrum of the abca4 gene . moreover , in our cohort , deep intronic variants in intron 30 and 36 likely play no or only a minor role in disease pathology . of note , our findings demonstrate a possible modifying effect of common sequence variants on abca4-associated disease .

Materials and Methods Patients Molecular Analysis Bioinformatic Analyses and Variant Classification Genetic Risk Score Calculation Minigene Assay Results Identification of Impact of Selected Disease-Associated ABCA4 Alleles in 335 STGD Patients Frequency of Deep Intronic Variants in Putative Alternative Exons of the The Contribution of Common Discussion Supplementary Material

a control group of 779 probands free of retinal disease was recruited from the lower franconian region in germany and used to assess the role of common variants in stgd1 pathology . for dna extractions , mutational analyses of the coding exons and the flanking intronic sequences [ 12 bp/+6 bp covering the key donor ( ( c|a ) a g g t ( a|g ) a g t ) and acceptor ( y10 n ( c|t ) a g ( g|a ) ) splice signals at the exon intron boundaries ] of the abca4 gene ( nm_00350.2 ) were mainly performed ( 186 patients ) by a custom - designed genechip customseq resequencing array ( retchip ) ( affymetrix , santa clara , ca , usa ) . for 149 patients , mutation testing was performed by next generation sequencing ( ngs ) on an ion torrent semiconductor personal sequencing machine ( life technologies , darmstadt , germany ) upon multiplex - pcr amplification of the fragments with the stargardt mastr kit ( multiplicom , niel , belgium ) . regions with a depth of sequence coverage below 30 were reanalyzed by sanger chain - terminating dideoxynucleotide sequencing on an abi 3130xl instrument ( applied biosystems , carlsbad , ca , usa ) . to test for deep intronic sequence variants in 236 patients , primer pairs abca4-exon30.1 fwd ( 5-tct gaa gtt ccc aca tag tac ttc c-3)/rev ( 5-gat tgt gtt tga gtg gct agg g-3 ) and abca4-exon36.1 fwd ( 5-act acc acc ttc ctg aca acc aa-3)/rev ( 5-cta cat ctc tct cca tag gct cag a-3 ) were used to amplify and sequence a 607- and 535-bp fragment within abca4 intervening sequences 30 and 36 , respectively . in addition , rare variants were further analyzed for a potential effect on correct splicing by using the interface provided by the software package alamut ( interactive biosoftware , rouen , france ; alamut visual 2.7.1 ) , which combines five algorithms ( splicesitefinder , maxentscan , nnsplice , genesplicer , and human splicing finder ) . in brief , logistic regression analysis was used to assess the association of common alleles ( maf > 0.01 ) present in the coding regions of the abca4 gene . a grs was calculated from significantly associated variants for all controls and patients from a multivariate logistic regression model . a control group of 779 probands free of retinal disease was recruited from the lower franconian region in germany and used to assess the role of common variants in stgd1 pathology . in accordance with the german genetic diagnostics act , written informed consent mutational analyses of the coding exons and the flanking intronic sequences [ 12 bp/+6 bp covering the key donor ( ( c|a ) a g g t ( a|g ) a g t ) and acceptor ( y10 n ( c|t ) a g ( g|a ) ) splice signals at the exon intron boundaries ] of the abca4 gene ( nm_00350.2 ) were mainly performed ( 186 patients ) by a custom - designed genechip customseq resequencing array ( retchip ) ( affymetrix , santa clara , ca , usa ) . for 149 patients , mutation testing was performed by next generation sequencing ( ngs ) on an ion torrent semiconductor personal sequencing machine ( life technologies , darmstadt , germany ) upon multiplex - pcr amplification of the fragments with the stargardt mastr kit ( multiplicom , niel , belgium ) . regions with a depth of sequence coverage below 30 were reanalyzed by sanger chain - terminating dideoxynucleotide sequencing on an abi 3130xl instrument ( applied biosystems , carlsbad , ca , usa ) . to test for deep intronic sequence variants in 236 patients , primer pairs abca4-exon30.1 fwd ( 5-tct gaa gtt ccc aca tag tac ttc c-3)/rev ( 5-gat tgt gtt tga gtg gct agg g-3 ) and abca4-exon36.1 fwd ( 5-act acc acc ttc ctg aca acc aa-3)/rev ( 5-cta cat ctc tct cca tag gct cag a-3 ) were used to amplify and sequence a 607- and 535-bp fragment within abca4 intervening sequences 30 and 36 , respectively . in addition , rare variants were further analyzed for a potential effect on correct splicing by using the interface provided by the software package alamut ( interactive biosoftware , rouen , france ; alamut visual 2.7.1 ) , which combines five algorithms ( splicesitefinder , maxentscan , nnsplice , genesplicer , and human splicing finder ) . in brief , logistic regression analysis was used to assess the association of common alleles ( maf > 0.01 ) present in the coding regions of the abca4 gene . a grs was calculated from significantly associated variants for all controls and patients from a multivariate logistic regression model . mutational analysis of the abca4 gene was performed in 335 stgd1 patients and identified 189 unique sequence variations , of which 171 were listed in the non - finnish european exac cohort as a rare variant with maf 0.005 . of the 171 rare abca4 variants , 148 were classified as pathogenic or likely pathogenic and 23 as vus ( supplementary table s2 ) . novel rare sequence variants in the abca4 gene the most common pathogenic or likely pathogenic mutations in our cohort were c.5882g > a ( 51 of 396 mutant alleles ) , c.[3113c > t(;)1622t > c ] ( 36/396 ) , c.2588g > c ( 28/396 ) , and c.5461 - 10t > c ( 23/396 ) , which is consistent with our earlier report of frequent mutations in 144 german stgd1 patients . functional analysis of putative splice mutations in the abca4 gene of the eight exonic sequence changes included in the minigene assay , three stop mutations and four missense mutations were predicted to create cryptic canonical splice sites , whereas the synonymous mutation c.3813g > a could weaken the donor splice site of exon 25 ( table 2 ) . two or more likely pathogenic or pathogenic changes in the abca4 gene were found in 151 of the 335 stgd1 patients . one pathogenic or likely pathogenic mutation was identified in 82 individuals , one vus in 13 cases resulting in a detection rate of 28.4% for carriers of a single abca4 mutation ( supplementary table s3 ) . to investigate the frequency of deep intronic variants in the abca4 locus in our patient cohort , we sequenced genomic regions encompassing putative alternative exons in intron 30 and 36 , namely , exon 30.1 , exon 36.01 , and exon 36.1 . ten different sequence variants were found in 236 patients , of which four ( c.4539 + 2028c > t , c.4540 - 2036c > g ) were defined as rare variants with a maf 0.005 in the 1000 genomes catalog , phase 3 ( table 3 ; supplementary fig . these changes were each identified once in the 472 alleles analyzed . deep intronic variants in the abca4 gene we noticed a striking difference in the frequencies of common abca4 variants ( maf 0.01 ) in the patient versus the control group ( supplementary table s2 ; fig . to estimate the independence of the associated variants , we fit a multivariate logistic regression model incorporating these variations . the remaining five risk - modulating variants were found to be independently associated with disease risk ( table 4 ) . repeating the analysis excluding the data from 52 samples of non - european or unknown ancestry , similar results were obtained , including the significant and independent association of the five risk - modulating variants ( c.1268a > g , c.5603a > t , c.5682g > c , c.5843c > t , c.6249c > t ) with similar effect sizes . frequency of common abca4 variants in patients with stargardt disease and european non - finnish controls . association of common abca4 variants with stargardt disease assuming that common pathogenic mutations emerged on common haplotypes tagged by associated variants , our analysis may measure their impact on disease risk rather than that of the common associated variant . we therefore evaluated the correlation between common mutations in our cohort ( c.1622t > c , c.2588g > c , c.3113c > t , c.4234c > t , c.5461 - 10t > c , c.5714 + 5g > a , c.5882g > a , c.5917delg ) and the five risk - modulating variants ( c.1268a > g , c.5603a > t , c.5682g > c , three frequent mutations ( c.2588g > c , c.5461 - 10t > c , c.5882g > a ) were found to be potentially correlated ( r > 0.05 ) with common stgd1-associated variants . to further evaluate the role of these common variants in retinal pathology , a grs was calculated for the stgd1 patients in our cohort and the 781 control individuals . this was computed as the sum of risk - increasing and protective alleles weighted by the relative effect size of each of the risk - modulating variants from the multivariate logistic regression model . 2 ) , implying a risk - increasing effect of common abca4 alleles when combined with a single defective copy of the abca4 gene . genetic risk score ( grs ) for patients and controls stratified according to the number of abca4 mutations or disease status . mutational analysis of the abca4 gene was performed in 335 stgd1 patients and identified 189 unique sequence variations , of which 171 were listed in the non - finnish european exac cohort as a rare variant with maf 0.005 . of the 171 rare abca4 variants , 148 were classified as pathogenic or likely pathogenic and 23 as vus ( supplementary table s2 ) . novel rare sequence variants in the abca4 gene the most common pathogenic or likely pathogenic mutations in our cohort were c.5882g > a ( 51 of 396 mutant alleles ) , c.[3113c > t(;)1622t > c ] ( 36/396 ) , c.2588g > c ( 28/396 ) , and c.5461 - 10t > c ( 23/396 ) , which is consistent with our earlier report of frequent mutations in 144 german stgd1 patients . functional analysis of putative splice mutations in the abca4 gene of the eight exonic sequence changes included in the minigene assay , three stop mutations and four missense mutations were predicted to create cryptic canonical splice sites , whereas the synonymous mutation c.3813g > a could weaken the donor splice site of exon 25 ( table 2 ) . two or more likely pathogenic or pathogenic changes in the abca4 gene were found in 151 of the 335 stgd1 patients . one pathogenic or likely pathogenic mutation was identified in 82 individuals , one vus in 13 cases resulting in a detection rate of 28.4% for carriers of a single abca4 mutation ( supplementary table s3 ) . in a total of 89 patients ( 26.6% ) to investigate the frequency of deep intronic variants in the abca4 locus in our patient cohort , we sequenced genomic regions encompassing putative alternative exons in intron 30 and 36 , namely , exon 30.1 , exon 36.01 , and exon 36.1 . ten different sequence variants were found in 236 patients , of which four ( c.4539 + 2028c > t , c.4540 - 2036c > a , c.5196 + 1013a > g , c.5196 + 1056a > g ) were defined as rare variants with a maf 0.005 in the 1000 genomes catalog , phase 3 ( table 3 ; supplementary fig . these changes were each identified once in the 472 alleles analyzed . we noticed a striking difference in the frequencies of common abca4 variants ( maf 0.01 ) in the patient versus the control group ( supplementary table s2 ; fig . to estimate the independence of the associated variants , we fit a multivariate logistic regression model incorporating these variations . the remaining five risk - modulating variants were found to be independently associated with disease risk ( table 4 ) . repeating the analysis excluding the data from 52 samples of non - european or unknown ancestry , similar results were obtained , including the significant and independent association of the five risk - modulating variants ( c.1268a > g , c.5603a > t , c.5682g > c , c.5843c > t , c.6249c > t ) with similar effect sizes . association of common abca4 variants with stargardt disease assuming that common pathogenic mutations emerged on common haplotypes tagged by associated variants , our analysis may measure their impact on disease risk rather than that of the common associated variant . we therefore evaluated the correlation between common mutations in our cohort ( c.1622t > c , c.2588g > c , c.3113c > t , c.4234c > t , c.5461 - 10t > c , c.5714 + 5g > a , c.5882g > a , c.5917delg ) and the five risk - modulating variants ( c.1268a > g , c.5603a > t , c.5682g > c , three frequent mutations ( c.2588g > c , c.5461 - 10t > c , c.5882g > a ) were found to be potentially correlated ( r > 0.05 ) with common stgd1-associated variants . to further evaluate the role of these common variants in retinal pathology , a grs was calculated for the stgd1 patients in our cohort and the 781 control individuals . this was computed as the sum of risk - increasing and protective alleles weighted by the relative effect size of each of the risk - modulating variants from the multivariate logistic regression model . 2 ) , implying a risk - increasing effect of common abca4 alleles when combined with a single defective copy of the abca4 gene . genetic risk score ( grs ) for patients and controls stratified according to the number of abca4 mutations or disease status . our study describes the findings of mutational profiling of the abca4 gene in the largest cohort of stgd1 patients published so far . a total of 171 unique rare sequence variants , 48 of which constitute novel , so far unreported , mutations , were identified in the coding region , and an additional four rare deep intronic variants were detected in intron 30 and 36 of the abca4 gene . in addition , small deletions / insertions may have been missed due to limitations in the detection accuracy of this type of mutation by the resequencing array and ion torrent semiconductor technology . an important question relates to the high proportion of carriers of single abca4 mutations consistently observed in numerous studies regardless of the ethnic background of the patients . a single mutant abca4 allele was found in almost 30% of the 335 individuals in our patient group , a number representing the upper end of the reported ranges . mutations in noncoding regions of the abca4 gene locus have been proposed as a common source for a second causative mutation . more specifically , several studies have described the occurrence of deep intronic variants in intron 30 and 36 in a significant proportion of patients ( 7.917.9% ) with single abca4 mutations . in our cohort , of 95 stgd patients with a single pathogenic / likely pathogenic abca4 mutation , only 2 were found to carry an additional rare intronic variant in intron 30 or 36 ( 2.1% ) . two other novel rare intronic variants were detected in a patient with two causative abca4 mutations and a patient with no abca4 mutations , respectively . from these data , we conclude that causative abca4 mutations in intron 30 and 36 may contribute to stgd1 pathogenesis only in a minority of cases . similarly , in a small german cohort of 46 patients with single heterozygous abca4 variants , no mutations were found in these intronic regions . to further add to the discussion on the impact of deep intronic variants to stgd1 disease , the full sequence analysis of the entire abca4 locus including exonic as well as intronic sequences will be needed independent of the patient 's mutation status . considering that 7.6% of stgd1 patients of a belgian cohort carried a founder deep intronic c.4539 + 2001g > a mutation , it is conceivable that a significant fraction of the german stgd1 patients carry as yet unknown mutations in the noncoding regions of the abca4 gene . to address the strikingly high frequency of carriers with only a single identified abca4 disease allele , we assessed the contribution of common abca4 variants on disease development . our data now facilitate a grs estimate that coalesces the total effect of protective and risk - increasing alleles similarly to the model developed to assess the genetic risk for amd . interestingly , the grs in the group of patients with only a single disease - associated abca4 mutation is significantly higher than in patients with no abca4 mutation , suggesting that common variants are involved in conferring risk for stgd1 . this could be due to linkage disequilibrium between certain pathogenic mutations and c.5682g > c , implying that the common abca4 allele either has no effect on the pathogenicity of the rare variant or adds to the damaging effect of the mutation . fourteen stgd1 patients in our study each harbor two null mutations in the abca4 gene . taken together , future studies aiming at investigation of the influence of common variations and rare mutations on disease risk are necessary , ideally in a prospective cohort setting . first , we expanded the mutational spectrum of stgd1 by identifying an additional 48 novel pathogenic / likely pathogenic gene variants in abca4 . our findings also add to the clarification of the role of deep intronic variants in putative alternative exons within intron 30 and 36 , which seem to represent only a minor proportion of disease - associated variants . finally , an in - depth analysis of common variants at the abca4 gene locus revealed risk - altering properties for several common variants , particularly in those patients who carry only a single disease - associated abca4 allele .